@article{weerarathne_maker_huang_taylor_cowan_hyatt_selvan_shatnawi_thomas_meinkoth_et al._2023, title={A Novel Vaccine Strategy to Prevent Cytauxzoonosis in Domestic Cats}, volume={11}, ISSN={["2076-393X"]}, DOI={10.3390/vaccines11030573}, abstractNote={Cytauxzoonosis is caused by Cytauxzoon felis (C. felis), a tick-borne parasite that causes severe disease in domestic cats in the United States. Currently, there is no vaccine to prevent this fatal disease, as traditional vaccine development strategies have been limited by the inability to culture this parasite in vitro. Here, we used a replication-defective human adenoviral vector (AdHu5) to deliver C. felis-specific immunogenic antigens and induce a cell-mediated and humoral immune response in cats. Cats (n = 6 per group) received either the vaccine or placebo in two doses, 4 weeks apart, followed by experimental challenge with C. felis at 5 weeks post-second dose. While the vaccine induced significant cell-mediated and humoral immune responses in immunized cats, it did not ultimately prevent infection with C. felis. However, immunization significantly delayed the onset of clinical signs and reduced febrility during C. felis infection. This AdHu5 vaccine platform shows promising results as a vaccination strategy against cytauxzoonosis.}, number={3}, journal={VACCINES}, author={Weerarathne, Pabasara and Maker, Rebekah and Huang, Chaoqun and Taylor, Brianne and Cowan, Shannon R. and Hyatt, Julia and Selvan, Miruthula Tamil and Shatnawi, Shoroq and Thomas, Jennifer E. and Meinkoth, James H. and et al.}, year={2023}, month={Mar} }
 @article{yang_ladouceur_baumgartner_marr_karounos_robertson_whitehurst_miller_birkenheuer_2023, title={A practical protocol to prepare paraffin-embedded whole tick histology sections}, volume={14}, ISSN={["1877-9603"]}, url={https://doi.org/10.1016/j.ttbdis.2023.102162}, DOI={10.1016/j.ttbdis.2023.102162}, abstractNote={Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases worldwide. With increasing occurrences of tick-borne diseases in both humans and veterinary species, there is a continuous need to further our understanding of ticks and the pathogens they transmit. Whole tick histology provides a full scope of the tick internal anatomy, allowing researchers to examine multiple organs of interest in a single section. This is in contrast to other techniques that are more commonly utilized in tick-borne disease research, such as electron microscopy and light microscopy of individual organs. There is a lack of literature describing a practical technique to process whole tick histologic sections. Therefore, the current study aims to provide researchers with a workable protocol to prepare high quality paraffin-embedded whole tick histology sections. Amblyomma americanum adults were used as an example species for this study. After a series of pilot experiments using a combination of various fixatives, softening agents and processing techniques, we elected to compare two common fixatives, 10% neutral-buffered formalin (NBF) and Bouin's solution for whole ticks. Equal numbers of A. americanum unfed adults (n = 10/fixative) were processed identically and their whole tick histology coronal sections were individually scored. Higher scores were assigned to whole tick sections that contained more internal organs that are crucial for tick-borne disease research (e.g. salivary glands and midgut), high integrity of tissues and exoskeleton on the section, and good fixation and staining quality of the tissues. The mean total scores for Bouin's-fixed ticks were significantly higher compared to NBF-fixed ticks (p = 0.001). To further assess our preferred technique, we also demonstrated the feasibility of producing high quality whole tick sections for three other common tick species of medical importance (Rhipicephalus sanguineus, Ixodes scapularis, and Dermacentor variabilis) using Bouin's solution. While this technique may require further optimization for other tick species, we described a feasible protocol that uses commonly available tools, reagents and standard histologic equipment. This should allow any investigator to easily make adjustments to this protocol as needed based on their experimental goals.}, number={4}, journal={TICKS AND TICK-BORNE DISEASES}, author={Yang, Tzushan S. and LaDouceur, Elise E. B. and Baumgartner, Wes A. and Marr, Henry S. and Karounos, Michael and Robertson, James and Whitehurst, Nathan and Miller, Laura S. and Birkenheuer, Adam J.}, year={2023}, month={Jul} }
 @article{yang_reichard_thomas_miller_marr_karounos_bell_birkenheuer_2023, title={Cytauxzoon felis in salivary glands of Amblyomma americanum}, volume={14}, ISSN={["1877-9603"]}, DOI={10.1016/j.ttbdis.2022.102056}, abstractNote={Cytauxzoon felis is a tick-borne piroplasmid hemoparasite that causes life-threatening disease in cats. Despite the critical role that ticks play in pathogen transmission, our knowledge regarding the C. felis life cycle remains limited to the feline hosts. Specific life stages of C. felis within the tick host have never been visualized microscopically and previous investigations have been limited to molecular detection by polymerase chain reaction (PCR). Sporozoites are the infectious stage of piroplasmids that are transmitted by ticks. In other tick-borne piroplasmids, sporozoite-based vaccines play a key role in disease prevention and management. We believe sporozoites have similar potential for cytauxzoonosis. Therefore, the objective of this study was to use different molecular and microscopic techniques to detect and evaluate C. felis sporozoites in tick salivary glands (SG). A total of 140 Amblyomma americanum adults that were fed on C. felis-infected cats as nymphs were included for this study. Specifically, dissected SGs were quartered and subjected to C. felis RT-PCR, RNAscope® in situ hybridization (ISH), histology, direct azure staining, and transmission electron microscopy (TEM). Cytauxzoon felis RT-PCR was also performed on half tick (HT) carcasses after SG dissection. Cytauxzoon felis RNA was detected in SGs of 17/140 ticks. Of these, 7/17 ticks had microscopic visualization via ISH and/or TEM. The remaining 10/17 ticks had only molecular detection of C. felis in SGs via RT-PCR without visualization. Cytauxzoon felis RNA was detected solely in HT carcasses via RT-PCR in 9/140 ticks. In ISH-positive tick SGs, hybridization signals were present in cytoplasms of SG acinar cells. TEM captured rare C. felis organisms with characteristic ultrastructural features of sporozoites. This study describes the first direct visualization of any developing stage of C. felis in ticks. Forthcoming studies should employ a combination of molecular and microscopic techniques to investigate the C. felis life cycle in A. americanum.}, number={1}, journal={TICKS AND TICK-BORNE DISEASES}, author={Yang, Tzushan S. and Reichard, Mason V and Thomas, Jennifer E. and Miller, Laura S. and Marr, Henry S. and Karounos, Michael and Bell, Aaron J. and Birkenheuer, Adam J.}, year={2023}, month={Jan} }
 @article{kao_spainhour_cowan_nafe_birkenheuer_reichard_miller_2022, title={A Serodiagnostic IgM ELISA to Detect Acute Cytauxzoonosis}, volume={11}, ISSN={["2076-0817"]}, DOI={10.3390/pathogens11101183}, abstractNote={Cytauxzoonosis is a tick-borne infectious disease affecting domestic cats with high mortality and limited treatment modalities. Because early diagnosis and therapeutic intervention are crucial to survival of infected cats, the objective of this study was to develop an ELISA capable of detecting cytauxzoonosis and differentiating acute vs. chronic infection in clinical feline blood samples. A microsphere immunoassay (MIA) was developed to evaluate the production of Cytauxzoon felis-specific IgM and IgG antibodies in serial plasma samples from cats with experimental C. felis infection by targeting a C. felis-specific transmembrane protein (c88). Recombinant c88 protein was utilized to develop indirect ELISAs to detect IgM and IgG antibodies in clinical plasma samples from: PCR-positive cats with acute C. felis infection (n = 36), C. felis-negative cats with pyrexia (n = 10), healthy C. felis-negative cats (n = 22), and chronic C. felis carriers (n = 4). Anti-c88 IgM antibodies were detectable at day 12 post-tick infestation in cats with experimental C. felis infection (within 24 hours of developing clinical signs), while anti-c88 IgG was detectable at day 15 post-tick infestation - indicating IgM could be used to detect early infection. Using a cut-off value of 19.85 percent positive, the C. felis IgM ELISA detected acute cytauxzoonosis in 94.44% (34/36) of cats presented with clinical signs of acute cytauxzoonosis with 100% specificity (indicating a "Strong Positive" result). When a lower cutoff of 8.60 percent positive was used, cytauxzoonosis was detected in the 2 remaining PCR-positive cats with 87.88% specificity (indicating of a "Weak Positive" result). One C. felis-negative, febrile cat had high IgG, and chronic carriers had variable IgM and IgG results. Combined interpretation of IgM and IgG ELISAs did not reliably differentiate acute vs. chronic infection. While further validation on assay performance is needed, the C. felis IgM ELISA is a promising test to detect acute cytauxzoonosis and can be utilized to develop a point-of-care test for clinical use.}, number={10}, journal={PATHOGENS}, author={Kao, Yun-Fan and Spainhour, Rebecca and Cowan, Shannon R. and Nafe, Laura and Birkenheuer, Adam and Reichard, Mason V and Miller, Craig A.}, year={2022}, month={Oct} }
 @article{dear_birkenheuer_2022, title={Babesia in North America An Update}, volume={52}, ISSN={["1878-1306"]}, DOI={10.1016/j.cvsm.2022.07.016}, abstractNote={Canine babesiosis results from infection of 1 of 5 identified protozoal species in the United States (Babesia conradae, Babesia sp. "coco," Babesia gibsoni, Babesia vogeli, and Babesia vulpes). They are part of the Apicomplexa family of protozoa and are obligate intraerythrocytic parasites. Domestic and wild canids are suspected of being intermediate hosts. This updated article aims to provide practical guidance about the clinical manifestations of disease, treatment options, and outcomes. In addition, the authors hope to provide some clarity about the taxonomy and nomenclature of these organisms, as they have undergone multiple changes since their initial discovery.}, number={6}, journal={VETERINARY CLINICS OF NORTH AMERICA-SMALL ANIMAL PRACTICE}, author={Dear, Jonathan D. and Birkenheuer, Adam}, year={2022}, month={Nov}, pages={1193–1209} }
 @article{cerreta_yang_ramsay_birkenheuer_rahoi_qurollo_wilson_cushing_2022, title={DETECTION OF VECTOR-BORNE INFECTIONS IN LIONS AND TIGERS AT TWO ZOOS IN TENNESSEE AND OKLAHOMA, USA}, volume={53}, ISSN={["1937-2825"]}, DOI={10.1638/2020-0199}, abstractNote={Protozoal and bacterial vector-borne infections are frequently diagnosed in domestic felids. However, with the exception of Mycoplasma haemofelis and Cytauxzoon felis, their occurrence in managed nondomestic felids housed in the United States is largely unknown. Following a case in February 2020 of fulminant cytauxzoonosis in an African lion (Panthera leo), EDTA-whole blood samples were collected opportunistically from February 2020 through June 2020 from 34 adult tigers (Panthera tigris) and eight adult African lions from the same sanctuary in eastern Tennessee as well as 14 adult tigers from a zoo in southern Oklahoma. Samples were analyzed for Cytauxzoon felis, Bartonella spp., hemotropic Mycoplasma, Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp., and Hepatozoon spp. DNA by PCR amplification. All animals were asymptomatic at the time of collection. None of the Oklahoma animals were positive for vector-borne organisms, but these pathogens were detected in tigers at the Tennessee facility, including Cytauxzoon felis (11.8%), "Candidatus Mycoplasma haemominutum" (5.9%), and Ehrlichia ewingii (2.9%). During the study period, two animals developed clinical signs of cytauxzoonosis and were assessed for vector-borne infections as part of their diagnostic evaluation. This study documents the presence of tick-borne diseases in managed nondomestic felids in the southeastern United States and underscores that ectoparasite control measures should be practiced to minimize exposure of carnivores in managed care.}, number={1}, journal={JOURNAL OF ZOO AND WILDLIFE MEDICINE}, author={Cerreta, Anthony J. and Yang, Tzushan S. and Ramsay, Edward C. and Birkenheuer, Adam J. and Rahoi, Dane and Qurollo, Barbara and Wilson, James and Cushing, Andrew C.}, year={2022}, month={Mar}, pages={50–59} }
 @article{yang_reichard_marr_cohn_nafe_whitehurst_birkenheuer_2022, title={Direct injection of Amblyomma americanum ticks with Cytauxzoon felis}, volume={13}, ISSN={["1877-9603"]}, DOI={10.1016/j.ttbdis.2021.101847}, abstractNote={Cytauxzoon felis is a tick-borne hemoprotozoan parasite that causes life-threatening disease in domestic cats in the United States. Currently, the platforms for C. felis research are limited to natural or experimental infection of domestic cats. This study aims to develop an alternative model by infecting Amblyomma americanum ticks with C. felis via direct injection. Amblyomma americanum adults were injected with C. felis-infected feline erythrocytes through two routes: directly into the digestive tract through the anal pore (IA injection), or percutaneously into the tick hemocoel (IH injection). RNAscope® in situ hybridization (ISH) was used to visualize the parasites within the ticks at different time points after injection. Four months after injection, ticks were divided into 3 infestation groups based on injection methods and inoculum type and fed on 3 naïve cats to assess the ticks' ability to transmit C. felis. Prior to the transmission challenge, selected ticks from each infestation group were tested for C. felis RNA via reverse transcription-PCR (RT-PCR). In both IA- and IH-injected ticks, ISH signals were observed in ticks up to 3 weeks after injection. The number of hybridization signals notably decreased over time, and no signals were detected by 4 months after injection. Prior to the transmission challenge, 37-57% of the sampled ticks were positive for C. felis RNA via RT-PCR. While the majority of injected ticks successfully attached and fed to repletion on all 3 cats during the transmission challenge, none of the cats became infected with C. felis. These results suggest that injected C. felis remained alive in ticks but was unable to progress to infective sporozoites after injection. It is unclear why this infection technique had been successful for other closely related tick-borne hemoprotozoa and not for C. felis. This outcome may be associated with uncharacterized differences in the C. felis life cycle, the lack of the feeding or molting in our model or absence of gametocytes in the inoculum. Nonetheless, our study demonstrated the potential of using ticks as an alternative model to study C. felis. Future improvement of a tick model for C. felis should consider other tick species for the injection model or utilize infection methods that more closely emulate the natural infection process.}, number={1}, journal={TICKS AND TICK-BORNE DISEASES}, author={Yang, Tzushan S. and Reichard, Mason V and Marr, Henry S. and Cohn, Leah A. and Nafe, Laura and Whitehurst, Nathan and Birkenheuer, Adam J.}, year={2022}, month={Jan} }
 @article{garrett_halseth_ruder_beasley_shock_birkenheuer_gabriel_fiorello_haire_olfenbuttel_et al._2022, title={Prevalence and genetic characterization of a Babesia microti-like species in the North American river otter (Lontra canadensis)}, volume={29}, ISSN={["2405-9390"]}, DOI={10.1016/j.vprsr.2022.100696}, abstractNote={A 4.5-month-old, male, North American river otter (Lontra canadensis) from Athens-Clarke County, Georgia, USA being temporarily housed at a rehabilitation facility, presented with a three-day history of lethargy, anorexia, and severe anemia. Antemortem blood smears revealed intraerythrocytic piroplasms. Supportive care and antiparasitic treatments were initiated, but the animal died three days following presentation. Gross necropsy revealed yellow discoloration of all adipose tissue throughout the carcass and a mildly enlarged, diffusely yellow to pale orange liver. Microscopically, moderate, centrilobular hepatocellular degeneration and necrosis were observed, consistent with hypoxia secondary to apparent hemolytic anemia. Piroplasms were frequently observed in red blood cells in histologic sections. The nearly full-length 18S rRNA gene sequence (1588 bp) was identical to a previously described piroplasm from North American river otters from North Carolina. Phylogenetically, based on the 18S rRNA gene sequence, the otter Babesia sp. was in a sister group with a clade that included several strains of Babesia microti-like species including Babesia sp. from badgers (Meles meles), Babesia vulpes, and Babesia sp. from raccoons (Procyon lotor). To better understand the distribution and genetic variability of this Babesia species, otters from four states in the eastern U.S. and California were tested. Overall, 30 of 57 (53%) otters were positive for Babesia sp. None of four otters from California were positive, but prevalences in eastern states were generally high, 5/9 (55%) in Georgia, 7/14 (50%) in South Carolina, 10/17 (59%) in North Carolina, and 8/13 (62%) in Pennsylvania). Partial 18S rRNA gene sequences from all populations were identical to the clinical case sequence. No Babesia sensu stricto infections were detected. There were six unique COI sequences (937 bp) detected in 18 positive otters. The most common lineage (A) was detected in 12 of 18 (67%) samples from Georgia, North Carolina, South Carolina, and Pennsylvania. Lineage B was found in two otters and the remaining lineage types were found in single otters. These six lineages were 99-99.8% similar to each other and were < 88% similar to related parasites such as B. vulpes, B. microti-like species of raccoons, B. microti, and B. rodhaini. Phylogenetically, the Babesia sp. of otters grouped together in a well-supported clade separate from a sister group including B. vulpes from fox (Vulpes vulpes) and domestic dogs. In conclusion, this report demonstrates that this piroplasm is a potential pathogen of North American river otters and the parasite is widespread in otter populations in the eastern United States.}, journal={VETERINARY PARASITOLOGY- REGIONAL STUDIES AND REPORTS}, author={Garrett, Kayla and Halseth, Ashlyn and Ruder, Mark G. and Beasley, James and Shock, Barbara and Birkenheuer, Adam J. and Gabriel, Mourad and Fiorello, Christine and Haire, M. Melanie and Olfenbuttel, Colleen and et al.}, year={2022}, month={Apr} }
 @article{hedgespeth_birkenheuer_friedenberg_olby_meurs_2021, title={A novel missense mutation of the NAT10 gene in a juvenile Schnauzer dog with chronic respiratory tract infections}, volume={35}, ISSN={["1939-1676"]}, url={https://doi.org/10.1111/jvim.16100}, DOI={10.1111/jvim.16100}, abstractNote={An 18-month-old intact male Schnauzer dog was evaluated for chronic, lifelong respiratory tract infections that were unresponsive to administration of a variety of antibiotics and corticosteroids. The dog developed persistent vomiting and diarrhea around 1 year of age that was minimally responsive to diet change, antibiotics, and corticosteroids. Despite supportive care, the dog was ultimately euthanized at 20 months of age due to persistent respiratory and gastrointestinal disease. Whole genome sequencing discovered a deleterious missense A/C mutation within the NAT10 gene, a gene essential for microtubule acetylation, appropriate ciliary development, and cytokinesis. Pipeline analysis of the genomes of 579 dogs from 55 breeds did not detect this mutation. Though never described in veterinary medicine, NAT10 mutation occurs in humans with ciliary aplasia, suggesting a pathophysiological mechanism for this dog and highlighting an associated mutation or possible novel genetic cause of chronic respiratory infections in dogs.}, number={3}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Hedgespeth, Barry A. and Birkenheuer, Adam J. and Friedenberg, Steven G. and Olby, Natasha J. and Meurs, Kathryn M.}, year={2021}, month={May}, pages={1542–1546} }
 @article{baneth_nachum-biala_birkenheuer_schreeg_prince_florin-christensen_schnittger_aroch_2020, title={A new piroplasmid species infecting dogs: morphological and molecular characterization and pathogeny of Babesia negevi n. sp.}, volume={13}, ISSN={["1756-3305"]}, DOI={10.1186/s13071-020-3995-5}, abstractNote={Abstract Introduction Babesiosis is a protozoan tick-borne infection associated with anemia and life-threatening disease in humans, domestic and wildlife animals. Dogs are infected by at least six well-characterized Babesia spp. that cause clinical disease. Infection with a piroplasmid species was detected by light microscopy of stained blood smears from five sick dogs from Israel and prompted an investigation on the parasite’s identity. Methods Genetic characterization of the piroplasmid was performed by PCR amplification of the 18S rRNA and the cytochrome c oxidase subunit 1 ( cox 1) genes, DNA sequencing and phylogenetic analysis. Four of the dogs were co-infected with Borrelia persica (Dschunkowsky, 1913), a relapsing fever spirochete transmitted by the argasid tick Ornithodoros tholozani Laboulbène & Mégnin. Co-infection of dogs with B. persica raised the possibility of transmission by O. tholozani and therefore, a piroplasmid PCR survey of ticks from this species was performed. Results The infected dogs presented with fever (4/5), anemia, thrombocytopenia (4/5) and icterus (3/5). Comparison of the 18S rRNA and cox 1 piroplasmid gene sequences revealed 99–100% identity between sequences amplified from different dogs and ticks. Phylogenetic trees demonstrated a previously undescribed species of Babesia belonging to the western group of Babesia ( sensu lato ) and closely related to the human pathogen Babesia duncani Conrad, Kjemtrup, Carreno, Thomford, Wainwright, Eberhard, Quick, Telfrom & Herwalt, 2006 while more moderately related to Babesia conradae Kjemtrup, Wainwright, Miller, Penzhorn & Carreno, 2006 which infects dogs. The piroplasm forms detected included tetrads (Maltese cross), merozoite and trophozoite stages whose average size was larger than stages of other canine Babesia spp. belonging to the Babesia ( s.l .) and B. gibsoni Patton, 1910, and smaller than other canine Babesia ( sensu stricto ) spp. Of 212 O. tholozani ticks surveyed, 11 (5.2%) harbored DNA of the new species of Babesia . Conclusions Babesia negevi n. sp. is described based on morphological and genetic characterization and phylogenetic analyses. The species is named after the Negev desert of southern Israel, where the first infected dog originated from. Despite co-infection in four dogs, the fifth dog had fatal disease attesting that B. negevi n. sp. infection requires clinical attention. Incriminating O. tholozani or another tick species as the vector of Babesia negevi n. sp., would require additional studies.}, number={1}, journal={PARASITES & VECTORS}, author={Baneth, Gad and Nachum-Biala, Yaarit and Birkenheuer, Adam Joseph and Schreeg, Megan Elizabeth and Prince, Hagar and Florin-Christensen, Monica and Schnittger, Leonhard and Aroch, Itamar}, year={2020}, month={Apr} }
 @article{hartley_marr_birkenheuer_2020, title={Cytauxzoon felis cytochrome b gene mutation associated with atovaquone and azithromycin treatment}, volume={34}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.15935}, abstractNote={Background Atovaquone and azithromycin (A&A) with supportive care improve survival rates in cats with cytauxzoonosis. Resistance to atovaquone via parasite cytochrome b gene (cytb) mutations occurs in other Apicomplexan protozoans but is not described in Cytauxzoon felis. Objective To serially characterize the C. felis cytb sequences from a cat that remained persistently infected after A&A treatment. Animal A cat with naturally occurring C. felis infection. Methods Case report of the anemic cat persistently infected with C. felis before, during and after A&A treatment. Cytauxzoon felis cytb genes were amplified and sequenced before, during and after A&A treatment. Results Cytauxzoon felis was detected before, during and after A&A treatment including samples collected 570 days after treatment. After A&A treatment, the cat's anemia improved slightly. Cytb sequencing revealed only wild-type cytb methionine (M128) in samples collected before treatment. In samples collected after treatment, the cytb coded for isoleucine (M128I) and valine (M128I) at 2- and 4-months after treatment. These M128I and M128V mutations persisted even after a repeat treatment course with a higher dose atovaquone combined with the standard dose of azithromycin. Conclusions and clinical importance This report documents C. felis atovaquone resistance associated with M128 cytb mutations. This study suggests parasites with mutations of cytb M128 can be selected and impart resistance to A&A treatment even with higher atovaquone dosing.}, number={6}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Hartley, Ashley N. and Marr, Henry S. and Birkenheuer, Adam J.}, year={2020}, month={Nov}, pages={2432–2437} }
 @article{birkenheuer_buch_beall_braff_chandrashekar_2020, title={Global distribution of canine Babesia species identified by a commercial diagnostic laboratory}, volume={22}, ISSN={["2405-9390"]}, DOI={10.1016/j.vprsr.2020.100471}, abstractNote={Babesia species are important canine pathogens with a nearly worldwide distribution. Our understanding of the distribution of these parasites is continually improving. This is in large part, due to improved molecular diagnostic capabilities. However, it can be difficult to assimilate and compare previous reports from various regions due to differences in molecular methods. In this report, we characterize the results of over 100,000 canine samples from 52 different countries and territories spanning 4 continents that were submitted to a commercial diagnostic laboratory for Babesia testing by polymerase chain reaction. The same diagnostic algorithm was used for all samples and is designed to identify and differentiate B. gibsoni, B. canis, B. vogeli, B. rossi and B. conradae. Overall 3.4% of the samples submitted tested positive for the presence of Babesia sp. DNA and were differentiated to the species level. Babesia gibsoni was the most commonly identified species (48.8% of the positive results) followed by B. canis (35.2%) then B. vogeli (15.3%). Babesia gibsoni and B. vogeli were more widely distributed than B. canis, which was primarily found in Europe. This is the largest study of its type and these data provide a global overview of which Babesia species veterinarians could expect to find in their practice area.}, journal={VETERINARY PARASITOLOGY- REGIONAL STUDIES AND REPORTS}, author={Birkenheuer, Adam J. and Buch, Jesse and Beall, Melissa J. and Braff, Jennifer and Chandrashekar, Ramaswamy}, year={2020}, month={Dec} }
 @article{birkenheuer_royal_cerreta_hemstreet_lunn_gookin_mcgarvey_2020, title={Perceptions and attitudes of Small Animal Internal Medicine specialists toward the publication requirement for board certification}, volume={34}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.15717}, abstractNote={Background The publication requirement for board certification in Small Animal Internal Medicine (SAIM) by the ACVIM is controversial. Objectives Directly and indirectly evaluate the perceptions SAIM Diplomates have on the publication requirement. A secondary objective was to compare the frequency with which publications submitted for credentialing purposes (CredPubs) were cited compared to control articles. Subjects One thousand two hundred forty-one SAIM Diplomates were sent an electronic survey. Methods A electronic survey was sent to all SAIM Diplomates. Practice websites were evaluated for reference to publication or research. An electronic database was searched to identify the number of times a subset of CredPubs were cited was compared to control articles. Results Five hundred six individuals responded. The majority of respondents (n = 428, 85.25%) stated the requirement should be retained either with no changes (n = 186, 37.05%) or with clarifications or modifications (n = 242, 48.21%). A minority of respondents (n = 74, 14.7%) felt it should be eliminated. “Understanding the scientific process” was the most commonly selected reason (n = 467, 92.48%) for the publication requirement. All websites that mentioned research or publication did so using a positive sentiment. With regard to relative citation rates; 17% of CredPubs were in the lower quartile, 59.1% of CredPubs were in the interquartile range, and 23.5% were in the upper quartile compared to control articles. Conclusions and Clinical Importance A majority of SAIM Diplomates favored the retention of the publication requirement in some form. CredPubs were cited at rates similar to control articles.}, number={2}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Birkenheuer, Adam J. and Royal, Kenneth D. and Cerreta, Anthony and Hemstreet, Daniel and Lunn, Katharine F. and Gookin, Jody L. and McGarvey, Stephanie}, year={2020}, month={Mar}, pages={574–580} }
 @article{cohn_shaw_shoemake_birkenheuer_2020, title={Second illness due to subsequent Cytauxzoon felis infection in a domestic cat}, DOI={10.1177/2055116920908963}, abstractNote={A castrated male domestic shorthair cat from a wooded area in Missouri had recovered from typical severe cytauxzoonosis at 4 years of age, after intensive in-hospital supportive care and administration of atovaquone and azithromycin. At 11 years of age, the same cat again experienced an acute febrile illness compatible with cytauxzoonosis. Intraerythrocytic piroplasms typical of Cytauxzoon felis were identified by cytology. The owners opted for euthanasia but allowed collection of splenic and hepatic tissue for histopathologic examination. Schizont-laden macrophages were identified in both tissue specimens, confirming active cytauxzoonosis at the time of the cat's death.Although cats that have recovered from cytauxzoonosis can harbor red blood cell piroplasms for many years without apparent clinical illness, repeat illness owing to either disease recrudescence or repeat infection has never been documented. In fact, recovered cats have been thought to be resistant to reinfection and subsequent illness. This report describes a cat that had recovered from documented cytauxzoonosis 7 years previously and then developed a subsequent clinical illness typical of cytauxzoonosis, which was accompanied not only by intraerythrocytic piroplasms, but also by schizont-laden tissue macrophages pathognomonic of clinical cytauxzoonosis.}, journal={Journal of Feline Medicine and Surgery Open Reports}, author={Cohn, Leah A. and Shaw, Dan and Shoemake, Catherine and Birkenheuer, Adam J.}, year={2020} }
 @article{easley_holowaychuk_lashnits_nordone_marr_birkenheuer_2020, title={Serum procalcitonin concentrations in dogs with induced endotoxemia}, volume={1}, url={https://doi.org/10.1111/jvim.15711}, DOI={10.1111/jvim.15711}, abstractNote={Background Procalcitonin (PCT) is an important biomarker for sepsis in human medicine, but there is little information regarding PCT as a biomarker for sepsis in dogs. There are no controlled studies evaluating serial concentrations of PCT in dogs. Hypothesis/objective That PCT would be rapidly detectable in serum after injection of LPS and would remain increased for at least 24 hours. Objective was to evaluate serial serum PCT concentrations in dogs after a single IV injection of LPS compared to placebo. Animals Six healthy mixed breed dogs. Methods A nonrandomized, placebo-controlled, crossover study was performed. Dogs were initially injected with placebo (0.9% NaCl; 1 mL, IV) and then experimental endotoxemia was induced by injecting lipopolysaccharide (LPS; 2 μg/kg, IV, once) after a 5-day washout period. Serial blood samples were collected for measurement of serum PCT after each injection. Difference in median PCT concentration between serial time points was assessed using a mixed effects model. Results After LPS administration, blood pressure decreased and body temperature increased along with the development of lethargy, vomiting, and diarrhea. Procalcitonin was significantly increased compared to baseline by 2 hours after injection of LPS (median = 67.9 versus 172.8, range = 46.0-74.1 versus 99.5-295.9, P = .0002) and remained significantly increased for 12 hours (median = 205.9, range = 119.9-297.4) with return to baseline by 48 hours. Procalcitonin was significantly higher than placebo 2, 4, 6, 8, 10, 12, and 24 hours after injection. There were no significant differences in PCT between time 0 and any of the subsequent time points in the saline group. Conclusions and clinical importance Procalcitonin expression is likely to be a clinically useful biomarker for sepsis in dogs and might have an additional role in prognostication and therapeutic decision-making.}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Easley, Frankie and Holowaychuk, Marie K. and Lashnits, Erin W. and Nordone, Shila K. and Marr, Henry and Birkenheuer, Adam J.}, year={2020} }
 @article{kendall_keenihan_kern_lindaberry_birkenheuer_moore_vaden_2020, title={Three-dimensional bladder ultrasound for estimation of urine volume in dogs compared with traditional 2-dimensional ultrasound methods}, volume={34}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.15959}, abstractNote={Background Although point-of-care volumetric assessments of the urinary bladder are not routinely performed in dogs, urine volume quantification can provide important clinical information including noninvasive urine output estimation. Hypothesis/objective Use of 3-dimensional (3D) ultrasound for determination of urinary bladder volume (UBV) in dogs will be accurate for different bladder volumes and will decrease the need for operator skill in measuring UBV compared to 2-dimensional (2D) ultrasound evaluation. Animals Ten laboratory-bred Beagle dogs. Methods Prospective, experimental study. Urinary bladders were infused with a calculated amount of sterile saline to represent small, medium, and large volumes. Each UBV was estimated and calculated by a board-certified veterinary radiologist using 3 different 2D ultrasound formulas followed by use of a 3D ultrasound device by a novice. Measured UBVs were compared to the instilled UBV for both 2D and 3D ultrasound methods. Time from start to end of examination was recorded for both ultrasound methods in a subset of dogs. Results The 3D ultrasound device underestimated UBV with a mean difference of -9.8 mL compared with 2D ultrasound that overestimated UBV with a difference of +4.2 to 20.3 mL dependent on the 2D formula used. The 3D ultrasound method took less time to measure UBV (mean of 80 seconds per measurement) compared to the 2D method (165 seconds per measurement; P = .02). Conclusions and clinical importance The tested 3D ultrasound device was found to be an accurate and rapid point-of-care tool for measuring UBV in dogs, providing a noninvasive method to estimate bladder volume in real time.}, number={6}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Kendall, Allison and Keenihan, Erin and Kern, Zachary T. and Lindaberry, Crystal and Birkenheuer, Adam and Moore, George E. and Vaden, Shelly L.}, year={2020}, month={Nov}, pages={2460–2467} }
 @article{neupane_sevala_balakrishnan_marr_wilson_maggi_birkenheuer_lappin_chomel_breitschwerdt_2020, title={Validation of Bartonella henselae Western Immunoblotting for Serodiagnosis of Bartonelloses in Dogs}, volume={58}, ISSN={["1098-660X"]}, DOI={10.1128/JCM.01335-19}, abstractNote={Bartonella spp. are etiological agents of life-threatening zoonotic diseases in dogs worldwide. Due to the poor sensitivity of immunofluorescent-antibody assays (IFAs), a reliable serodiagnostic test for canine bartonelloses is of clinical importance. The utility of Western blotting (WB) for the serodiagnosis of canine bartonelloses has not been critically investigated. The objective of this study was to characterize WB immunodominant proteins that could be used to confirm a serodiagnosis of bartonelloses.}, number={4}, journal={JOURNAL OF CLINICAL MICROBIOLOGY}, author={Neupane, Pradeep and Sevala, Sindhura and Balakrishnan, Nandhakumar and Marr, Henry and Wilson, James and Maggi, Ricardo and Birkenheuer, Adam and Lappin, Michael and Chomel, Bruno and Breitschwerdt, Edward B.}, year={2020}, month={Apr} }
 @article{garden_kidd_mexas_chang_jeffery_blois_fogle_macneill_lubas_birkenheuer_et al._2019, title={ACVIM consensus statement on the diagnosis of immune-mediated hemolytic anemia in dogs and cats}, volume={33}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.15441}, abstractNote={Immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune-mediated erythrocyte destruction, and adverse consequences of long-term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence-based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.}, number={2}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Garden, Oliver A. and Kidd, Linda and Mexas, Angela M. and Chang, Yu-Mei and Jeffery, Unity and Blois, Shauna L. and Fogle, Jonathan E. and MacNeill, Amy L. and Lubas, George and Birkenheuer, Adam and et al.}, year={2019}, pages={313–334} }
 @article{divincenti_garner_thomas_birkenheuer_2019, title={Babesia sp. infection in a zoo-housed polar bear (Ursus maritimus)}, DOI={10.1016/j.vprsr.2019.100350}, abstractNote={A 28-year-old female polar bear (Ursus maritimus) housed in a zoo in Upstate New York presented with acute inappetence and lethargy. The bear's condition rapidly deteriorated, and because laboratory testing indicated severe hepatic and renal disease, the bear was humanely euthanized. Examination of a blood smear from a sample collected just prior to euthanasia revealed the presence of intra-erythrocytic inclusions, which were identified as Babesia sp. by PCR. Although it is unclear if babesiosis contributed to this bear's clinical signs, this is the first report of Babesia sp. infection in this species. Zoological institutions exhibiting polar bears and located in tick-endemic areas, as well as managers of wild populations, should be aware of this species' susceptibility to babesiosis.}, journal={Veterinary Parasitology: Regional Studies and Reports}, author={DiVincenti, Louis and Garner, Michael and Thomas, Brittany and Birkenheuer, Adam}, year={2019} }
 @article{naor_lindemann_schreeg_marr_birkenheuer_carpenter_ryseff_2019, title={Clinical, morphological, and molecular characterization of an undetermined Babesia species in a maned wolf (Chrysocyon brachyurus)}, volume={10}, ISSN={["1877-9603"]}, DOI={10.1016/j.ttbdis.2018.09.005}, abstractNote={A possible novel Babesia species infection of a maned wolf (Chrysocyon brachyurus) was first reported in 2012. The current case details a confirmed report of a maned wolf with infection by an undetermined species of Babesia. As the mortality and morbidity of babesiosis is high, this may become a significant concern to captive maned wolves, which are considered a near-threatened species by the World Association of Zoos and Aquariums. The aim of this study is to report the clinical, morphological and molecular characterization of this Babesia species. A 2.5-year-old, intact female maned wolf was found laterally recumbent with pale mucous membranes and jaundice the morning of presentation. Hematological and serum biochemical data were consistent with babesiosis and showed a regenerative severe anemia, leukocytosis, thrombocytopenia, hyperbilirubinemia, azotemia, increased creatine phosphokinase and increase alanine aminotransferase. On blood film review, inclusion bodies were seen in the red blood cells with cytomorphological features that were most consistent with a small form Babesia species. A blood sample was sent for polymerase chain reaction (PCR) testing and multi-locus sequence analyses. These findings suggested a unique Babesia species that is most closely related to a Babesia species (Babesia sp. AJB-2006) that has been found to infect raccoons (Procyon lotor) in North America. Although the cytomorphological features of the piroplasms and the clinical presentation were similar in both the current and 2012 case, when comparing the 18S melt curve temperature of the two Babesia isolates, the peak temperature was different. Unfortunately, genetic material from the 2012 case was not available so comparison of multi-locus gene sequences could not be performed, excluding the possibility to definitively state if the Babesia spp. from both cases were distinct from each other. The maned wolf was treated with a whole blood transfusion, dexamethazone (0.28 mg/kg IM), azithromycin (10 mg/kg in NaCl SC), atavaquone (1.5 cc PO), and 2 imidocarb (6.6 mg/kg IM) injections, and clinically improved. These findings demonstrate the need to further characterize the molecular and epidemiological differences of the Babesia species in this case report and the Babesia species known to infect raccoons.}, number={1}, journal={TICKS AND TICK-BORNE DISEASES}, author={Naor, Adi Wasserkrug and Lindemann, Dana M. and Schreeg, Megan E. and Marr, Henry S. and Birkenheuer, Adam J. and Carpenter, James W. and Ryseff, Julia K.}, year={2019}, month={Jan}, pages={124–126} }
 @article{barash_thomas_birkenheuer_breitschwerdt_lemler_qurollo_2019, title={Prevalence of
            Babesia
            spp. and clinical characteristics of
            Babesia vulpes
            infections in North American dogs}, volume={7}, ISSN={0891-6640 1939-1676}, url={http://dx.doi.org/10.1111/jvim.15560}, DOI={10.1111/jvim.15560}, abstractNote={Background Babesiosis is an important cause of thrombocytopenia and hemolytic anemia in dogs. Babesia vulpes, reported in European dogs and North American foxes, rarely has been reported in domestic North American dogs. Newly optimized polymerase chain reaction (PCR) primers facilitate more sensitive amplification of B. vulpes DNA. Objectives To determine the prevalence of Babesia sp. infections in dogs being tested for Babesia infection, and to describe co-infections and clinicopathologic abnormalities in B. vulpes positive dogs. Animals Dog blood or tissue samples (n = 9367) submitted to a diagnostic laboratory between June 2015 and June 2018 were tested using an optimized Babesia PCR assay. Methods Comprehensive canine vector-borne disease diagnostic testing was performed on convenience samples. Results Babesia sp. DNA was amplified from 269/9367 (2.9%) North American dogs. Babesia sp. infections included B. gibsoni monoinfection (157; 1.7%), B. vulpes monoinfection (19; 0.20%), and B. gibsoni and B. vulpes coinfection (29; 0.31%). Forty-three of the 48 total B. vulpes-infected dogs were American Pit Bull Terrier-type breeds, of which 36 historically were involved with dog fights. Coinfections with Mycoplasma, Dirofilaria immitis, or Wolbachia and coexposures to Bartonella, Ehrlichia, and Rickettsia spp. were documented in B. vulpes-infected dogs. Clinicopathologic data in B. vulpes-infected dogs both with and without coinfections included anemia, thrombocytopenia, hyperglobulinemia, hypoalbuminemia, and proteinuria. Conclusions and clinical importance Babesia vulpes infection in domestic North American dogs is commonly found in conjunction with other coinfections, including B. gibsoni and hemotropic Mycoplasma. Similar to B. gibsoni, dog-to-dog transmission of B. vulpes may be a frequent mode of transmission.}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Barash, Nanelle R. and Thomas, Brittany and Birkenheuer, Adam J. and Breitschwerdt, Edward B. and Lemler, Erica and Qurollo, Barbara A.}, year={2019}, month={Jul} }
 @article{barash_birkenheuer_vaden_jacob_2018, title={Agreement between Parallel Canine Blood and Urine Cultures: Is Urine Culture the Poor Man's Blood Culture?}, volume={56}, ISSN={["1098-660X"]}, DOI={10.1128/JCM.00506-18}, abstractNote={Bloodstream infections are a significant cause of morbidity and mortality in critically ill dogs, but due to cost and difficulties in sample acquisition, blood cultures are infrequently obtained. In ill dogs, urine cultures may be recommended as surrogates for blood cultures.}, number={9}, journal={JOURNAL OF CLINICAL MICROBIOLOGY}, author={Barash, Nanelle R. and Birkenheuer, Adam J. and Vaden, Shelly L. and Jacob, Megan E.}, year={2018}, month={Sep} }
 @article{barash_birkenheuer_vaden_jacob_2018, title={Agreement between parallel canine blood and urine cultures: Is urine culture the poor man's blood culture?}, DOI={10.1128/JCM.00506-1810}, journal={Journal of Clinical Microbiology}, author={Barash, Nanelle R. and Birkenheuer, Adam J. and Vaden, Shelly L. and Jacob, Megan E.}, year={2018} }
 @article{ullal_birkenheuer_vaden_2018, title={Azotemia and Proteinuria in Dogs Infected with Babesia gibsoni}, volume={54}, ISSN={["1547-3317"]}, DOI={10.5326/jaaha-ms-6693}, abstractNote={ABSTRACT Babesiosis is a hemoprotozoal tick-borne disease that is commonly associated with thrombocytopenia and anemia; however, renal involvement has been documented in dogs. The purpose of this retrospective study was to document azotemia and proteinuria in dogs infected with Babesia sp. and to describe the response to antiprotozoal therapy. The electronic database of the North Carolina State University Vector Borne Disease Laboratory was searched to identify dogs who were diagnosed with babesiosis and to determine if they had proteinuria and/or azotemia. Dogs were excluded if they had coinfections or comorbidities known to cause glomerular injury. Of 35 dogs identified during the initial search, 5 were included; however, only 4 of these dogs had both pre- and posttreatment data. All five dogs were American pit bull terriers or American pit bull terrier-mixed breed dogs, were infected with Babesia gibsoni, and had hypoalbuminemia and proteinuria. Three dogs had azotemia. Responses to antiprotozoal treatment included normalization of (three) or increase in (one) serum albumin, resolution (one) or improvement (one) of azotemia, and reduction in proteinuria (two). Laboratory findings consistent with glomerular disease can be found in Babesia gibsoni-infected dogs, and treatment can lead to improvement of the azotemia and proteinuria.}, number={3}, journal={JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION}, author={Ullal, Tarini and Birkenheuer, Adam and Vaden, Shelly}, year={2018}, pages={156–160} }
 @article{birkenheuer_marr_wilson_breitschwerdt_qurollo_2018, title={Babesia gibsoni
 cytochrome b mutations in canine blood samples submitted to a US veterinary diagnostic laboratory}, volume={32}, ISSN={0891-6640}, url={http://dx.doi.org/10.1111/jvim.15300}, DOI={10.1111/jvim.15300}, abstractNote={Background Babesiosis caused by Babesia gibsoni is recognized throughout the world and can be difficult to treat. Resistance to atovaquone is associated with mutations in the B. gibsoni mitochondrial genome, specifically the M128 position of cytochrome b (cytb). The prevalence of cytb mutations in North America has not been reported. Hypothesis/objectives The objective of our study was to describe the prevalence of cytb M128 mutations in B. gibsoni in canine blood samples submitted to a US veterinary diagnostic laboratory. A secondary objective was to determine whether or not some dogs had wild-type cytb in our initial samples then had M128 mutations detected in follow-up samples. Animals One-Hundred seventy-four dogs that tested positive for the presence of B. gibsoni between 2012 and 2017. Methods Case series of consecutive samples submitted to a veterinary diagnostic laboratory. Partial B. gibsoni cytb genes were amplified by polymerase chain reaction and screened for the presence of mutations at the M128 position. Results The overall prevalence of M128 mutants was 3.5% (6/173 dogs) in the initial samples. The incidence of new cytb mutants in dogs that tested positive for B. gibsoni, which then had follow-up testing, was 12.1% (5/41). Conclusions and Clinic Importance: Our study reaffirms that B. gibsoni infection is widespread and most commonly detected in American Staffordshire Terrier/American Pit Bull Terrier dogs (128/174, 74% of the infected dogs in our study). The prevalence of cytb mutations does not warrant pretreatment genotyping.}, number={6}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Birkenheuer, Adam J. and Marr, Henry S. and Wilson, James M. and Breitschwerdt, Edward B. and Qurollo, Barbara A.}, year={2018}, month={Oct}, pages={1965–1969} }
 @article{neupane_hegarty_marr_maggi_birkenheuer_breitschwerdt_2018, title={Evaluation of cell culture-grown Bartonella
 antigens in immunofluorescent antibody assays for the serological diagnosis of bartonellosis in dogs}, volume={32}, ISSN={0891-6640}, url={http://dx.doi.org/10.1111/jvim.15301}, DOI={10.1111/jvim.15301}, abstractNote={BACKGROUND Because of poor sensitivity and questionable specificity of immunofluorescent antibody assays (IFAs), serological diagnosis of Bartonella species infections in dogs remains challenging. Despite limitations, IFA testing is the historical gold standard for Bartonella serodiagnosis in animals and humans. Because most diagnostic laboratories test against only 1 or 2 Bartonella spp., testing against a broader panel of Bartonella antigens may enhance diagnostic sensitivity and specificity. OBJECTIVE To evaluate the sensitivity and specificity of Bartonella IFA using 8 cell culture-grown Bartonella spp. isolates. ANIMALS Archived serum samples from 34 Bartonella spp. naturally exposed, polymerase chain reaction (PCR)-positive dogs and from 26 PCR-negative and IFA-negative dogs. METHODS Bartonella IFA sensitivity and specificity were assessed using cell culture-grown whole cell antigens derived from 3 Bartonella henselae (Bh) strains (Bh Houston 1, Bh San Antonio Type 2, Bh California 1), 3 Bartonella vinsonii subsp. berkhoffii genotypes (Bvb I, II, and III), Bartonella koehlerae (Bk), and Bartonella quintana (Bq). RESULTS Only 62% of 34 Bartonella spp. PCR-positive dogs were seroreactive to any of the 8 Bartonella IFA antigens, indicating low IFA sensitivity. PCR-positive dogs were most often IFA seroreactive to Bq (n = 15), to Bvb II (n = 13), or to both (n = 9) antigens. Of the 26 previously IFA-negative/PCR-negative dogs, 4 (15%) were seroreactive using the expanded antigen panel. CONCLUSION AND CLINICAL IMPORTANCE Despite IFA testing of dogs against 8 different Bartonella isolates, IFA sensitivity remained poor, and specificity was only 85%. Development of a reliable serological assay is needed to facilitate the diagnosis of Bartonella infection in dogs.}, number={6}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Neupane, Pradeep and Hegarty, Barbara C. and Marr, Henry S. and Maggi, Ricardo G. and Birkenheuer, Adam J. and Breitschwerdt, Edward B.}, year={2018}, month={Oct}, pages={1958–1964} }
 @article{khana_peterson_stanton_schreeg_birkenheuer_tarigo_2018, title={Genetic conservation of Cytauxzoon felis antigens and mRNA expression in the schizont life-stage}, volume={263}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2018.10.007}, abstractNote={Cytauxzoonosis is a highly fatal disease of domestic cats caused by the apicomplexan protozoan Cytauxzoon felis, which is most closely related to Theileria spp. The growing prevalence, high morbidity and mortality, and treatment cost of cytauxzoonosis emphasize the need for vaccine development. Traditional approaches for vaccine development, however, have been hindered by the inability to culture C. felis in vitro. Recent availability of the annotated C. felis genome combined with genome-based vaccine design and protein microarray immunoscreening allowed for high-throughput identification of C. felis antigens that could serve as vaccine candidates. This study assessed the suitability of three of these vaccine candidates (cf30, cf63, cf58) in addition to a previously reported vaccine candidate (cf76) based on two criteria: genetic conservation among diverse C. felis geographic isolates and expression in tissues containing the C. felis schizont life stage, which has been previously associated with the development of a protective immune response. A comparison of seventeen C. felis isolates across seven states demonstrated high sequence identity (99-100%) for cf30, cf63, and cf58, similar to the degree of conservation previously reported for cf76. RNAscope® in situ hybridization using acutely infected feline splenic tissue revealed robust levels of all transcripts in the schizont life stage of the parasite. These data support the suitability of these three antigens for further investigation as vaccine candidates against cytauxzoonosis.}, journal={VETERINARY PARASITOLOGY}, author={Khana, Daven B. and Peterson, David S. and Stanton, James B. and Schreeg, Megan E. and Birkenheuer, Adam J. and Tarigo, Jaime L.}, year={2018}, month={Nov}, pages={49–53} }
 @article{schreeg_marr_tarigo_sherrill_outi_scholl_bird_vigil_hung_nakajima_et al._2018, title={Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis}, volume={15}, ISSN={["1559-0275"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85059281263&partnerID=MN8TOARS}, DOI={10.1186/s12014-018-9218-9}, abstractNote={Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development.Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model.Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis.Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.}, number={1}, journal={CLINICAL PROTEOMICS}, author={Schreeg, Megan E. and Marr, Henry S. and Tarigo, Jaime L. and Sherrill, Meredith K. and Outi, Hilton K. and Scholl, Elizabeth H. and Bird, David M. and Vigil, Adam and Hung, Chris and Nakajima, Rie and et al.}, year={2018}, month={Dec} }
 @article{kovačević filipović_beletić_ilić božović_milanović_tyrrell_buch_breitschwerdt_birkenheuer_chandrashekar_2018, title={Molecular and Serological Prevalence of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeenses, E. ewingii, Borrelia burgdorferi, Babesia canis, B. gibsoni and B. vogeli among Clinically Healthy Outdoor Dogs in Serbia}, volume={14}, ISSN={2405-9390}, url={http://dx.doi.org/10.1016/J.VPRSR.2018.10.001}, DOI={10.1016/J.VPRSR.2018.10.001}, abstractNote={Data concerning combined molecular and serological prevalence of emerging canine tick-borne pathogens in Serbia are lacking. A large population of outdoor living dogs in Belgrade, Serbia's' capital, present an excellent population for epidemiology study. Blood samples were collected from 111 dogs, including 46 shelter, 31 free roaming, and 34 hunting dogs. Species-specific real-time polymerase chain reaction (PCR) (IDEXX Laboratories, Inc., Westbrook Maine, USA) was applied for the molecular detection of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, Babesia canis, B. gibsoni and B. vogeli. A research based SNAP assay (SNAP® M-A, IDEXX Laboratories, Inc., Westbrook Maine, USA) that uses genus and species-specific peptides was used to asses Anaplasma spp., A. phagocytophilum, A. platys, Ehrlichia spp., E. canis, E. chaffeensis, E. ewingii and Borrelia burgdorferi antibody status. B. canis, B. gibsoni and B. vogeli antibody status was assessed with an indirect immunofluorescence test (MegaCor Diagnostic, Horbranz, Austria). Anaplasma spp. and Ehrlichia spp. DNA was not amplified. One quarter of the dogs were A. phagocytophilum, one dog was A. platys, one was E. ewingii and two dogs were B. burgdorferi seroreactive with the SNAP® M-A. Babesia canis or B. gibsoni DNA was amplified by PCR from 16.2% of dogs, whereas 67.6% were seroreactive to one or more Babesia spp. Babesia vogeli was not PCR amplified. We conclude that outdoor dogs in this territory are reservoirs for B. canis and B. gibsoni and are frequently co-exposed to combinations of Anaplasma and Babesia spp.}, journal={Veterinary Parasitology: Regional Studies and Reports}, publisher={Elsevier BV}, author={Kovačević Filipović, Milica M. and Beletić, Anđelo D. and Ilić Božović, Anja V. and Milanović, Zorana and Tyrrell, Phyllis and Buch, Jesse and Breitschwerdt, Edward B. and Birkenheuer, Adam J. and Chandrashekar, Ramaswamy}, year={2018}, month={Dec}, pages={117–122} }
 @article{mylonakis_schreeg_chatzis_pearce_marr_saridomichelakis_birkenheuer_2018, title={Molecular detection of vector-borne pathogens in Greek cats}, volume={9}, ISSN={["1877-9603"]}, DOI={10.1016/j.ttbdis.2017.08.013}, abstractNote={Infectious diseases have been increasingly recognized in cats worldwide. The objective of this study was the molecular investigation of the prevalence of selected pathogens in healthy and sick cats from Greece, a country highly endemic for several canine vector-borne pathogens. Blood and/or bone marrow samples from 50 clinically healthy and 50 sick adult (>1 year-old) cats were retrospectively examined for the amplification of Bartonella spp., haemoplasmas, Ehrlichia spp., Anaplasma spp., Babesia spp., and Cytauxzoon spp. DNA. Overall, 14.9% of the cats were found to be infected or co-infected by haemoplasmas, including Candidatus Mycoplasma haemominutum and M. haemofelis. In addition, 8.5% of the cats were infected by Bartonella henselae, Bartonella clarridgeiae or Bartonella koehlerae. In contrast, DNA of Ehrlichia spp., Anaplasma spp., Babesia spp. and Cytauxzoon spp. was not amplified from the blood or bone marrow of any cat. There was no significant difference in either haemoplasma or Bartonella infection rates when comparing healthy and sick cats. This study represents the first description of Bartonella koehlerae in Greek cats.}, number={2}, journal={TICKS AND TICK-BORNE DISEASES}, author={Mylonakis, Mathios E. and Schreeg, Megan and Chatzis, Manolis K. and Pearce, Julian and Marr, Henry S. and Saridomichelakis, Manolis N. and Birkenheuer, Adam J.}, year={2018}, month={Feb}, pages={171–175} }
 @article{lashnits_correa_hegarty_birkenheuer_breitschwerdt_2017, title={Bartonella
 Seroepidemiology in Dogs from North America, 2008-2014}, volume={32}, ISSN={0891-6640}, url={http://dx.doi.org/10.1111/jvim.14890}, DOI={10.1111/jvim.14890}, abstractNote={Background Improved understanding of Bartonella species seroepidemiology in dogs may aid clinical decision making and enhance current understanding of naturally occurring arthropod vector transmission of this pathogen. Objectives To identify demographic groups in which Bartonella exposure may be more likely, describe spatiotemporal variations in Bartonella seroreactivity, and examine co-exposures to other canine vector-borne diseases (CVBD). Animals A total of 15,451 serology specimens from dogs in North America were submitted to the North Carolina State University, College of Veterinary Medicine Vector Borne Disease Diagnostic Laboratory between January 1, 2008, and December 31, 2014. Methods Bartonella henselae, Bartonella koehlerae, and Bartonella vinsonii subspecies berkhoffii indirect fluorescent antibody (IFA) serology results, as well as results from a commercial assay kit screening for Dirofilaria immitis antigen and Ehrlichia species, Anaplasma phagocytophilum, and Borrelia burgdorferi antibodies, and Ehrlichia canis, Babesia canis, Babesia gibsoni, and Rickettsia species IFA results were reviewed retrospectively. Results Overall, 3.26% of dogs were Bartonella spp. seroreactive; B. henselae (2.13%) and B. koehlerae (2.39%) were detected more frequently than B. vinsonii subsp. berkhoffii (1.42%, P < 0.0001). Intact males had higher seroreactivity (5.04%) than neutered males (2.87%, P < 0.0001) or intact or spayed females (3.22%, P = 0.0003). Mixed breed dogs had higher seroreactivity (4.45%) than purebred dogs (3.02%, P = 0.0002). There was no trend in seasonal seroreactivity; geographic patterns supported broad distribution of exposure, and co-exposure with other CVBD was common. Conclusions and Clinical Importance Bartonella spp. exposure was documented throughout North America and at any time of year. Male intact dogs, mixed breed dogs, and dogs exposed to other CVBD have higher seroreactivity to multiple Bartonella species.}, number={1}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Lashnits, E. and Correa, M. and Hegarty, B.C. and Birkenheuer, A. and Breitschwerdt, E.B.}, year={2017}, month={Dec}, pages={222–231} }
 @article{levine_cianciolo_linder_bizikova_birkenheuer_brooks_salous_nordone_bellinger_marr_et al._2017, title={Endothelial alterations in a canine model of immune thrombocytopenia}, volume={30}, ISSN={0953-7104 1369-1635}, url={http://dx.doi.org/10.1080/09537104.2017.1378807}, DOI={10.1080/09537104.2017.1378807}, abstractNote={Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.}, number={1}, journal={Platelets}, publisher={Informa UK Limited}, author={LeVine, Dana N. and Cianciolo, Rachel E. and Linder, Keith E. and Bizikova, Petra and Birkenheuer, Adam J. and Brooks, Marjory B. and Salous, Abdelghaffar K. and Nordone, Shila K. and Bellinger, Dwight A. and Marr, Henry and et al.}, year={2017}, month={Nov}, pages={88–97} }
 @article{qurollo_archer_schreeg_marr_birkenheuer_haney_thomas_breitschwerdt_2017, title={Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA}, volume={10}, ISSN={1756-3305}, url={http://dx.doi.org/10.1186/s13071-017-2064-1}, DOI={10.1186/s13071-017-2064-1}, abstractNote={Babesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp.Using 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments (lsu5-lsu4) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR.The LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia. sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA qPCR positive.We have developed a more sensitive qPCR assay with a more expansive range of Babesia spp. detection by targeting a highly conserved region of mtDNA, when compared to an established 18S qPCR.}, number={1}, journal={Parasites & Vectors}, publisher={Springer Nature}, author={Qurollo, Barbara A. and Archer, Nikole R. and Schreeg, Megan E. and Marr, Henry S. and Birkenheuer, Adam J. and Haney, Kaitlin N. and Thomas, Brittany S. and Breitschwerdt, Edward B.}, year={2017}, month={Mar} }
 @article{manning_birkenheuer_briley_montgomery_harris_vanone_gookin_2016, title={Intermittent At-Home Suctioning of Esophageal Content for Prevention of Recurrent Aspiration Pneumonia in 4 Dogs with Megaesophagus}, volume={30}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.14527}, abstractNote={Megaesophagus carries a poor to guarded prognosis due to death from aspiration pneumonia. Options for medical management of regurgitation are limited to strategic oral or gastrostomy tube feeding.To describe the use and efficacy of intermittent esophageal suctioning to prevent regurgitation and associated episodes of aspiration pneumonia in dogs with megaesophagus.Four dogs with acquired idiopathic megaesophagus and recurrent aspiration pneumonia.Retrospective review of medical records of dogs with megaesophagus in which intermittent suctioning of esophageal content was employed for management of recurrent aspiration pneumonia.Intermittent suctioning of the esophagus was initiated in 4 dogs after failure of strict gastrostomy tube feeding failed to prevent regurgitation and repeated episodes of aspiration pneumonia. Suctioning was accomplished by esophagostomy tube in 3 dogs and per os in 1 dog. After initiation of esophageal suctioning, dogs survived for a median of 13.5 additional months (range, 10-30 months) during which time 2 dogs had no additional episodes of aspiration pneumonia and 2 dogs had infrequent episodes of pneumonia, but aspiration was suspected to be a contributing factor in their death. Complications included clogging of the esophagostomy tube, esophagostomy site infections, and esophagitis.Use of intermittent esophageal suctioning in dogs with megaesophagus that continue to regurgitate despite gastrostomy tube feedings can reduce or abolish clinical episodes of aspiration pneumonia.}, number={5}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Manning, K. and Birkenheuer, A. J. and Briley, J. and Montgomery, S. A. and Harris, J. and Vanone, S. L. and Gookin, J. L.}, year={2016}, pages={1715–1719} }
 @article{schreeg_marr_tarigo_cohn_bird_scholl_levy_wiegmann_birkenheuer_2016, title={Mitochondrial Genome Sequences and Structures Aid in the Resolution of Piroplasmida phylogeny}, volume={11}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84994744879&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0165702}, abstractNote={The taxonomy of the order Piroplasmida, which includes a number of clinically and economically relevant organisms, is a hotly debated topic amongst parasitologists. Three genera (Babesia, Theileria, and Cytauxzoon) are recognized based on parasite life cycle characteristics, but molecular phylogenetic analyses of 18S sequences have suggested the presence of five or more distinct Piroplasmida lineages. Despite these important advancements, a few studies have been unable to define the taxonomic relationships of some organisms (e.g. C. felis and T. equi) with respect to other Piroplasmida. Additional evidence from mitochondrial genome sequences and synteny should aid in the inference of Piroplasmida phylogeny and resolution of taxonomic uncertainties. In this study, we have amplified, sequenced, and annotated seven previously uncharacterized mitochondrial genomes (Babesia canis, Babesia vogeli, Babesia rossi, Babesia sp. Coco, Babesia conradae, Babesia microti-like sp., and Cytauxzoon felis) and identified additional ribosomal fragments in ten previously characterized mitochondrial genomes. Phylogenetic analysis of concatenated mitochondrial and 18S sequences as well as cox1 amino acid sequence identified five distinct Piroplasmida groups, each of which possesses a unique mitochondrial genome structure. Specifically, our results confirm the existence of four previously identified clades (B. microti group, Babesia sensu stricto, Theileria equi, and a Babesia sensu latu group that includes B. conradae) while supporting the integration of Theileria and Cytauxzoon species into a single fifth taxon. Although known biological characteristics of Piroplasmida corroborate the proposed phylogeny, more investigation into parasite life cycles is warranted to further understand the evolution of the Piroplasmida. Our results provide an evolutionary framework for comparative biology of these important animal and human pathogens and help focus renewed efforts toward understanding the phylogenetic relationships within the group.}, number={11}, journal={PLOS ONE}, author={Schreeg, Megan E. and Marr, Henry S. and Tarigo, Jaime L. and Cohn, Leah A. and Bird, David M. and Scholl, Elizabeth H. and Levy, Michael G. and Wiegmann, Brian M. and Birkenheuer, Adam J.}, year={2016}, month={Nov} }
 @article{schreeg_marr_griffith_tarigo_bird_reichard_cohn_levy_birkenheuer_2016, title={PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S)}, volume={225}, ISSN={["1873-2550"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84975504702&partnerID=MN8TOARS}, DOI={10.1016/j.vetpar.2016.06.013}, abstractNote={Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections.}, journal={VETERINARY PARASITOLOGY}, author={Schreeg, Megan E. and Marr, Henry S. and Griffith, Emily H. and Tarigo, Jaime L. and Bird, David M. and Reichard, Mason V. and Cohn, Leah A. and Levy, Michael G. and Birkenheuer, Adam J.}, year={2016}, month={Jul}, pages={123–130} }
 @article{wardrop_birkenheuer_blais_callan_kohn_lappin_sykes_2016, title={Update on Canine and Feline Blood Donor Screening for Blood-Borne Pathogens}, volume={30}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.13823}, abstractNote={An update on the 2005 American College of Veterinary Internal Medicine (ACVIM) Consensus Statement on blood donor infectious disease screening was presented at the 2015 ACVIM Forum in Indianapolis, Indiana, followed by panel and audience discussion. The updated consensus statement is presented below. The consensus statement aims to provide guidance on appropriate blood-borne pathogen testing for canine and feline blood donors in North America.}, number={1}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Wardrop, K. J. and Birkenheuer, A. and Blais, M. C. and Callan, M. B. and Kohn, B. and Lappin, M. R. and Sykes, J.}, year={2016}, pages={15–35} }
 @article{conner_hanel_brooks_cohn_birkenheuer_2015, title={Coagulation abnormalities in 5 cats with naturally occurring cytauxzoonosis}, volume={25}, ISSN={["1476-4431"]}, DOI={10.1111/vec.12326}, abstractNote={To characterize hemostasis and determine if disseminated intravascular coagulation (DIC) is present in cats with cytauxzoonosis.Cross-sectional study.University teaching hospital.Five client-owned cats with cytologic and PCR-confirmed cytauxzoonosis.None.Admission samples were collected for hemostasis testing including platelet count, activated partial thromboplastin time, prothrombin time, fibrinogen, antithrombin (AT), d-dimer, protein C, plasminogen, antiplasmin, factors VII, VIII, IX, X, and XI, von Willebrand factor, and thromboelastography. Results were compiled for combined criteria used to define DIC, and all 5 cats satisfied criteria using a previously described modified scoring system for DIC in cats. The abnormalities found in all 5 cats included thrombocytopenia, low protein C activity, and prolonged prothrombin time; however, none of the cats had low AT activity. None of the cats had clinical signs of hemorrhage despite thrombocytopenia, coagulation factor deficiency (5/5 cats), and thromboelastographic evidence of hypocoagulability (2/5 cats). Three of 5 cats survived to hospital discharge. The nonsurvivors had disseminated cytauxzoonosis with schizont-laden macrophages in vessels of various organs.This is the first report that comprehensively describes the hemostastic status of cats with naturally occurring infection with Cytauxzoon felis. All 5 cats had laboratory evidence of overt DIC. Unlike human and canine models of sepsis-induced DIC, AT deficiency was not found in this series of cats. Further research is warranted to investigate therapeutic strategies targeting thrombotic DIC to improve survival in cats with cytauxzoonosis.}, number={4}, journal={JOURNAL OF VETERINARY EMERGENCY AND CRITICAL CARE}, author={Conner, Bobbi J. and Hanel, Rita M. and Brooks, Marjory B. and Cohn, Leah A. and Birkenheuer, Adam J.}, year={2015}, pages={538–545} }
 @article{vandersea_birkenheuer_litaker_vaden_renschler_gookin_2015, title={Identification of Parabodo caudatus (class Kinetoplastea) in urine voided from a dog with hematuria}, volume={27}, ISSN={["1943-4936"]}, DOI={10.1177/1040638714562827}, abstractNote={A voided urine sample, obtained from a 13-year-old intact male dog residing in a laboratory animal research facility, was observed to contain biflagellate protozoa 5 days following an episode of gross hematuria. The protozoa were identified as belonging to the class Kinetoplastea on the basis of light microscopic observation of Wright–Giemsa-stained urine sediment in which the kinetoplast was observed basal to 2 anterior flagella. A polymerase chain reaction (PCR) assay using primers corresponding with conserved regions within the 18S ribosomal RNA gene of representative kinetoplastid species identified nucleotide sequences with 100% identity to Parabodo caudatus. Parabodo caudatus organisms were unable to be demonstrated cytologically or by means of PCR in samples collected from the dog’s environment. The dog had a history of 50 complete urinalyses performed over the 12-year period preceding detection of P. caudatus, and none of these were noted to contain protozoa. Moreover, the gross hematuria that was documented 5 days prior to detection of P. caudatus had never before been observed in this dog. Over the ensuing 2.5 years of the dog’s life, 16 additional complete urinalyses were performed, none of which revealed the presence of protozoa. Bodonids are commonly found in soil as well as in freshwater and marine environments. However, P. caudatus, in particular, has a 150-year-long, interesting, and largely unresolved history in people as either an inhabitant or contaminant of urine. This historical conundrum is revisited in the current description of P. caudatus as recovered from the urine of a dog.}, number={1}, journal={JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION}, author={Vandersea, Mark W. and Birkenheuer, Adam J. and Litaker, R. Wayne and Vaden, Shelly L. and Renschler, Janelle S. and Gookin, Jody L.}, year={2015}, month={Jan}, pages={117–120} }
 @article{rizzi_reichard_cohn_birkenheuer_taylor_meinkoth_2015, title={Prevalence of Cytauxzoon felis infection in healthy cats from enzootic areas in Arkansas, Missouri, and Oklahoma}, volume={8}, ISSN={["1756-3305"]}, DOI={10.1186/s13071-014-0618-z}, abstractNote={BackgroundInfection with Cytauxzoon felis in domestic cats can cause fever, lethargy, depression, inappetence, icterus, and often death. With a high mortality rate, cytauxzoonosis was historically considered a fatal disease. Within the last 15 years, cats with or without treatment have been recognized as chronically infected survivors of C. felis infection. Our objective was to determine the prevalence of C. felis in healthy domestic cats from Arkansas, Missouri, and Oklahoma.MethodsInfection with C. felis was determined using DNA extracted from anticoagulated whole blood and PCR amplification using C. felis-specific primers. Chi-square, Fisher’s exact tests, and odds ratios were used to compare proportions of cats infected with C. felis.ResultsBlood samples were collected from 902 healthy domestic cats between October 2008 and April 2012. DNA from Cytauxzoon felis was detected in 56 of 902 (6.2%; 95% confidence interval, 4.7–7.9) samples. The highest prevalence of C. felis infection (15.5%; 10.3–21.7) was observed in cats from Arkansas, followed by cats from Missouri (12.9%; 6.1–24.0), and cats from Oklahoma (3.4%; 2.2–5.1). Cats sampled in Arkansas and Missouri were 5.1 and 4.2, respectively, times more likely to be chronically infected with C. felis than cats from Oklahoma.ConclusionsInfection with C. felis is common in domestic cats through Arkansas, Missouri, and Oklahoma. The high prevalence of C. felis reported herein suggests that infected domestic cats are likely reservoirs of infection for naive felines. The high prevalence of C. felis substantiates the importance for the use of approved acaricides on cats to prevent cytauxzoonosis.}, journal={PARASITES & VECTORS}, author={Rizzi, Theresa E. and Reichard, Mason V. and Cohn, Leah A. and Birkenheuer, Adam J. and Taylor, Jared D. and Meinkoth, James H.}, year={2015}, month={Jan} }
 @article{schreeg_marr_tarigo_cohn_levy_birkenheuer_2015, title={Rapid High-Resolution Melt Analysis of Cytauxzoon felis Cytochrome b To Aid in the Prognosis of Cytauxzoonosis}, volume={53}, ISSN={["1098-660X"]}, DOI={10.1128/jcm.00635-15}, abstractNote={Cytauxzoon felis is a virulent, tick-transmitted, protozoan parasite that infects felines. Cytauxzoonosis was previously thought to be uniformly fatal in domestic cats. Treatment combining atovaquone and azithromycin (A&A) has been associated with survival rates of over 60%. Atovaquone, a ubiquinone analogue, targets C. felis cytochrome b (cytb), of which 30 unique genotypes have been identified. The C. felis cytb genotype cytb1 is associated with increased survival rates in cats treated with A&A. The purpose of this study was to design a PCR panel that could distinguish C. felis cytb1 from other cytochrome b genotypes. Primer pairs were designed to span five different nucleotide positions at which single-nucleotide polymorphisms in the C. felis cytb gene had been identified. Through the use of high-resolution melt analysis, this panel was predicted to distinguish cytb1 from other cytb genotypes. Assays were validated using samples from 69 cats with cytauxzoonosis for which the C. felis cytb genotypes had been characterized previously. The PCR panel identified C. felis cytb1 with 100% sensitivity and 98.2% specificity. High-resolution melt analysis can rapidly provide prognostic information for clients considering A&A treatment in cats with cytauxzoonosis.}, number={8}, journal={JOURNAL OF CLINICAL MICROBIOLOGY}, author={Schreeg, Megan E. and Marr, Henry S. and Tarigo, Jaime L. and Cohn, Leah A. and Levy, Michael G. and Birkenheuer, Adam J.}, year={2015}, month={Aug}, pages={2517–2524} }
 @article{levine_birkenheuer_brooks_nordone_bellinger_jones_fischer_oglesbee_frey_brinson_et al._2014, title={A novel canine model of immune thrombocytopenia: has immune thrombocytopenia (ITP) gone to the dogs?}, volume={167}, ISSN={0007-1048}, url={http://dx.doi.org/10.1111/bjh.13005}, DOI={10.1111/bjh.13005}, abstractNote={Canine immune thrombocytopenia (ITP) is analogous to human ITP, with similar platelet counts and heterogeneity in bleeding phenotype among affected individuals. With a goal of ultimately investigating this bleeding heterogeneity, a canine model of antibody-mediated ITP was developed. Infusion of healthy dogs with 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP) resulted in profound, dose-dependent thrombocytopenia. Model dogs developed variable bleeding phenotypes, e.g. petechiae and haematuria, despite similar degrees of thrombocytopenia. 2F9 infusion was not associated with systemic inflammation, consumptive coagulopathy, or impairment of platelet function. Unexpectedly however, evaluation of cytokine profiles led to the identification of platelets as a potential source of serum interleukin-8 (IL8) in dogs. This finding was confirmed in humans with ITP, suggesting that platelet IL8 may be a previously unrecognized modulator of platelet-neutrophil crosstalk. The utility of this model will allow future study of bleeding phenotypic heterogeneity including the role of neutrophils and endothelial cells in ITP.}, number={1}, journal={British Journal of Haematology}, publisher={Wiley}, author={LeVine, Dana N. and Birkenheuer, Adam J. and Brooks, Marjory B. and Nordone, Shila K. and Bellinger, Dwight A. and Jones, Sam L. and Fischer, Thomas H. and Oglesbee, Stephen E. and Frey, Kahlina and Brinson, Nicole S. and et al.}, year={2014}, month={Jul}, pages={110–120} }
 @article{maggi_birkenheuer_hegarty_bradley_levy_breitschwerdt_2014, title={Comparison of serological and molecular panels for diagnosis of vector-borne diseases in dogs}, volume={7}, ISSN={1756-3305}, url={http://dx.doi.org/10.1186/1756-3305-7-127}, DOI={10.1186/1756-3305-7-127}, abstractNote={BackgroundCanine vector-borne diseases (CVBD) are caused by a diverse array of pathogens with varying biological behaviors that result in a wide spectrum of clinical presentations and laboratory abnormalities. For many reasons, the diagnosis of canine vector-borne infectious diseases can be challenging for clinicians. The aim of the present study was to compare CVBD serological and molecular testing as the two most common methodologies used for screening healthy dogs or diagnosing sick dogs in which a vector-borne disease is suspected.MethodsWe used serological (Anaplasma species, Babesia canis, Bartonella henselae, Bartonella vinsonii subspecies berkhoffii, Borrelia burgdorferi, Ehrlichia canis, and SFG Rickettsia) and molecular assays to assess for exposure to, or infection with, 10 genera of organisms that cause CVBDs (Anaplasma, Babesia, Bartonella, Borrelia, Ehrlichia, Francisella, hemotropic Mycoplasma, Neorickettsia, Rickettsia, and Dirofilaria). Paired serum and EDTA blood samples from 30 clinically healthy dogs (Group I) and from 69 sick dogs suspected of having one or more canine vector-borne diseases (Groups II-IV), were tested in parallel to establish exposure to or infection with the specific CVBDs targeted in this study.ResultsAmong all dogs tested (Groups I-IV), the molecular prevalences for individual CVBD pathogens ranged between 23.3 and 39.1%. Similarly, pathogen-specific seroprevalences ranged from 43.3% to 59.4% among healthy and sick dogs (Groups I-IV). Among these representative sample groupings, a panel combining serological and molecular assays run in parallel resulted in a 4-58% increase in the recognition of exposure to or infection with CVBD.ConclusionsWe conclude that serological and PCR assays should be used in parallel to maximize CVBD diagnosis.}, number={1}, journal={Parasites & Vectors}, publisher={Springer Nature}, author={Maggi, Ricardo G and Birkenheuer, Adam J and Hegarty, Barbara C and Bradley, Julie M and Levy, Michael G and Breitschwerdt, Edward B}, year={2014}, pages={127} }
 @article{pritchard_birkenheuer_hanel_wood_2014, title={Copperhead (Agkistrodon contortrix) envenomation of dogs: 52 cases (2004-2011)}, DOI={10.5326/JAAHA-MS-6131}, abstractNote={Copperhead envenomation is common within the US, and no studies exist describing the clinical course of copperhead envenomation in dogs. Almost all treatment decisions regarding those bites are extrapolated from retrospective studies evaluating the clinical course of rattlesnake bites. Because copperheads and rattlesnakes produce venom with different potency, assumptions that treatment of the different envenomations should be similar may be incorrect. The purpose of this retrospective study was to evaluate the clinical course of copperhead envenomation in dogs and administered treatments. Medical records of 52 dogs treated for copperhead envenomation were reviewed, and owners were contacted regarding outcome. The most common clinical signs associated with copperhead envenomation included swelling, pain, and ecchymosis. Clinicopathological abnormalities (e.g., thrombocytopenia, elevated clotting times, leukocytosis) were mild, and red blood cell morphology changes and coagulopathies were rare. Most dogs were treated with antimicrobials, analgesics, and fluid therapy. No dogs in this study required the use of antivenin and all survived to discharge. This study found that the clinical course after copperhead envenomation is generally limited to local rather than systemic illness. Copperhead envenomation in dogs is largely self-limiting and responsive to supportive care with hospitalization for monitoring.}, journal={Journal of the American Animal Hospital Association}, author={Pritchard, Jessica C. and Birkenheuer, Adam J. and Hanel, Rita M. and Wood, Michael W.}, year={2014} }
 @article{lewis_cohn_marr_birkenheuer_2014, title={Failure of efficacy and adverse events associated with dose-intense diminazene diaceturate treatment of chronic Cytauxzoon felis infection in five cats}, volume={16}, ISSN={["1532-2750"]}, DOI={10.1177/1098612x13502974}, abstractNote={Cytauxzoon felis is a hemoprotozoan parasite of cats. While many infected cats die of acute illness, some enter a chronic carrier state. To date, no treatment has been documented to clear the chronic carrier state, leaving recovered cats to act as a potential indirect source of infection via a tick vector. Diminazene diaceturate is an anti-protozoal therapy that has been suggested for use in the treatment of acute cytauxzoonosis, but which failed to clear the carrier state at the dose used in acute illness. We hypothesized that a dose-intensified regimen of diminazene could reduce or eliminate parasitemia from five domestic cats naturally infected with C felis. Cats were administered 4 mg/kg of diminazene diaceturate intramuscularly for 5 consecutive days. Clearance of the organism was assessed via semi-quantitative polymerase chain reaction and light microscopy 1, 3, 6 and 10 weeks after starting treatment. Additionally, cats were monitored for adverse drug reactions by daily observation and examination. Complete blood count, biochemical profile and urinalysis were performed at 1, 3 and 10 weeks. Adverse events were common and included profuse salivation and nausea at the time of injection, monoparesis in the injected leg, proteinuria and potential hepatotoxicity. Severity of parasitemia was not reduced. Diminazene diaceturate cannot be recommended for elimination of the carrier state of C felis infection.}, number={2}, journal={JOURNAL OF FELINE MEDICINE AND SURGERY}, author={Lewis, Kristin M. and Cohn, Leah A. and Marr, Henry S. and Birkenheuer, Adam J.}, year={2014}, month={Feb}, pages={157–163} }
 @article{floras_holowaychuk_hodgins_marr_birkenheuer_sharif_bersenas_bienzle_2014, title={Investigation of a Commercial ELISA for the Detection of Canine Procalcitonin}, volume={28}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.12309}, abstractNote={Background

Rapid identification of sepsis enables prompt administration of antibiotics and is essential to improve patient survival. Procalcitonin (PCT) is a biomarker used to diagnose sepsis in people. Commercial assays to measure canine PCT peptide have not been validated.



Objective

To investigate the validity of a commercially available enzyme-linked immunosorbent assay (ELISA) marketed for the measurement of canine PCT.



Animals

Three dogs with sepsis, 1 healthy dog, 1 dog with thyroid carcinoma.



Methods

Experimental study. The ELISA's ability to detect recombinant and native canine PCT was investigated and intra-assay and interassay coefficients of variability were calculated. Assay validation including mass spectrometry of the kit standard solution was performed.



Results

The ELISA did not consistently detect recombinant canine PCT. Thyroid lysate yielded a positive ELISA signal. Intra-assay variability ranged from 18.9 to 77.4%, while interassay variability ranged from 56.1 to 79.5%. Mass spectrometry of the standard solution provided with the evaluated ELISA kit did not indicate presence of PCT.



Conclusions and Clinical Importance

The results of this investigation do not support the use of this ELISA for the detection of PCT in dogs.}, number={2}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Floras, A. N. K. and Holowaychuk, M. K. and Hodgins, D. C. and Marr, H. S. and Birkenheuer, A. and Sharif, S. and Bersenas, A. M. E. and Bienzle, D.}, year={2014}, month={Mar}, pages={599–602} }
 @article{balakrishnan_pritchard_ericson_grindem_phillips_jennings_mathews_tran_birkenheuer_breitschwerdt_et al._2014, title={Prostatitis, Steatitis, and Diarrhea in a Dog following Presumptive Flea-Borne Transmission of Bartonella henselae}, volume={52}, ISSN={0095-1137 1098-660X}, url={http://dx.doi.org/10.1128/JCM.00942-14}, DOI={10.1128/jcm.00942-14}, abstractNote={Bartonella henselae is increasingly associated with a variety of pathological entities, which are often similar in dogs and human patients. Following an acute flea infestation, a dog developed an unusual clinical presentation for canine bartonellosis. Comprehensive medical, microbiological, and surgical interventions were required for diagnosis and to achieve a full recovery.}, number={9}, journal={Journal of Clinical Microbiology}, publisher={American Society for Microbiology}, author={Balakrishnan, N. and Pritchard, J. and Ericson, M. and Grindem, C. and Phillips, K. and Jennings, S. and Mathews, K. and Tran, H. and Birkenheuer, A. J. and Breitschwerdt, Edward and et al.}, editor={Munson, E.Editor}, year={2014}, month={Jun}, pages={3447–3452} }
 @article{yancey_hegarty_qurollo_levy_birkenheuer_weber_diniz_breitschwerdt_2014, title={Regional Seroreactivity and Vector-Borne Disease Co-Exposures in Dogs in the United States from 2004–2010: Utility of Canine Surveillance}, volume={14}, ISSN={1530-3667 1557-7759}, url={http://dx.doi.org/10.1089/vbz.2014.1592}, DOI={10.1089/vbz.2014.1592}, abstractNote={Vector-borne disease (VBD) pathogens remain an emerging health concern for animals and humans throughout the world. Surveillance studies of ticks and humans have made substantial contributions to our knowledge of VBD epidemiology trends, but long-term VBD surveillance data of dogs in the United States is limited. This seroreactivity study assessed US temporal and regional trends and co-exposures to Anaplasma, Babesia, Bartonella, Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia spp., and spotted fever group Rickettsia in dogs from 2004-2010. Dog serum samples (N=14,496) were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Disease Diagnostic Laboratory for vector-borne pathogens diagnostic testing using immunofluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) assays. These convenience samples were retrospectively reviewed and analyzed. The largest proportion of samples originated from the South (47.6%), with the highest percent of seroreactive samples observed in the Midatlantic (43.4%), compared to other US regions. The overall seroreactivity of evaluated VBD antigens were Rickettsia rickettsia (10.4%), B. burgdorferi (5.2%), Ehrlichia spp. (4.3%), Bartonella henselae (3.8%), Anaplasma spp. (1.9%), Bartonella vinsonii subsp. berkhoffii (1.5%), Babesia canis (1.1%), and D. immitis (0.8%). Significant regional and annual seroreactivity variation was observed with B. burgdorferi, Ehrlichia, and Rickettsia exposures. Seasonal seroreactivity variation was evident with Rickettsia. Seroreactivity to more than one antigen was present in 16.5% of exposed dogs. Nationally, the most prevalent co-exposure was Rickettsia with Ehrlichia spp. (5.3%), and the highest odds of co-exposure was associated with Anaplasma spp. and B. burgdorferi (odds ratio=6.6; 95% confidence interval 5.0, 8.8). Notable annual and regional seroreactivity variation was observed with certain pathogens over 7 years of study, suggesting canine surveillance studies may have value in contributing to future VBD knowledge.}, number={10}, journal={Vector-Borne and Zoonotic Diseases}, publisher={Mary Ann Liebert Inc}, author={Yancey, Caroline B. and Hegarty, Barbara C. and Qurollo, Barbara A. and Levy, Michael G. and Birkenheuer, Adam J. and Weber, David J. and Diniz, Pedro P.V.P. and Breitschwerdt, Edward B.}, year={2014}, month={Oct}, pages={724–732} }
 @article{li_d’annibale-tolhurst_adler_fang_yin_birkenheuer_levy_jones_sung_hawkins_et al._2013, title={A Myristoylated Alanine-Rich C Kinase Substrate–Related Peptide Suppresses Cytokine mRNA and Protein Expression in LPS-Activated Canine Neutrophils}, volume={48}, ISSN={1044-1549 1535-4989}, url={http://dx.doi.org/10.1165/rcmb.2012-0278OC}, DOI={10.1165/rcmb.2012-0278oc}, abstractNote={Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C substrate that has emerged as a potential therapeutic target for the amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. MARCKS also plays a key role in regulating the adhesion, migration, and degranulation of neutrophils. Moreover, given its biological role in epithelial and immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion by neutrophils. Because the amino terminus of MARCKS is highly conserved across vertebrate species, we successfully applied the well-characterized human MARCKS inhibitory peptide, myristoylated N-terminal sequence (MANS), to attenuate the function of MARCKS in isolated canine neutrophils. Pretreatment of canine neutrophils with MANS peptide significantly reduced both mRNA and protein expression in a broad range of LPS-induced cytokines, including IL-8, a chemokine (C-X-C motif) ligand-1 orthologue, and TNF-α, in comparison with untreated cells or those treated with a control peptide. This reduction in cytokine expression was observed even when neutrophils were treated with MANS 2 hours after LPS exposure. The observed reduction in cytokine secretion was not attributable to protein retention or cell death, but was associated with reduced cytokine transcript synthesis. These observations identify MARCKS protein as a promising therapeutic target in the treatment of inflammatory diseases or syndromes attributed to neutrophil influx and inflammatory cytokine production, such as sepsis, acute lung injury, and acute respiratory distress syndrome.}, number={3}, journal={American Journal of Respiratory Cell and Molecular Biology}, publisher={American Thoracic Society}, author={Li, Jingjing and D’Annibale-Tolhurst, Melissa A. and Adler, Kenneth B. and Fang, Shijing and Yin, Qui and Birkenheuer, Adam J. and Levy, Michael G. and Jones, Samuel L. and Sung, Eui Jae and Hawkins, Eleanor C. and et al.}, year={2013}, month={Mar}, pages={314–321} }
 @article{tarigo_scholl_bird_brown_cohn_dean_levy_doolan_trieu_nordone_et al._2013, title={A Novel Candidate Vaccine for Cytauxzoonosis Inferred from Comparative Apicomplexan Genomics}, volume={8}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84882652524&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0071233}, abstractNote={Cytauxzoonosis is an emerging infectious disease of domestic cats (Felis catus) caused by the apicomplexan protozoan parasite Cytauxzoon felis. The growing epidemic, with its high morbidity and mortality points to the need for a protective vaccine against cytauxzoonosis. Unfortunately, the causative agent has yet to be cultured continuously in vitro, rendering traditional vaccine development approaches beyond reach. Here we report the use of comparative genomics to computationally and experimentally interpret the C. felis genome to identify a novel candidate vaccine antigen for cytauxzoonosis. As a starting point we sequenced, assembled, and annotated the C. felis genome and the proteins it encodes. Whole genome alignment revealed considerable conserved synteny with other apicomplexans. In particular, alignments with the bovine parasite Theileria parva revealed that a C. felis gene, cf76, is syntenic to p67 (the leading vaccine candidate for bovine theileriosis), despite a lack of significant sequence similarity. Recombinant subdomains of cf76 were challenged with survivor-cat antiserum and found to be highly seroreactive. Comparison of eleven geographically diverse samples from the south-central and southeastern USA demonstrated 91-100% amino acid sequence identity across cf76, including a high level of conservation in an immunogenic 226 amino acid (24 kDa) carboxyl terminal domain. Using in situ hybridization, transcription of cf76 was documented in the schizogenous stage of parasite replication, the life stage that is believed to be the most important for development of a protective immune response. Collectively, these data point to identification of the first potential vaccine candidate antigen for cytauxzoonosis. Further, our bioinformatic approach emphasizes the use of comparative genomics as an accelerated path to developing vaccines against experimentally intractable pathogens.}, number={8}, journal={PLOS ONE}, author={Tarigo, Jaime L. and Scholl, Elizabeth H. and Bird, David McK and Brown, Corrie C. and Cohn, Leah A. and Dean, Gregg A. and Levy, Michael G. and Doolan, Denise L. and Trieu, Angela and Nordone, Shila K. and et al.}, year={2013}, month={Aug} }
 @article{qurollo_davenport_sherbert_grindem_birkenheuer_breitschwerdt_2013, title={Infection with Panola MountainEhrlichiasp. in a Dog with Atypical Lymphocytes and Clonal T-Cell Expansion}, volume={27}, ISSN={0891-6640}, url={http://dx.doi.org/10.1111/jvim.12148}, DOI={10.1111/jvim.12148}, abstractNote={An 11-year-old, castrated male Scottish Terrier from Raleigh, NC that lived predominantly indoors and had no travel history was referred to the North Carolina State University Veterinary Health Complex (NCSU-VHC) in October 2012 for routine reevaluation of hepatobiliary disease. The dog's previous 3-year medical history included biliary mucocele (2010); neutrophilic hepatitis (2011); recurrent Escherichia coli urinary tract infections, esophageal dysmotility, aspiration pneumonia, and transient thrombocytopenia (2012); and food responsive enteropathy (2009–2012). All of these medical problems were well controlled at the time of examination and no clinical abnormalities were reported by the dog's owners. In the months before the present examination, ticks occasionally were noted and fleas were commonly found on the dog despite reported use of preventive therapies. On physical examination, the dog was obese (body condition score, 7 out of 9) and had mild hepatomegaly, both of which had been present for more than a year. Notable CBC findings included mild thrombocytopenia (platelet count, 143,000/μL; reference interval [RI], 190,000–468,000/μL) and an increased number of atypical lymphocytes (1,773/μL), with normal appearing lymphocytes within the laboratory reference range (1,854/μL; RI, 594–3,305/μL) and an otherwise normal differential cell count. A review of the blood smear by a pathologist identified a population of immature lymphocytes with angular nuclei and cell shape, multiple nucleoli, and deeply basophilic, vacuolated cytoplasm that had a tendency to mold into surrounding cells (Fig 1). These morphologic abnormalities raised the suspicion of possible lymphoid neoplasia, although these changes can be seen secondary to reactive processes such as chronic inflammatory or infectious diseases. Serum biochemical abnormalities included an increase in alkaline phosphatase activity (ALP; 817 IU/L; RI, 16–140 IU/L) and alanine aminotransferase activity (ALT; 60 IU/L; RI, 12–54). During the previous 12-month period, ALT activity varied between normal and 80 IU/L and ALP activity varied between 359 and 534 IU/L. Because these hematological and serum biochemical abnormalities were present 2 weeks later, abdominal ultrasound examination was repeated and identified mottled splenic parenchyma and several previously identified changes including hyperechoic nodular hepatomegaly; mild dystrophic mineralization of the spleen, liver, kidneys, and prostate; and, a thickened urinary bladder apex. Splenic cytology identified a mixed lymphoid population and mild extramedullary hematopoiesis. Liver cytology identified normal hepatocytes and an expansion of intermediate lymphocytes, raising concern for lymphoma. Cytology of a palpably normal popliteal lymph node identified expansion of intermediate to large lymphocytes, most consistent with lymphoma (Fig 2). T-cell clonality was documented in a liver aspirate after submission to NCSU-CVM Clinical Immunology Laboratory for polymerase chain reaction (PCR) to identify antigen receptor rearrangements (PARR). Aseptically collected ethylenediamine tetraacetic acid (EDTA)-anticoagulated whole blood and serum were submitted to the NCSU-CVM Vector Borne Diseases Diagnostic Laboratory (VBDDL) for a vector-borne pathogen serology panel and Bartonella alpha-Proteobacteria Growth Medium (BAPGM) enrichment blood culture PCR platform. The dog was seronegative for antibodies to Anaplasma phagocytophilum, Anaplasma platys, and Borrelia burgdorferi and for Dirofilaria immitis antigen using a commercial enzyme-linked immunosorbent assay based kit (SNAP® 4Dx® Plusa); the dog was seronegative for antibodies to Babesia canis, Babesia gibsoni, Bartonella henselae, and Bartonella vinsonii subsp. berkhoffii, using indirect immunofluorescent antibody (IFA) testing. By IFA, the dog was seroreactive to Rickettsia rickettsii (1:64; laboratory cut-off value, 1:64) and Ehrlichia canis (1:1028; laboratory cut-off value, 1:64) antigens, but seronegative to Ehrlichia spp. peptides in the SNAP 4Dx Plusa kit, which is known to detect antibodies against E. canis, E. chaffeensis, and E. ewingii. Bartonella enrichment blood culture and PCR were negative. Nine months before this presentation, the dog was seronegative in the NCSU-CVM-VBDDL to the vector-borne pathogens tested above. Because of the discrepancy between the E. canis IFA and SNAP 4Dx Plusa test results, Ehrlichia spp. PCR was performed on DNA extracted from the dog's EDTA-anticoagulated whole blood using a previously described 16S ribosomal RNA PCR assay.1 A 351 base pair portion of the 16S rRNA gene was amplified, sequenced, and investigated in the NCBI GenBank nucleotide database. The partial 16S rRNA gene identified infection with the Panola Mountain Ehrlichia sp. (PME) with 100% coverage (331 bp) and 100% identity to PME 16S ribosomal RNA gene (DQ324367.1). To confirm these results, alternative PME genes, including gltA and map1 were targeted. Furthermore, sodb, a gene not previously sequenced for PME, was amplified using newly developed primers designed to amplify Ehrlichia spp. sodb genes. Primers designed to amplify a 311 bp portion of PME gltA (EPM-gltA-Forward, 5′-CGTGTTTTTCTGCCTTAGCTGCAC and EPM-gltA-Reverse, 5′-CGGCCCAGAAGAACC TGTCA) based on a published PME gltA sequence (DQ363995.1) and a second set of primers designed to target a 480 bp portion of PME map1 (EPM-map1-Forward-3, 5′-CGAGAGCCAACGTTTACAT and EPM-map1-Reverse-2, 5′-GTACCAATACCTGCACATAC) based on published PME map1 sequences (DQ324368.1, EU272373.1 and EU272355.1) were used. Primers designed to amplify a 300 bp portion of sodb (sodb-Forward, 5′-TTTAATAATGCTGGTCAAGTATGGAATCAT and sodb-Reverse, 5′-AAGCGTGTTCCCATACATCCATAG) based on published Ehrlichia spp. sodb sequences (AF392615.1, CP000107.1) were used. Reactions contained 5 μL of DNA extract, 12.5 μL of MyTaqHS-2Xb, 0.125 μM (or 0.25 μM for sodb primers) of each primerc and RNAse-free, molecular-grade water to a final volume of 25 μL. All reactions were performed in a thermocyclerd with an aluminum block under the following conditions, with respective primer annealing temperatures specified: initial temperature at 94°C for 3 minutes, 55 cycles consisting of denaturation at 94°C for 10 seconds, annealing at 68°C (gltA), 62°C (map1), 58°C (sodb) for 10 seconds, extension at 72°C for 15 seconds, and a final extension at 72°C for 30 seconds. Negative controls (RNAse-free, molecular-grade water and uninfected canine genomic DNA) were included in all assays. Amplified PCR products were sequenced directlye and alignments were made with GenBank sequences using AlignX software.f Sequence identities for the partial gltA and map1 genes are as follows, all with 100% coverage: gltA, 100% similar (269 bp) to PME partial gltA gene from an infected goat (DQ363995.1) and Amblyomma americanum tick (EU272374.1); map1, 100% similar (460 bp) to PME partial map1 gene from an infected goat (DQ324368.1) and A. americanum tick (EU272373.1). Before this study, there was no sequence for PME sodb deposited in GenBank. The highest sequence identities assigned by BLASTg for the partial gene amplified using Ehrlichia spp. sodb primers are as follows, all with 100% coverage: sodb, 89% (264/295 bp) similar to multiple strains of E. ruminantium sodb gene, with the highest Max scores reported for Senegal (DQ647026.1), Pokoase (DQ647024.1), and Kumm1 (DQ647023.1) strains, 83% (245/295 bp) similar to E. canis strain Jake sodb (CP000107.1) and 80% (236/295 bp) similar to E. chaffeensis strain Arkansas sodb (AF392615.1). The PME partial sodb gene sequence was submitted to Genbank (Accession number KC702804). Primer sets specific for Rickettsia ompA, E. canis p30, E. ewingii p28, and E. chaffeensis p28 did not amplify DNA from the PME-infected blood sample, suggesting this dog was not likely coinfected with Rickettsia or other Ehrlichia species. Retrospective PCRs using DNA extracted from predoxycycline splenic cytologic smears were negative (16S rRNA, gltA, map1, sodb, E. canis p30, E. ewingii p28, and E. chaffeensis p28) for pathogen DNA. Treatment for suspected lymphoma was not initiated because of the possibility that lymphocytosis and other lymphocytic abnormalities were due to a reactive process secondary to ehrlichiosis.2-4 Treatment with doxycycline (approximately 5 mg/kg PO q12h) was commenced for 30 days. After starting doxycycline treatment, occasional vomiting was noted. Resolution of the thrombocytopenia and lymphocytosis occurred 1 week after starting the treatment. ALP and ALT activities remained increased at 1,989 IU/L and 119 IU/L, respectively. One week after completion of doxycycline, repeat aspiration cytology identified a mixed lymphoid population with expansion of intermediate lymphocytes and mild extramedullary hematopoiesis within the spleen, a mixed lymphoid population within the popliteal lymph node, and an expansion of intermediate lymphocytes and mild vacuolar change within the liver. Flow cytometry of the liver showed a population of large, granular cells with positive intracellular staining for CD3, consistent with T-cell lymphoma. When cytology was repeated again 4 weeks after completion of doxycycline, there was resolution of the abnormal lymphoid population in liver and lymph nodes, but there were increased numbers of lymphoblasts and mild persistent extramedullary hematopoiesis within the spleen (Fig 3). ALP and ALT activities had decreased to 1,352 IU/L and 66 IU/L, respectively. Throughout the 3-month time period described in this case report, the owners reported no clinical abnormalities other than occasional gastrointestinal signs, but felt in retrospect that the dog may have been slightly lethargic as they reported it was more energetic after completion of the doxycycline treatment regimen. Furthermore, 5 subsequent CBCs performed over the next 6 months remained normal. EDTA-anticoagulated whole blood and convalescent serum samples collected from the dog approximately 2 weeks after starting doxycycline treatment were PCR negative for PME (16S rRNA, gltA, map1 and sodb) and the SNAP 4Dx Plus was positive for anti-Ehrlichia spp. antibodies, respectively. Convalescent serum, collected approximately 5 weeks after completion of antibiotic treatment, was minimally reactive (1:32) to R. rickettsii antigens, whereas the dog remained E. canis seroreactive by both IFA (1:512) and SNAP 4Dx Plusa (weak Ehrlichia spp. positive). EDTA whole blood collected at this time remained PCR negative (16S rRNA, gltA, map1 and sodb) for PME. In this report, we provide molecular evidence of PME in a thrombocytopenic dog with abnormal lymphocytosis and clonal T-cell expansion. Treatment with doxycycline resulted in resolution of thrombocytopenia, abnormal lymphocytosis, and abnormal lymphoid cells in liver and lymph nodes, supporting a potential role for PME as a cause of host immune dysregulation. These findings also support a potential pathogenic role for PME as a cause of thrombocytopenia in dogs from the United States. Cytopathology and flow cytometry in this case identified a large number of atypical lymphocytes in the peripheral blood and in hepatic and splenic tissue aspirates with a high number of intermediate lymphocytes, granular CD3+ cells, and clonal T-cell expansion. Intracellular infections that induce cell-mediated immunity can cause cytological changes similar to malignancy.5, 6 A study characterizing peripheral blood smears in human ehrlichiosis patients infected with either E. chaffeensis or E. ewingii documented prominent large granular lymphocytes with atypical, folded, hyperchromatic nuclei that might be confused with neoplastic NK or NK-like T-cells.6 Immunophenotypes compared between dogs with naturally acquired canine monocytic ehrlichiosis (CME) and healthy dogs found that dogs with CME had higher relative numbers of CD3+ T-cells in peripheral blood than did healthy dogs.7 Additional studies found that dogs experimentally infected with E. canis had transitory CD8+ lymphocytosis in both peripheral blood and lymph nodes.8, 9 Additional studies describe clinically ill, E. canis-seropositive dogs with an increasing percentage of peripheral blood CD8+ lymphocytes.3, 10 PARR results from the dog in this case demonstrated clonal T-cell expansion, typically associated with malignancy. At least 2 reports, however, describe this phenomenon in dogs with E. canis infections.2, 4 In a previous report, an E. canis-seropositive dog with pancytopenia and clonal T-cell expansion was treated with doxycycline, which resolved the pancytopenia and the dog remained healthy 2 years later.2 Further investigation is needed to determine if expanded T-cells in E. canis infections are transitory or potentially could develop into lymphoid malignancy, particularly in association with chronic undiagnosed infections. As current evidence suggests, a role for Ehrlichia spp. in immune dysregulation is likely, but the extent may be subject to host factors, species and strain virulence, as well as phase of the disease. CME has acute, subclinical, and chronic phases representing different infection durations and variations in clinical disease manifestations. One recent study, however, found no statistically significant differences among CD3+, CD8+, and, CD4+ cells in peripheral blood samples from dogs with clinical or subclinical CME.11 Signs of lymphoid malignancy continue to be monitored in the dog of this report. Resolution of the immunologic abnormalities noted in liver, lymph node, and blood after doxycycline treatment, however, strongly supports an infectious etiology. To the authors’ knowledge, this is the first report of PME infection in a dog. Genetically and antigenically similar to E. ruminantium, PME was first identified by PCR in a goat experimentally infested with A. americanum ticks collected from Panola Mountain State Park, Georgia.12 Goats infected with PME developed serous nasal discharge and febrile illness with hematologic changes consisting of decreased ALP activities and neutropenia; rare morulae in mononuclear cells were identified in 1 goat.12, 13 PME also was detected by PCR in whole blood from a man in Atlanta, GA who developed myalgia after being bitten by a nymphal A. americanum tick.14 No hematologic abnormalities were reported, and clinical signs resolved after doxycycline treatment. The role, if any, of PME in the overall pathogenesis of the various disease manifestations described in the dog of this report over its 3 year history of illness is impossible to assess. At the time PME was identified, the dog was asymptomatic and hematological abnormalities were limited to thrombocytopenia, increased numbers of atypical lymphocytes, and progressively increasing ALP activity (after initiation of doxycycline). The most notable characteristics of illness that potentially were related to PME infection in this dog included thrombocytopenia, cytological changes in the liver, lymph node and spleen, and clonal T-cell expansion. After treatment with doxycycline, sequential resolution of most of these abnormalities occurred. It is not clear when the dog became infected with PME, but serum screened for exposure to vector-borne pathogens 8 months earlier was negative, and the dog's owners reported flea and tick exposure over the previous 6-month time period. PME exposure presumably occurred in central NC because the dog had no travel history outside of the state. This is not a surprising finding, given documentation of PME in A. americanum from the eastern United States, with PME-positive ticks detected in FL, GA, KY, NJ, and NY.15 In addition, deer from AR, NC, and VA were PCR positive for PME and were shown to be competent reservoirs for the pathogen.16 Documentation of PME in this dog supports the possibility that this tick-borne organism may represent an unrecognized human or ruminant pathogen in NC and surrounding states. Currently, serological diagnostics specific for PME are not available. Serum from goats infected with PME was weakly IFA seropositive to E. chaffeensis and seropositive by ELISA to the MAP1 protein from E. ruminantium.12, 13 Serum from the dog of this report reacted strongly with E. canis antigen by IFA, but less strongly with the synthetic antigens used in a commercial ELISA (SNAP 4Dx Plus). Antibodies to other Ehrlichia spp. such as E. chaffeensis have been shown to cross-react with E. canis antigens, and it is likely that the reactivity from this PME-infected dog also represented a cross-reaction.17 Whereas E. canis, E. ewingii, and E. chaffeensis were not detected by PCR, we cannot rule out the possibility that seroreactivity was because of previous exposure with one of these pathogens. In addition to highlighting the emergence of vector-borne pathogens in a novel host species, the findings in this case report underscore the challenges faced in diagnosing canine lymphocytic malignancies and reinforce the need to better understand the immunopathology of canine ehrlichiosis, which can be caused by E. canis, E. chaffeensis, E. ewingii, E. muris, and PME in dogs in North America. Documentation of abnormal or expanded lymphocyte populations in the blood or tissues of dogs should prompt diagnostic consideration of infection with an intracellular pathogen, including Ehrlichia spp. Conflict of Interest: Authors disclose no conflict of interest.}, number={5}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Qurollo, B.A. and Davenport, A.C. and Sherbert, B.M. and Grindem, C.B. and Birkenheuer, A.J. and Breitschwerdt, E.B.}, year={2013}, month={Jul}, pages={1251–1255} }
 @article{schreeg_marr_tarigo_cohn_levy_birkenheuer_2013, title={Pharmacogenomics of Cytauxzoon felis Cytochrome b: Implications for Atovaquone and Azithromycin Therapy in Domestic Cats with Cytauxzoonosis}, volume={51}, ISSN={["0095-1137"]}, DOI={10.1128/jcm.01407-13}, abstractNote={Cytauxzoon felis, an emerging virulent protozoan parasite that infects domestic cats, is treated with atovaquone and azithromycin (A&A). Atovaquone targets parasite cytochrome b. We characterized the C. felis cytochrome b gene (cytb) in cats with cytauxzoonosis and found a cytb genotype that was associated with survival in A&A-treated cats.}, number={9}, journal={JOURNAL OF CLINICAL MICROBIOLOGY}, author={Schreeg, Megan E. and Marr, Henry S. and Tarigo, Jaime and Cohn, Leah A. and Levy, Michael G. and Birkenheuer, Adam J.}, year={2013}, month={Sep}, pages={3066–3069} }
 @article{palerme_brown_marks_birkenheuer_2013, title={Splenosystemic Shunts in Cats: A Retrospective of 33 Cases (2004-2011)}, volume={27}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.12188}, abstractNote={Portosystemic shunts are uncommonly reported in cats. The majority of reports describe congenital shunts in young cats originating from the left gastric vein. Although they are only rarely reported, acquired portosystemic shunts in cats appear to be more variable in their anatomic location.To describe the signalment and disease conditions found in cats with splenosystemic shunts.Thirty-three client-owned cats with documented splenosystemic shunts.Retrospective study. All cats with vascular communications between the splenic and left renal veins or the splenic vein and caudal vena cava diagnosed ultrasonographically between 2004 and 2011 were included. Collected data included age, breed, sex, presenting complaints, clinicopathologic data, as well as clinical diagnosis when available.Splenosystemic shunts were identified in 1.3% of the cats that had an abdominal ultrasound performed during the study period. Older, spayed female cats were found to be significantly overrepresented when compared with the total population of cats having undergone ultrasound over the same time period. A large proportion of cats (42%) had a hepatopathy with the potential for associated portal hypertension.Neither the signalment of cats in this report nor the anatomy of their portovascular anomalies shared similarities with those cats previously identified with single-vessel shunts. The relevance and etiology of these newly described splenosystemic shunts remain elusive and warrantsfurther investigation.}, number={6}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Palerme, J-S. and Brown, J. C. and Marks, S. L. and Birkenheuer, A. J.}, year={2013}, month={Nov}, pages={1347–1353} }
 @article{owens_downey_pressler_birkenheuer_chandler_scott-moncrieff_2012, title={Congenital Adrenal Hyperplasia Associated with Mutation in an 11 ss-Hydroxylase-Like Gene in a Cat}, volume={26}, ISSN={["0891-6640"]}, DOI={10.1111/j.1939-1676.2012.00971.x}, number={5}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Owens, S. L. and Downey, M. E. and Pressler, B. M. and Birkenheuer, A. J. and Chandler, D. W. and Scott-Moncrieff, J. C.}, year={2012}, pages={1221–1226} }
 @article{lewis_cohn_marr_birkenheuer_2012, title={Diminazene Diaceturate for Treatment of Chronic Cytauxzoon felis Parasitemia in Naturally Infected Cats}, DOI={10.1111/j.1939-1676.2012.01003.x}, abstractNote={Background Cytauxzoon felis is a hemoprotozoal parasite that causes substantial morbidity and mortality during the acute phase of infection in cats. However, cats that survive the acute illness remain persistently infected and may serve as a reservoir for the tick-transmitted pathogen. Objective We investigated the ability of the antiprotozoal compound diminazene diaceturate to eliminate the pathogen from naturally infected C. felis carriers. Animals Seven healthy, chronically infected domestic cats housed in a research setting. Methods Prospective clinical trial. Cats were treated in a masked fashion with diminazene diaceturate (3 mg/kg) or placebo IM in a series of 2 injections 7 days apart. Clearance of the organism was assessed by light microscopy and real-time polymerase chain reaction (PCR) at 0, 3, 6, and 10 weeks. In addition, cats were monitored for behavioral changes or for changes on physical examination, CBC, plasma biochemical profile, and urinalysis periodically. Cats that remained parasitemic at the end of 10 weeks were switched to the alternative treatment and similarly monitored for an additional 10 weeks. Results Adverse events associated with treatment were limited to self-resolving hypersalivation and injection site soreness; the former was ameliorated by premedication with atropine. Parasite burden, as assayed by both light microscopy and real-time PCR, was similar between diminazene- and placebo-treated cats. Conclusions and Clinical Relevance Diminazene diaceturate was unable to eliminate the pathogen or decrease parasite burden in healthy, chronically infected cats.}, journal={Journal of Veterinary Internal Medicine}, author={Lewis, K. M. and Cohn, L. A. and Marr, H. S. and Birkenheuer, A. J.}, year={2012} }
 @article{lewis_cohn_downey_whitney_birkenheuer_2012, title={Evaluation of Cytauxzoon felis infection status in captive-born wild felids housed in an area endemic for the pathogen}, volume={241}, ISSN={["0003-1488"]}, DOI={10.2460/javma.241.8.1088}, abstractNote={Abstract Objective —To determine whether apparently healthy captive-born wild felids that were not native to North America and were housed in an area endemic for Cytauxzoon felis harbored the pathogen. Design —Prospective observational case series. Animals —11 captive-born wild felids that were (1 bobcat [ Lynx rufus ] and 1 cougar [ Puma concolor ]) or were not (1 lion [ Panthera leo ] and 8 tigers [ Panthera tigris ]) native to North America and 6 domestic cats (5 pets and 1 feral). Procedures —Blood was collected, and a PCR assay for C felis was performed. The C felis 18S rRNA gene sequence was characterized in samples that tested positive. Blood smears were evaluated microscopically for intraerythrocytic organisms consistent with C felis . Blood smears from an additional 6 feral domestic cats found dead on the study premises were also evaluated. Results —4 tigers and 6 domestic cats without clinical signs of disease tested positive for C felis infection via PCR assay; intraerythrocytic organisms consistent with C felis were identified in smears from 1 C felis —infected tiger (which also had azotemia) and in smears from 11 of 12 domestic cats. Possible erythrocytic inclusions were identified in 1 tiger that tested negative for C felis . Sequences of C felis 18S rRNA amplicons from all infected tigers shared > 99.8% identity with reported C felis sequences from North American domestic cats and were identical to amplicons from domestic cats on the premises. Conclusions and Clinical Relevance —Captive tigers without clinical signs of disease tested positive for C felis . The PCR assay for C felis appeared to be more reliable than cytologic detection of piroplasms in tigers.}, number={8}, journal={JAVMA-JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION}, author={Lewis, Kristin M. and Cohn, Leah A. and Downey, Megan E. and Whitney, Marlyn S. and Birkenheuer, Adam J.}, year={2012}, month={Oct}, pages={1088–1092} }
 @article{holowaychuk_birkenheuer_li_marr_boll_nordone_2012, title={Hypocalcemia and Hypovitaminosis D in Dogs with Induced Endotoxemia}, volume={26}, ISSN={["0891-6640"]}, DOI={10.1111/j.1939-1676.2012.00886.x}, abstractNote={Background Hypocalcemia is a documented electrolyte disturbance in people and animals with sepsis, but its mechanism is poorly understood. Objective To investigate mechanisms of hypocalcemia in dogs with experimentally induced endotoxemia. Animals Six healthy mixed breed dogs were included in this nonrandomized, placebo-controlled, crossover study. Methods Dogs initially were injected with placebo (0.9% NaCl; 1 mL, IV) and then lipopolysaccharide (LPS; 2 μg/kg, IV) after a 5-day washout period. Blood and urine samples were collected for measurement of serum total calcium (tCa), ionized calcium (iCa), total magnesium (tMg), ionized magnesium (iMg), parathyroid hormone (PTH), 25-hydroxyvitamin D (vitamin D), venous blood gases, and fractional excretion (FE) of calcium. Results After LPS administration, body temperature increased and blood pressure decreased. Both iCa and tCa decreased (P < .01), but iMg was not significantly different between control and LPS treatments. PTH concentrations increased (P < .01) and vitamin D concentrations decreased (P < .01). Venous pH, bicarbonate, base excess, and blood glucose also decreased (P < .01). Urine tCa concentration was below the limit of detection for all dogs after LPS administration. Conclusions Hypocalcemia occurs during endotoxemia in dogs and is associated with hypovitaminosis D. Hypomagnesemia, hypoparathyroidism, alkalosis, and increased calciuresis are not associated with hypocalcemia in endotoxemic dogs.}, number={2}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Holowaychuk, M. K. and Birkenheuer, A. J. and Li, J. and Marr, H. and Boll, A. and Nordone, S. K.}, year={2012}, pages={244–251} }
 @article{lewis_cohn_birkenheuer_2012, title={Lack of evidence for perinatal transmission of Cytauxzoon felis in domestic cats}, volume={188}, ISSN={["0304-4017"]}, DOI={10.1016/j.vetpar.2012.02.019}, abstractNote={Cytauxzoon felis is a hemoprotozoan parasite of cats capable of causing severe, often fatal disease during acute infection, but cats that survive the acute stage of disease become chronic carriers. These otherwise healthy carriers are capable of transmitting the infection to other cats via the bite of a vector tick. A variety of other hematoprotozoan parasites are capable of vertical transmission from mother to offspring. If this were possible for C. felis, it could be an important part of the explanation for the apparent emergence of this disease with an increased incidence in an expanding geographic area. We investigated the possibility of perinatal transmission of C. felis from chronically infected cats to their offspring. Two queens produced a total of 14 healthy kittens in three litters. All kittens tested negative for C. felis by microscopic slide review and PCR until they were adopted to private homes at approximately 12 weeks of age. While this does not rule out the possibility of perinatal transmission, it is unlikely to be a common phenomenon.}, number={1-2}, journal={VETERINARY PARASITOLOGY}, author={Lewis, Kristin M. and Cohn, Leah A. and Birkenheuer, Adam J.}, year={2012}, month={Aug}, pages={172–174} }
 @article{stowe_birkenheuer_grindem_2012, title={Pathology in practice. Intraerythrocytic infection with organisms consistent with a  large Babesia sp.}, volume={241}, DOI={10.2460/javma.241.8.1029}, number={8}, journal={Journal of the American Veterinary Medical Association}, author={Stowe, Devorah Marks and Birkenheuer, Adam and Grindem, Carol B}, year={2012}, month={Oct}, pages={1029–1031} }
 @article{lewis_cohn_birkenheuer_papich_2012, title={Pharmacokinetics of diminazene diaceturate in healthy cats}, volume={35}, ISSN={["0140-7783"]}, DOI={10.1111/j.1365-2885.2011.01359.x}, abstractNote={Journal of Veterinary Pharmacology and TherapeuticsVolume 35, Issue 6 p. 608-610 SHORT COMMUNICATION Pharmacokinetics of diminazene diaceturate in healthy cats K. M. LEWIS, K. M. LEWIS University of Missouri College of Veterinary Medicine, Columbia, MOSearch for more papers by this authorL. A. COHN, L. A. COHN University of Missouri College of Veterinary Medicine, Columbia, MOSearch for more papers by this authorA. J. BIRKENHEUER, A. J. BIRKENHEUER North Carolina State University College of Veterinary Medicine, Raleigh, NC, USASearch for more papers by this authorM. G. PAPICH, M. G. PAPICH North Carolina State University College of Veterinary Medicine, Raleigh, NC, USASearch for more papers by this author K. M. LEWIS, K. M. LEWIS University of Missouri College of Veterinary Medicine, Columbia, MOSearch for more papers by this authorL. A. COHN, L. A. COHN University of Missouri College of Veterinary Medicine, Columbia, MOSearch for more papers by this authorA. J. BIRKENHEUER, A. J. BIRKENHEUER North Carolina State University College of Veterinary Medicine, Raleigh, NC, USASearch for more papers by this authorM. G. PAPICH, M. G. PAPICH North Carolina State University College of Veterinary Medicine, Raleigh, NC, USASearch for more papers by this author First published: 29 December 2011 https://doi.org/10.1111/j.1365-2885.2011.01359.xCitations: 3 Leah A. Cohn, Department of Veterinary Medicine and Surgery, University of Missouri - College of Veterinary Medicine, 900 E. Campus, Dr., Columbia, MO 65211, USA. E-mail:cohnl@missouri.edu This work was previously presented as a poster at the 2011 ACVIM Forum in Denver, CO. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume35, Issue6December 2012Pages 608-610 RelatedInformation}, number={6}, journal={JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS}, author={Lewis, K. M. and Cohn, L. A. and Birkenheuer, A. J. and Papich, M. G.}, year={2012}, month={Dec}, pages={608–610} }
 @article{di cicco_downey_beeler_marr_cyrog_kidd_diniz_cohn_birkenheuer_2012, title={Re-emergence of Babesia conradae and effective treatment of infected dogs with atovaquone and azithromycin}, volume={187}, ISSN={["0304-4017"]}, DOI={10.1016/j.vetpar.2012.01.006}, abstractNote={Babesia conradae ( B. conradae ) causes hemolytic anemia in dogs. This organism has not been reported clinically since it was originally described in southern California in 1991. To date, no anti-protozoal therapies have been associated with clearance of B. conradae . This report describes the use of atovaquone and azithromycin for the treatment of dogs naturally infected with B. conradae and report the re-emergence of B. conradae in southern California. Twelve dogs naturally infected with B. conradae were identified by practicing veterinarians and public health officials in southern California. Treatments consisted of a 10 day course of atovaquone (13.3 mg/kg PO q 8 h) and azithromycin (10–12.5 mg/kg PO q 24 h). Four dogs were treated in a randomized blinded placebo-controlled fashion, four additional cases were treated in a non-random, non-blinded fashion and one dog received no treatment. All dogs were tested for B. conradae DNA by polymerase chain reaction (PCR) initially and then once or 3 times post treatment (60–210 days). B. conradae infected dogs that received treatment did not have any detectable Babesia DNA by PCR after treatment. In contrast, dogs receiving placebo had detectable Babesia DNA by PCR throughout the study period. Combination therapy with atovaquone and azithromycin appears to be effective for acute and chronic babesiosis caused by B. conradae .}, number={1-2}, journal={VETERINARY PARASITOLOGY}, author={Di Cicco, Michael F. and Downey, Megan E. and Beeler, Emily and Marr, Henry and Cyrog, Peter and Kidd, Linda and Diniz, Pedro Paulo V. P. and Cohn, Leah A. and Birkenheuer, Adam J.}, year={2012}, month={Jun}, pages={23–27} }
 @article{shock_birkenheuer_patton_olfenbuttel_beringer_grove_peek_butfiloski_hughes_lockhart_et al._2012, title={Variation in the ITS-1 and ITS-2 rRNA genomic regions of Cytauxzoon felis from bobcats and pumas in the eastern United States and comparison with sequences from domestic cats}, volume={190}, ISSN={0304-4017}, url={http://dx.doi.org/10.1016/j.vetpar.2012.06.010}, DOI={10.1016/j.vetpar.2012.06.010}, abstractNote={Cytauxzoon felis, a tick-borne protozoan parasite, is the causative agent of cytauxzoonosis in domestic cats in the United States. The natural reservoir for this parasite is the bobcat (Lynx rufus), which typically does not develop clinical signs. Although not likely important reservoirs, C. felis has also been detected in pumas (Puma concolor) in Florida and Louisiana. Recent studies suggest that specific genotypes of C. felis that circulate in domestic cats may be associated with variable clinical outcomes and specific spatial locations. In the current study, we investigated the intraspecific variation of the C. felis internal transcribed spacer (ITS)-1 and ITS-2 rRNA regions from 145 wild felids (139 bobcats and six pumas) from 11 states (Florida, Georgia, Kansas, Kentucky, Louisiana, Missouri, North Carolina, North Dakota, South Carolina, Oklahoma, and Pennsylvania). Unambiguous ITS-1 and ITS-2 data were obtained for 144 and 112 samples, respectively, and both ITS-1 and ITS-2 sequences were obtained for 111 (77%) samples. For the ITS-1 region, sequences from 65 samples collected from wild felids were identical to those previously reported in domestic cats, while the other 79 sequences were unique. C. felis from 45 bobcats and one puma had ITS-1 sequences identical to the most common sequence reported from domestic cats. Within the ITS-2 region, sequences from 49 bobcats were identical to those previously reported in domestic cats and 63 sequences were unique (with some occurring in more than one bobcat). The most common ITS-2 sequence from domestic cats was also common in wild felids (31 bobcats and a puma). Samples from three pumas from Florida and two bobcats from Missouri had a 40- or 41-bp insert in the ITS-2 similar to one described previously in a domestic cat from Arkansas. Additionally, a previously undescribed 198- or 199-bp insert was detected in the ITS-2 sequence from four bobcats. Collectively, based on combined ITS-1 and ITS-2 sequences, five different genotypes were detected in the wild felids. Genotype ITSa was the most common genotype (11 bobcats and one puma) and fewer numbers of ITSb, ITSe, ITSg, and ITSi were detected in bobcats. These data indicate that, based on ITS-1 and ITS-2 sequences, numerous C. felis strains may circulate in wild felids.}, number={1-2}, journal={Veterinary Parasitology}, publisher={Elsevier BV}, author={Shock, Barbara C. and Birkenheuer, Adam J. and Patton, Laura L. and Olfenbuttel, Colleen and Beringer, Jeff and Grove, Daniel M. and Peek, Matt and Butfiloski, Joseph W. and Hughes, Daymond W. and Lockhart, J. Mitchell and et al.}, year={2012}, month={Nov}, pages={29–35} }
 @article{cohn_birkenheuer_brunker_ratcliff_craig_2011, title={Efficacy of Atovaquone and Azithromycin or Imidocarb Dipropionate in Cats with Acute Cytauxzoonosis}, volume={25}, ISSN={["0891-6640"]}, DOI={10.1111/j.1939-1676.2010.0646.x}, abstractNote={Background: Imidocarb or a combination of atovaquone and azithromycin (A&A) has been suggested for treatment of cats with cytauxzoonosis, but neither has been prospectively evaluated for efficacy. Hypothesis/Objectives: That survival to hospital discharge is improved by treatment with A&A as compared with imidocarb. Animals: Eighty acutely ill cats with Cytauxzoon felis infection treated at one of 18 veterinary clinics in 5 states. Methods: An open-label, randomized prospective study compared survival in cats treated with atovaquone (15 mg/kg PO q8h) and azithromycin (10 mg/kg PO q24h) or imidocarb (3.5 mg/kg IM). All received heparin, fluids, and supportive care. Clinical and clinicopathologic data from initial presentation were collated. Parasitemia was quantified (n = 79) and pathogens genotyped (n = 60). Logistic regression was used to determine the impact of treatment group on the primary endpoint, survival to hospital discharge or death. Covariants were analyzed by rank-sum testing. Results: Of 53 cats treated with A&A, 32 (60%) survived to discharge while only 7 of 27 cats (26%) treated with imidocarb survived (P= .0036; odds ratio 7.2, 95% CI 2.2, 24). Cats with a lower parasitemia were more likely to survive, as were cats with higher white blood cell counts and lower total bilirubin. Unique pathogen genotypes were identified from 15 cats, while genotype isolated from 21 cats had been described previously. There were multiple pathogen genotypes identified in 24 cats. Conclusions and Clinical Importance: Survival to discharge was more likely in cats treated with A&A as compared with imidocarb, although case fatality rate remained high.}, number={1}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Cohn, L. A. and Birkenheuer, A. J. and Brunker, J. D. and Ratcliff, E. R. and Craig, A. W.}, year={2011}, pages={55–60} }
 @article{li_birkenheuer_marr_levy_yoder_nordone_2011, title={Expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) on canine neutrophils}, volume={35}, ISSN={0145-305X}, url={http://dx.doi.org/10.1016/j.dci.2011.03.021}, DOI={10.1016/j.dci.2011.03.021}, abstractNote={The dog is both a valued veterinary species and a widely used translational model for sepsis research. However, relatively little work has been performed evaluating potential biomarkers present during canine infection. Triggering receptor expressed on myeloid cells-1 (TREM-1) has shown promise as a biomarker for infection and pneumonia in humans. Here we describe, for the first time, the expression and function of the canine orthologue of TREM-1. Expression of TREM-1 on canine neutrophils is significantly up-regulated by stimulation with microbial agonists of TLR2/6, TLR1/2, and TLR4/MD2. Kinetics of TREM-1 protein up-regulation are rapid, with significant increases observed within 2 hr of neutrophil activation. Functionally, canine TREM-1 synergistically enhances LPS-induced production of IL-8, TNF-α and a canine orthologue of CXCL1. Collectively, these data suggest that TREM-1 expression in dogs, as it is in humans, is an amplifier of pro-inflammatory responses to microbial products. These results have direct application to veterinary diagnostics as well as the potential to enhance the utility of canine disease models in the assessment of potential therapeutics in the treatment of human sepsis.}, number={8}, journal={Developmental & Comparative Immunology}, publisher={Elsevier BV}, author={Li, Jingjing and Birkenheuer, Adam J. and Marr, Henry S. and Levy, Michael G. and Yoder, Jeffrey A. and Nordone, Shila K.}, year={2011}, month={Aug}, pages={872–880} }
 @article{levy_lappin_glaser_birkenheuer_anderson_edinboro_2011, title={Prevalence of infectious diseases in cats and dogs rescued following Hurricane Katrina}, volume={238}, ISSN={["1943-569X"]}, DOI={10.2460/javma.238.3.311}, abstractNote={Abstract Objective —To determine the prevalence of infectious diseases of animal and zoonotic importance in cats and dogs rescued and transferred from the Gulf Coast region following Hurricane Katrina. Design —Cross-sectional study. Animals —414 dogs and 56 cats rescued and transferred from the Gulf Coast region within 4 months after the hurricane. Procedures —EDTA-anticoagulated blood and serum samples were tested via PCR and serologic assays for infectious diseases. Results —In dogs, prevalence was highest for anti-West Nile virus (WNV) antibodies (218/390 [55.9%]), Dirofilaria immitis antigen (195/400 [48.8%]), anti- Toxoplasma gondii antibodies (92/366 [25.1%]), and hemotropic mycoplasma DNA (40/345 [11.9%]). The DNA of Bartonella spp, Ehrlichia spp, or Babesia spp or anti-canine influenza virus antibodies were identified in < 2% of dogs. In cats, prevalence was highest for antibodies against Bartonella spp and DNA of Bartonella spp combined (49/55 [89.1 %]), anti– T gondii antibodies (13/55 [23.6%]), hemotropic mycoplasma DNA (5/47 [10.6%]), anti-WNV antibodies (5/48 [10.4%]), D immitis antigen (4/50 [8.0%]), and anti–FIV antibodies (4/56 [7.1%]). A total of 308 (74.4%) dogs and 52 (92.9%) cats had evidence of previous or current vector-borne infections. Conclusions and Clinical Relevance —Cats and dogs rescued from the disaster region had evidence of multiple infectious diseases. The dispersal of potentially infectious animals to other regions of North America where some infections were not typically found could have contributed to new geographic ranges for these organisms or to underdiagnosis in affected animals because of a low index of suspicion in regions with low disease prevalence.}, number={3}, journal={JAVMA-JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION}, author={Levy, Julie K. and Lappin, Michael R. and Glaser, Amy L. and Birkenheuer, Adam J. and Anderson, Tara C. and Edinboro, Charlotte H.}, year={2011}, month={Feb}, pages={311–317} }
 @article{hummel_grooters_davidson_jennings_nicklas_birkenheuer_2011, title={Successful management of gastrointestinal pythiosis in a dog using itraconazole, terbinafine, and mefenoxam}, volume={49}, ISSN={["1369-3786"]}, DOI={10.3109/13693786.2010.543705}, abstractNote={Medical therapy for pythiosis is hampered by a lack of efficacious drugs. The present report describes a case of canine gastrointestinal pythiosis in which lesions were resolved through the administration of itraconazole, terbinafine, and the agricultural fungicide mefenoxam. No substantial adverse effects occurred in association with administration of the latter compound. Additional studies are needed to evaluate the pharmacokinetics of mefenoxam and to further assess its tolerability and potential efficacy for the treatment of pythiosis in dogs.}, number={5}, journal={MEDICAL MYCOLOGY}, author={Hummel, James and Grooters, Amy and Davidson, Gigi and Jennings, Samuel and Nicklas, Jodi and Birkenheuer, Adam}, year={2011}, month={Jul}, pages={539–542} }
 @article{clancey_horney_burton_birkenheuer_mcburney_tefft_2010, title={Babesia (Theileria) annae in a Red Fox (Vulpes vulpes) from Prince Edward Island, Canada}, volume={46}, ISSN={0090-3558}, url={http://dx.doi.org/10.7589/0090-3558-46.2.615}, DOI={10.7589/0090-3558-46.2.615}, abstractNote={A 4-6-mo-old female red fox (Vulpes vulpes) was presented to the Atlantic Veterinary College (AVC) Teaching Hospital, Prince Edward Island, Canada. On presentation, the fox was weak and had pale mucous membranes. A complete blood count and a serum biochemistry profile were performed. Blood smear examination revealed low numbers of erythrocytes containing centrally to paracentrally located, single, rarely multiple, approximately 1 x 2 microm, oval to round organisms with morphology similar to Babesia microti. Polymerase chain reaction testing and DNA sequencing of the Babesia species 18S rRNA gene were performed on DNA extracted from whole blood. Results were positive for a Babesia microti-like parasite genetically identical to Babesia (Theileria) annae. The fox was euthanized due to poor prognosis for recovery. Necropsy examination revealed multifocal to locally extensive subacute nonsuppurative meningoencephalitis, an eosinophilic broncho-pneumonia, a moderate diffuse vacuolar hepatopathy, and lesions associated with blunt trauma to the left abdominal region. This is the first reported case of a red fox in Canada infected with a piroplasm. It remains uncertain whether the presence of this hemoparasite in this fox was pathogenic or an incidental finding. The potential for competent vectors of Babesia species on Prince Edward Island, the potential for this Babesia microti-like parasite to infect other wild and domestic canids, and the significance of this parasite to the health of infected individuals are yet to be determined.}, number={2}, journal={Journal of Wildlife Diseases}, publisher={Wildlife Disease Association}, author={Clancey, Noel and Horney, Barbara and Burton, Shelley and Birkenheuer, Adam and McBurney, Scott and Tefft, Karen}, year={2010}, month={Apr}, pages={615–621} }
 @article{kamani_sannusi_dogo_tanko_egwu_tafarki_ogo_kemza_onovoh_shamaki_et al._2010, title={Babesia canis and Babesia rossi co-infection in an untraveled Nigerian dog}, volume={173}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2010.06.040}, abstractNote={A sexually intact 6-month-old female Alsatian dog was presented to the Veterinary Clinic of the National Veterinary Research Institute, Vom, Plateau State, Nigeria, for the following complaints: anorexia, hemoglobinuria, fever, tick infestation and general malaise. Microscopy revealed piroplasms with a wide range of sizes (1-5 μm in length) in red blood cells, raising a suspicion of a co-infection with two or more Babesia species. Specific PCR assays for canine Babesia spp. and DNA sequencing revealed the presence of Babesia canis and Babesia rossi co-infection. This study constitutes the first report of co-infection with B. canis and B. rossi in the West African sub-region and the first report of autochthonous B. canis on the African continent. Practitioners should be aware of potential changes in the species/sub-species of Babesia causing canine babesiosis in this region.}, number={3-4}, journal={VETERINARY PARASITOLOGY}, author={Kamani, Joshua and Sannusi, Abdulrahim and Dogo, A. Goni and Tanko, James T. and Egwu, Kinsley O. and Tafarki, Agbadu E. and Ogo, Isaac N. and Kemza, Sarah and Onovoh, Emmanuel and Shamaki, David and et al.}, year={2010}, month={Oct}, pages={334–335} }
 @article{birkenheuer_horney_bailey_scott_sherbert_catto_marr_camacho_ballman_2010, title={Babesia microti-like infections are prevalent in North American foxes}, volume={172}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2010.05.020}, abstractNote={Babesia microti-like organisms have recently been identified as a cause of hemolytic anemia and azotemia in European dogs. A genetically and morphologically similar B. microti-like parasite has been identified in two foxes from North America. In order to assess the prevalence of this parasite in North American wild canids we screened blood samples from coyotes (Canis latrans) and red foxes (Vulpes vulpes) from eastern Canada and red foxes and gray foxes (Urocyon cinereoargenteus) from North Carolina, USA for the presence B. microti-like DNA by polymerase chain reaction. Thirty-nine percent (50/127) of the red fox samples, 26% (8/31) of the gray fox samples and none (0/12) from the coyote samples tested positive for the presence of B. microti-like DNA. Partial 18S ribosomal ribonucleic acid and beta-tubulin genes from the North American B. microti-like parasites of foxes were sequenced and samples from six domestic dogs from Spain that were infected with a B. microti-like parasite were analyzed for comparison. Partial 18S ribosomal ribonucleic acid and beta-tubulin gene sequences from the North American B. microti-like parasites of foxes were nearly identical to those previously reported from foxes as well as those from domestic dogs from Spain characterized in this study. Interestingly, partial beta-tubulin gene sequences characterized from the B. microti-like parasites of domestic dogs from Spain in this study were different from those previously reported from a Spanish domestic dog sample which is believed to be a pseudogene. The ability of the North American B. microti-like parasite to infect and induce disease in domestic dogs remains unknown. Further studies investigating the pathogenic potential of the North American B. microti-like parasite in domestic dogs are indicated.}, number={3-4}, journal={VETERINARY PARASITOLOGY}, author={Birkenheuer, Adam J. and Horney, Barbara and Bailey, Matthew and Scott, McBurney and Sherbert, Brittany and Catto, Victoria and Marr, Henry S. and Camacho, Angel-Tomas and Ballman, Anne E.}, year={2010}, month={Sep}, pages={179–182} }
 @article{sikorski_birkenheuer_holowaychuk_mccleary-wheeler_davis_littman_2010, title={Babesiosis Caused by a Large Babesia Species in 7 Immunocompromised Dogs}, volume={24}, ISSN={["1939-1676"]}, DOI={10.1111/j.1939-1676.2009.0440.x}, abstractNote={A large unnamed Babesia species was detected in a dog with lymphoma. It was unknown if this was an underrecognized pathogen.Report the historical and clinicopathologic findings in 7 dogs with babesiosis caused by a large unnamed Babesia species characterize the 18S ribosomal ribonucleic acid (rRNA) genes.Seven immunocompromised dogs from which the Babesia was isolated.Retrospective case review. Cases were identified by a diagnostic laboratory, the attending clinicians were contacted and the medical records were reviewed. The Babesia sp. 18S rRNA genes were amplified and sequenced.Six of 7 dogs had been splenectomized; the remaining dog was receiving oncolytic drugs. Lethargy, anorexia, fever, and pigmenturia were reported in 6/7, 6/7, 4/7, and 3/7 dogs. Laboratory findings included mild anemia (7/7) and severe thrombocytopenia (6/7). Polymerase chain reaction (PCR) assays used to detect Babesia sensu stricto species were all positive, but specific PCR assays for Babesia canis and Babesia gibsoni were negative in all dogs. The 18S rRNA gene sequences were determined to be identical to a large unnamed Babesia sp. previously isolated. Cross-reactive antibodies against other Babesia spp. were not always detectable. Five dogs were treated with imidocarb dipropionate and 1 dog with atovaquone/azithromycin; some favorable responses were noted. The remaining dog was untreated and remained a clinically stable carrier.Dogs with pigmenturia, anemia, and thrombocytopenia should be tested for Babesia sp. by PCR. Serology is not sufficient for diagnosis of this Babesia sp. Asplenia, chemotherapy, or both might represent risk factors for persistent infection, illness, or both.}, number={1}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Sikorski, L. E. and Birkenheuer, A. J. and Holowaychuk, M. K. and McCleary-Wheeler, A. L. and Davis, J. M. and Littman, M. P.}, year={2010}, pages={127–131} }
 @article{barber_li_diniz_porter_breitschwerdt_claiborne_birkenheuer_levine_levine_chandler_et al._2010, title={Evaluation of Brain Tissue or Cerebrospinal Fluid with Broadly Reactive Polymerase Chain Reaction for Ehrlichia, Anaplasma, Spotted Fever Group Rickettsia, Bartonella, and Borrelia Species in Canine Neurological Diseases (109 Cases)}, volume={24}, ISSN={0891-6640 1939-1676}, url={http://dx.doi.org/10.1111/j.1939-1676.2009.0466.x}, DOI={10.1111/j.1939-1676.2009.0466.x}, abstractNote={Vector-transmitted microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, Bartonella, and Borrelia are commonly suspected in dogs with meningoencephalomyelitis (MEM), but the prevalence of these pathogens in brain tissue and cerebrospinal fluid (CSF) of dogs with MEM is unknown.To determine if DNA from these genera is present in brain tissue and CSF of dogs with MEM, including those with meningoencephalitis of unknown etiology (MUE) and histopathologically confirmed cases of granulomatous (GME) and necrotizing meningoencephalomyelitis (NME).Hundred and nine dogs examined for neurological signs at 3 university referral hospitals.Brain tissue and CSF were collected prospectively from dogs with neurological disease and evaluated by broadly reactive polymerase chain reaction (PCR) for Ehrlichia, Anaplasma, Spotted Fever Group Rickettsia, Bartonella, and Borrelia species. Medical records were evaluated retrospectively to identify MEM and control cases.Seventy-five cases of MUE, GME, or NME, including brain tissue from 31 and CSF from 44 cases, were evaluated. Brain tissue from 4 cases and inflammatory CSF from 30 cases with infectious, neoplastic, compressive, vascular, or malformative disease were evaluated as controls. Pathogen nucleic acids were detected in 1 of 109 cases evaluated. Specifically, Bartonella vinsonii subsp. berkhoffii DNA was amplified from 1/6 dogs with histopathologically confirmed GME.The results of this investigation suggest that microorganisms in the genera Ehrlichia, Anaplasma, Rickettsia, and Borrelia are unlikely to be directly associated with canine MEM in the geographic regions evaluated. The role of Bartonella in the pathogenesis of GME warrants further investigation.}, number={2}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Barber, R.M. and Li, Q. and Diniz, P.P.V.P. and Porter, B.F. and Breitschwerdt, E.B. and Claiborne, M.K. and Birkenheuer, A.J. and Levine, J.M. and Levine, G.J. and Chandler, K. and et al.}, year={2010}, month={Mar}, pages={372–378} }
 @article{chinnadurai_birkenheuer_blanton_maggi_belfiore_marr_breitschwerdt_stoskopf_2010, title={Prevalence of Selected Vector-borne Organisms and Identification of Bartonella Species DNA in North American River Otters (Lontra canadensis)}, volume={46}, ISSN={0090-3558}, url={http://dx.doi.org/10.7589/0090-3558-46.3.947}, DOI={10.7589/0090-3558-46.3.947}, abstractNote={Trapper-killed North American river otters (Lontra canadensis) in North Carolina, USA, were screened for multiple vector-borne bacteria known to be pathogenic to mammals. Blood was collected from 30 carcasses in 2006, from 35 in 2007, and from one live otter in 2008. Samples were screened using conventional polymerase chain reaction (PCR) tests for DNA from Bartonella spp., Ehrlichia spp., and spotted fever group Rickettsia spp. All samples were negative for Rickettsia spp. Twelve of 30 samples from 2006 produced amplicons using the assay designed to detect Ehrlichia spp., but sequencing revealed that the amplified DNA fragment was from a novel Wolbachia sp., thought to be an endosymbiote of a Dirofilaria sp. Between 2006 and 2007, DNA from a novel Bartonella sp. was detected in 19 of 65 animals (29%). Blood from one live otter captured in 2008 was found positive for this Bartonella sp. by both PCR and culture. The pathogenicity of this Bartonella species in river otters or other mammals is unknown.}, number={3}, journal={Journal of Wildlife Diseases}, publisher={Wildlife Disease Association}, author={Chinnadurai, Sathya K. and Birkenheuer, Adam J. and Blanton, Hunter L. and Maggi, Ricardo G. and Belfiore, Natalia and Marr, Henry S. and Breitschwerdt, Edward B. and Stoskopf, Michael K.}, year={2010}, month={Jul}, pages={947–950} }
 @article{bartlett_abou-madi_messick_birkenheuer_kollias_2009, title={DIAGNOSIS AND TREATMENT OF BABESIA ODOCOILEI IN CAPTIVE REINDEER (RANGIFER TARANDUS TARANDUS) AND RECOGNITION OF THREE NOVEL HOST SPECIES}, volume={40}, ISSN={["1937-2825"]}, DOI={10.1638/2008-0011.1}, abstractNote={Two captive reindeer (Rangifer tarandus tarandus) at a New York zoological institution were diagnosed with Babesia odocoilei. Clinical signs consistent with acute babesiosis included fever, hemoglobinuria, and hemolytic anemia. Both episodes were precipitated by stressful events that may have compromised their immunocompetence. The diagnosis was confirmed by visualization of intraerythrocytic parasites on stained blood smears, polymerase chain reaction, and speciation of the Babesia by sequencing a hypervariable region of the 18S rRNA gene. One reindeer died with gross and histopathologic lesions, including pigmentary nephrosis with severe acute tubular degeneration and necrosis secondary to intravascular hemolysis. A second reindeer was successfully treated with supportive care and an antiprotozoal, imidocarb dipropionate (Imizol, 12%, Schering-Plough Animal Health, Union, New Jersey 07083, USA) at 3 mg/kg s.c. or i.m. s.i.d. on days 1, 2, 6, 9, and 21. Two other reindeer in the exhibit tested negative for Babesia by polymerase chain reaction but were treated with imidocarb dipropionate as prophylaxis while final testing results were pending. Additionally, B. odocoilei was identified in three novel asymptomatic host species within the collection: yak (Bos grunniens), muntjac (Muntiacus reevesi), and markhor goat (Caprafalconeri). Due to the high morbidity and mortality associated with acute babesiosis, captive reindeer should receive tick prevention, be tested for subclinical infections in endemic areas, and receive aggressive treatment for acute infections when clinical babesiosis is suspected.}, number={1}, journal={JOURNAL OF ZOO AND WILDLIFE MEDICINE}, author={Bartlett, Susan L. and Abou-Madi, Noha and Messick, Joanne B. and Birkenheuer, Adam and Kollias, George V.}, year={2009}, month={Mar}, pages={152–159} }
 @article{levine_zhou_ghiloni_fields_birkenheuer_gookin_roberston_malloy_feldman_2009, title={Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets in a Pomeranian Dog Caused by a Novel Mutation in the Vitamin D Receptor Gene}, volume={23}, ISSN={["1939-1676"]}, DOI={10.1111/j.1939-1676.2009.0405.x}, number={6}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={LeVine, D. N. and Zhou, Y. and Ghiloni, R. J. and Fields, E. L. and Birkenheuer, A. J. and Gookin, J. L. and Roberston, I. D. and Malloy, P. J. and Feldman, D.}, year={2009}, pages={1278–1283} }
 @article{duncan_marr_birkenheuer_maggi_williams_correa_breitschwerdt_2008, title={Bartonella DNA in the Blood and Lymph Nodes of Golden Retrievers with Lymphoma and in Healthy Controls}, volume={22}, ISSN={0891-6640 1939-1676}, url={http://dx.doi.org/10.1111/j.1939-1676.2007.0018.x}, DOI={10.1111/j.1939-1676.2007.0018.x}, abstractNote={Although lymphoma is the most common neoplastic process reported in dogs, its precise etiology is unknown. Golden Retrievers are more likely to develop lymphoma, suggesting a breed predisposition; however, other factors, including environment, immunity, and infection, are likely contributors to oncogenesis.We hypothesized that the development of lymphoma in Golden Retrievers may be associated with vector-borne infections, specifically Bartonella, Anaplasma, or Ehrlichia species infections.Golden Retrievers with lymphoma and healthy Golden Retrievers from across the United States were recruited for study participation.A matched, case-control study was performed to determine the association of lymphoma and the presence of Bartonella, Anaplasma, and Ehrlichia species in serum, blood, and lymph node aspirates.Using PCR analyses and DNA sequencing, single and coinfections with Bartonella henselae, Bartonella elizabethae, Bartonella quintana, and/or Bartonella vinsonii (berkhoffii) were detected in the blood and lymph node aspirates of Golden Retrievers with lymphoma (5/28 dogs, 18%) and in healthy Golden Retrievers (10/56 dogs, 18%); no Anaplasma or Ehrlichia DNA was detected in any dog. When compared with dogs with lymphoma, a higher (P <.001) proportion of healthy Golden Retrievers were receiving monthly acaricide treatments (2.6 times higher).Bartonella DNA can be detected in blood and lymph nodes; importantly, in this report, Bartonella was detected in the same proportion of clinically healthy dogs and dogs with lymphoma. Longitudinal studies should be conducted to determine the mode of transmission of Bartonella in dogs, whether lymphatic infection is persistent, or whether these bacteria may contribute to the development of lymphoma.}, number={1}, journal={Journal of Veterinary Internal Medicine}, publisher={Wiley}, author={Duncan, A.W. and Marr, H.S. and Birkenheuer, A.J. and Maggi, R.G. and Williams, L.E. and Correa, M.T. and Breitschwerdt, E.B.}, year={2008}, month={Jan}, pages={89–95} }
 @article{birkenheuer_marr_warren_acton_mucker_humphreys_tucker_2008, title={Cytauxzoon felis infections are present in bobcats (Lynx rufus) in a region where cytauxzoonosis is not recognized in domestic cats}, volume={153}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2008.01.020}, abstractNote={This study was performed to determine the prevalence of Cytauxzoon felis (C. felis) infections in bobcats (Lynx rufus) from a region where C. felis is recognized in domestic cats, North Carolina (NC), and a region where C. felis is not recognized in domestic cats, Pennsylvania (PA). Samples from NC (n=32) were obtained post-mortem via cardiac puncture from legally trapped bobcats. Samples from PA (n=70) were collected post-mortem onto Nobuto blood collecting strips by the PA Game Commission. Each sample was tested using a C. felis specific PCR assay as well as a PCR assay targeting host DNA to rule out the presence of PCR inhibitors. Three samples were excluded due to the presence of PCR inhibitors. Thirty-three percent (10/30) of the samples from NC and 7% (5/69) of the samples from PA tested positive for the presence of C. felis. The proportion of C. felis positive bobcats from NC was significantly different than that from PA (P<0.005). Despite the lower prevalence of C. felis infections in bobcats from PA this finding is unique and indicates the potential for C. felis infections in domestic cats in the northeastern USA if the appropriate tick vectors are present. Veterinary practitioners in PA should be on alert for cytauxzoonosis in domestic cats. Further studies about the epidemiology and transmission of C. felis infections among both domestic cats and bobcats are needed.}, number={1-2}, journal={VETERINARY PARASITOLOGY}, author={Birkenheuer, Adam J. and Marr, Henry S. and Warren, Camille and Acton, Anne E. and Mucker, Eric M. and Humphreys, Jan G. and Tucker, Melissa D.}, year={2008}, month={May}, pages={126–130} }
 @article{stauffer_birkenheuer_levy_marr_gookin_2008, title={Evaluation of four DNA extraction methods for the detection of Tritrichomonas foetus in feline stool specimens by polymerase chain reaction}, volume={20}, ISSN={["1943-4936"]}, DOI={10.1177/104063870802000518}, abstractNote={Feces are increasingly valued as practical samples for molecular diagnosis of infectious disease. However, extraction of polymerase chain reaction (PCR) quality DNA from fecal samples can be challenging because of coextraction of PCR inhibitors. Because the type and quantity of PCR inhibitors is influenced by diet, endogenous flora, and concurrent disease, it is unlikely that extraction method performance with human feces can be directly extrapolated to that of domestic cats. In the present study, 4 commercially available DNA extraction methods were examined for their influence on the sensitivity of PCR for the detection of Tritrichomonas foetus in feline stool. DNA was extracted from serially diluted feline-origin T. foetus trophozoites in the absence or presence of feline feces. The ZR Fecal DNA kit was identified as affording the greatest analytical sensitivity and reproducibility and was able to detect ≥10 T. foetus organisms per 100 mg feces in 100% of PCR reactions. Further, the identified extraction method could be completed in the shortest time of all kits tested.}, number={5}, journal={JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION}, author={Stauffer, Stephen H. and Birkenheuer, Adam J. and Levy, Michael G. and Marr, Henry and Gookin, Jody L.}, year={2008}, month={Sep}, pages={639–641} }
 @article{hawkins_johnson_guptill_marr_breitschwerdt_birkenheuer_2008, title={Failure to identify an association between serologic or molecular evidence ofBartonellainfection and idiopathic rhinitis in dogs}, volume={233}, ISSN={0003-1488}, url={http://dx.doi.org/10.2460/javma.233.4.597}, DOI={10.2460/javma.233.4.597}, abstractNote={Abstract Objective —To determine whether infection with or exposure to Bartonella spp was associated with idiopathic rhinitis in dogs. Design —Case-control study. Animals —44 dogs with idiopathic nasal discharge and 63 age- and weight-matched control dogs without nasal discharge and no clinical signs of bartonellosis. Procedures —Serum was tested for antibodies against Bartonella henselae and Bartonella vinsonii subsp berkhoffii with indirect fluorescent antibody assays. Blood was tested for Bartonella DNA with a PCR assay. Results —Results of the antibody and PCR assays were negative for all 44 dogs with idiopathic nasal discharge. One control dog had antibodies against B henselae ; a second control dog had positive PCR assay results. We did not detect a significant association between assay results and group designation. Conclusions and Clinical Relevance —The present study failed to confirm an association between idiopathic rhinitis and exposure to or infection with Bartonella spp in dogs. Findings do not rule out the possibility that Bartonella infection may cause nasal discharge in some dogs, but the failure to find any evidence of exposure to or infection with Bartonella spp in dogs with idiopathic nasal discharge suggested that Bartonella infection was not a common cause of the disease.}, number={4}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Hawkins, Eleanor C. and Johnson, Lynelle R. and Guptill, Lynn and Marr, Henry S. and Breitschwerdt, Edward B. and Birkenheuer, Adam J.}, year={2008}, month={Aug}, pages={597–599} }
 @article{lehtinen_birkenheuer_droleskey_holman_2008, title={In vitro cultivation of a newly recognized Babesia sp in dogs in North Carolina}, volume={151}, ISSN={["0304-4017"]}, DOI={10.1016/j.vetpar.2007.10.022}, abstractNote={A novel large Babesia sp. from an infected dog was cultivated in vitro by microaerophilous stationary phase culture methodology. A primary culture initiated in enriched RPMI-1640 medium supplemented with 40% canine serum and incubated in a 2% oxygen environment supported parasite growth in vitro. Subsequent subcultures into enriched HL-1 medium with 20% fetal bovine serum also supported parasite propagation. Cultures were successfully introduced to 5% carbon dioxide in air atmosphere at passage 4. To date, the parasites have been continuously cultured through 35 passages, although the parasitemias are low, ranging from 0.2 to 0.3%. Parasites cultured in RPMI with canine serum were cryopreserved and successfully recovered from liquid nitrogen storage. The small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived parasites, differing in a single base position from the previously reported sequence for this Babesia sp. The ultrastructure of the parasite was consistent with that of other large Babesia spp., except that the spherical body contained numerous round particles unlike the inclusions previously described in Babesia spp.}, number={2-4}, journal={VETERINARY PARASITOLOGY}, author={Lehtinen, Lauren E. and Birkenheuer, Adam J. and Droleskey, Robert E. and Holman, Patricia J.}, year={2008}, month={Feb}, pages={150–157} }
 @article{birkenheuer_marr_hladio_acton_2008, title={Molecular evidence of prevalent dual piroplasma infections in North American raccoons (Procyon lotor)}, volume={135}, ISSN={["1469-8161"]}, DOI={10.1017/S0031182007003538}, abstractNote={Based on 18S rRNA sequence analyses 2 distinct genotypes of piroplasms have been described in raccoons. One genotype resides in the Babesia sensu stricto clade and the other in the Babesia microti-like clade. Since these organisms appear morphologically indistinguishable, it is unclear which strain is responsible for the majority of the infections in raccoons. In order to overcome these limitations we performed a molecular survey of raccoons using polymerase chain reaction assays specific for each genotype. We tested blood samples from 41 wild raccoons trapped in eastern North Carolina using PCR assays and found that 95% (39/41) had detectable piroplasm DNA. Ninety percent (37/41) of the samples contained Babesia sensu stricto DNA and 83% (34/41) samples contained Babesia microti-like DNA. DNA from both genotypes was present in 76% (31/41) samples suggesting a very high rate of co-infections. The presence of dual piroplasma infections in carnivores appears to be an uncommon finding. This study highlights the need for molecular assays for the accurate identification of piroplasma. Further studies are indicated to investigate the ability of these parasites to infect domestic animals as well as their zoonotic potential.}, number={1}, journal={PARASITOLOGY}, author={Birkenheuer, A. J. and Marr, H. S. and Hladio, N. and Acton, A. E.}, year={2008}, month={Jan}, pages={33–37} }
 @article{small_atkins_gordon_birkenheuer_booth-sayer_keene_fujii_miller_2008, title={Use of a nitinol gooseneck snare catheter for removal of adult Dirofilaria immitis in two cats}, volume={233}, ISSN={["0003-1488"]}, DOI={10.2460/javma.233.9.1441}, abstractNote={2 cats were examined because of congestive heart failure secondary to heartworm infection.One cat had severe abdominal distention and the other had dyspnea secondary to chylothorax. Both had loud right-sided heart murmurs, precordial thrills, and jugular distension. Thoracic radiography revealed cardiomegaly and enlarged caudal pulmonary arteries. Echocardiography revealed tricuspid regurgitation and multiple hyperechoic structures consistent with adult Dirofilaria immitis within the right atrium, right ventricle, and main pulmonary artery. Pulmonary hypertension was documented by means of Doppler echocardiography in 1 cat.Cats were anesthetized, and a nitinol gooseneck snare catheter was introduced into the right side of the heart via a jugular venotomy. In the first cat, the snare was used to retrieve 5 female and 2 male adult D immitis. The catheter was then passed into the main pulmonary artery in an unsuccessful attempt to retrieve remaining heartworms. In the second cat, 2 adult female D immitis were removed from the right atrium with the nitinol snare. In both cats, clinical signs resolved within 4 weeks after the procedure.Findings suggested that use of a nitinol gooseneck snare catheter may be a safe and effective technique for removing adult D immitis from the right atrium and ventricle in cats and that successful removal of adult heartworms in infected cats may resolve clinical signs of right-sided congestive heart failure and chylothorax. In addition, findings in 1 cat suggested that removal of all adult heartworms may not be necessary for clinical signs to resolve.}, number={9}, journal={JAVMA-JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION}, author={Small, Merrilee T. and Atkins, Clarke E. and Gordon, Sonya G. and Birkenheuer, Adam J. and Booth-Sayer, Margaret A. and Keene, Bruce W. and Fujii, Yoko and Miller, Matthew W.}, year={2008}, month={Nov}, pages={1441–1445} }
 @article{puskar_lemons_papich_vaden_birkenheuer_2007, title={Antibiotic-resistant Corynebacterium jeikeium urinary tract infection in a cat}, volume={43}, ISSN={["0587-2871"]}, DOI={10.5326/0430061}, abstractNote={A 10-year-old, castrated male, domestic longhaired cat with a history of urinary tract disease and perineal urethrostomy was presented for evaluation of persistent urinary tract inflammation. Prior to referral, diphtheroid organisms had been cultured from a urine sample obtained by cystocentesis, and they were interpreted as sample contamination. Subsequent urine culture and gene sequencing identified Corynebacterium jeikeium, which was resistant to antibiotics and appeared to be the cause of the urinary tract infection.}, number={1}, journal={JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION}, author={Puskar, Michelle and Lemons, Carol and Papich, Mark G. and Vaden, Shelley L. and Birkenheuer, Adam}, year={2007}, pages={61–64} }
 @article{valentine_harms_cadenas_birkenheuer_marr_braun-mcneill_maggi_breitschwerdt_2007, title={Bartonella DNA in Loggerhead Sea Turtles}, volume={13}, ISSN={1080-6040 1080-6059}, url={http://dx.doi.org/10.3201/eid1306.061551}, DOI={10.3201/eid1306.061551}, abstractNote={To the Editor: Bartonella are fastidious, aerobic, gram-negative, facultative, intracellular bacteria that infect erythrocytes, erythroblasts, endothelial cells, monocytes, and dendritic cells, and are transmitted by arthropod vectors or by animal scratches or bites (1–6). Currently, 20 species or subspecies of Bartonella have been characterized, of which 8 are known zoonotic pathogens (7). B. henselae has been recently identified from canine blood (8) and from harbor porpoises (9). Pathogenic bacteria are an important threat in terrestrial and marine environments, and in the case of B. henselae, reservoir hosts may be more diverse than currently recognized.}, number={6}, journal={Emerging Infectious Diseases}, publisher={Centers for Disease Control and Prevention (CDC)}, author={Valentine, K. Hope and Harms, Craig A. and Cadenas, Maria B. and Birkenheuer, Adam J. and Marr, Henry S. and Braun-McNeill, Joanne and Maggi, Ricardo G. and Breitschwerdt, Edward B.}, year={2007}, month={Jun}, pages={949–950} }
 @article{hawkins_birkenheuer_marr_rogala_large_adler_2007, title={Quantification of mucin gene expression in tracheobronchial epithelium of healthy dogs and dogs with chronic bronchitis}, volume={68}, ISSN={["1943-5681"]}, DOI={10.2460/ajvr.68.4.435}, abstractNote={To develop a real-time PCR assay for the quantification of mucin gene expression in tracheobronchial brushing specimens from dogs and compare mucin gene expression in specimens from dogs with naturally occurring chronic bronchitis with that in specimens from healthy dogs.7 healthy dogs and 5 dogs with chronic bronchitis.Primers that were designed to span the predicted intron-exon boundaries of a canine MUC5AC-like gene were used to develop a real-time PCR assay for quantification of expression of that gene. Total mRNA was isolated from tracheobronchial brushing specimens obtained from dogs with and without bronchitis during anesthesia; MUC5AC-like gene expression in those samples was quantified by use of the real-time PCR assay.The PCR assay was sensitive and specific for the target sequence, the predicted amino acid sequence of which had greatest homology with human, porcine, and rat MUC5AC. The assay was able to quantify the target over a wide dynamic range. Dogs with chronic bronchitis had a 3.0-fold increase in the quantity of MUC5AC-like mRNA, compared with healthy dogs.The ability to measure mucin gene expression from tracheobronchial brushing specimens collected from client-owned dogs during routine bronchoscopy should prove to be a useful tool for the study of bronchitis in dogs and expand the usefulness of airway inflammation in dogs as a model for bronchitis in humans.}, number={4}, journal={AMERICAN JOURNAL OF VETERINARY RESEARCH}, author={Hawkins, Eleanor C. and Birkenheuer, Adam J. and Marr, Henry S. and Rogala, Allison R. and Large, Edward E. and Adler, Kenneth B.}, year={2007}, month={Apr}, pages={435–440} }
 @article{haber_tucker_marr_levy_burgess_lappin_birkenheuer_2007, title={The detection of Cytauxzoon felis in apparently healthy free-roaming cats in the USA}, volume={146}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2007.02.029}, abstractNote={Cytauxzoon felis typically causes fatal disease in domestic cats. Survival after infection and persistent parasitemia without clinical illness has been documented in a few cases. To our knowledge there are no prevalence studies of C. felis in domestic cats. The purpose of this study was to estimate the prevalence of C. felis infected cats that were presented to trap-neuter-return programs in Florida, North Carolina and Tennessee. Cats that were presented to trap-neuter-return programs were tested using a C. felis-specific PCR assay. A total of 961 domestic cats were tested (494 from Florida; 392 from North Carolina; 75 from Tennessee). Prevalence of C. felis infection in this population was 0.3%. Two cats from Florida and one cat from Tennessee tested positive for the presence of C. felis DNA. These amplicons were sequenced and confirmed to be C. felis. The cat from Tennessee was alive without evidence of illness 2 months post-surgery. The other two cats were alive 24 h post-surgery, but were then lost to follow-up. This is the first report documenting C. felis infections in free-roaming cats. Despite the low prevalence rate, the presence of apparently healthy infected free-roaming cats suggests that they may have the capacity to serve as an additional reservoir host for C. felis. Further investigations should evaluate the potential vector competence of domestic cats as well as the role of chronically infected cats in areas in which cytauxzoonosis appears hyperendemic.}, number={3-4}, journal={VETERINARY PARASITOLOGY}, author={Haber, Marion D. and Tucker, Melissa D. and Marr, Henry S. and Levy, Julie K. and Burgess, Jill and Lappin, Michael R. and Birkenheuer, Adam J.}, year={2007}, month={May}, pages={316–320} }
 @article{birkenheuer_harms_neel_marr_tucker_acton_tuttle_stoskopf_2007, title={The identification of a genetically unique piroplasma in North American river otters (Lontra canadensis)}, volume={134}, ISSN={["1469-8161"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-34249725062&partnerID=MN8TOARS}, DOI={10.1017/S0031182006002095}, abstractNote={SUMMARY During a routine health check of a wild-caught North American river otter ( Lontra canadensis ) small piroplasms were noted within erythrocytes. Analyses of the 18S ribosomal ribonucleic acid (rRNA) gene sequences determined that this was a genetically unique organism most closely related to Babesia microti -like parasites found in other small carnivores. Subsequently 39 wild-trapped North American river otters from North Carolina were tested for the presence of piroplasma deoxyribonucleic acid (DNA) via polymerase chain reaction and piroplasma DNA was detected in 82% (32/39) of these samples. Sequencing of partial 18S rRNA genes from selected cases determined that they were identical to the sentinel case. This report documents the existence of a genetically unique piroplasma in North American river otters and indicates that the prevalence of piroplasma in North Carolina otters is quite high. The pathogenic potential of this organism for otters or other species remains unknown.}, number={5}, journal={PARASITOLOGY}, author={Birkenheuer, A. J. and Harms, C. A. and Neel, J. and Marr, H. S. and Tucker, M. D. and Acton, A. E. and Tuttle, A. D. and Stoskopf, M. K.}, year={2007}, month={May}, pages={631–635} }
 @article{lennon_boyle_hutchins_friedenthal_correa_bissett_moses_papich_birkenheuer_2007, title={Use of basal serum or plasma cortisol concentrations to rule out a diagnosis of hypoadrenocorticism in dogs: 123 cases (2000-2005)}, volume={231}, ISSN={["0003-1488"]}, DOI={10.2460/javma.231.3.413}, abstractNote={To determine whether basal serum or plasma cortisol concentration can be used as a screening test to rule out hypoadrenocorticism in dogs.Retrospective case-control study.110 dogs with nonadrenal gland illnesses and 13 dogs with hypoadrenocorticism.Sensitivity and specificity of basal serum or plasma cortisol concentrations of either <or= 1 microg/dL or <or= 2 microg/dL to detect dogs with hypoadrenocorticism were estimated by use of the ACTH stimulation test as the gold standard.Basal cortisol concentrations of <or= 1 microg/dL had excellent sensitivity (100%) and specificity (98.2%) for detecting dogs with hypoadrenocorticism. For basal cortisol concentrations of <or= 2 microg/dL, sensitivity was 100% but specificity was 78.2%.On the basis of sensitivity and specificity, basal serum or plasma cortisol concentrations had high negative predictive values over a wide range of prevalence rates and can be used to rule out a diagnosis of hypoadrenocorticism. Dogs with basal cortisol concentrations > 2 microg/dL that are not receiving corticosteroids, mitotane, or ketoconazole are highly unlikely to have hypoadrenocorticism. However, if the basal cortisol concentration is <or= 2 microg/dL, little to no information regarding adrenal gland function can be obtained and an ACTH stimulation test should be performed.}, number={3}, journal={JAVMA-JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION}, author={Lennon, Elizabeth M. and Boyle, Tonya E. and Hutchins, Rae Grace and Friedenthal, Arit and Correa, Maria T. and Bissett, Sally A. and Moses, Lorra S. and Papich, Mark G. and Birkenheuer, Adam J.}, year={2007}, month={Aug}, pages={413–416} }
 @article{birkenheuer_le_valenzisi_tucker_levy_breitschwerdt_2006, title={Cytauxzoon felisinfection in cats in the mid-Atlantic states: 34 cases (1998–2004)}, volume={228}, ISSN={0003-1488}, url={http://dx.doi.org/10.2460/javma.228.4.568}, DOI={10.2460/javma.228.4.568}, abstractNote={Abstract Objective —To describe the demographic and clinical characteristics of feline cytauxzoonosis in the midAtlantic states and compare the Cytauxzoon felis 18S rRNA gene sequences from affected cats with sequences reported from affected cats in other regions. Design —Retrospective case series. Animals —34 cats with C felis infection. Procedure —Medical records of cats in which C felis infection was diagnosed from May 1998 through June 2004 were reviewed; data collected included signalment, month of diagnosis, geographic location, clinicopathologic abnormalities, medical treatments, outcome, and necropsy findings when applicable. Cytauxzoon felis DNA was amplified, cloned, and sequenced from 4 of these cats and compared with previously reported C felis DNA sequences. Results —Of 34 C felis –infected cats, 28 resided in North Carolina, 3 resided in South Carolina, and 3 resided in Virginia; in 32 cats, a diagnosis of C felis infection was made in April through September. Pancytopenia and icterus were the most common clinicopathologic abnormalities. Thirty-two cats either died or were euthanatized, and 2 cats survived. At 5 veterinary hospitals, multiple cases were identified, and 4 multicat households had > 1 cat infected with C felis . The 18S rRNA gene sequences characterized in organisms obtained from 4 cats were nearly identical to C felis DNA sequences reported from other US regions. Conclusions and Clinical Relevance —Data indicate that veterinarians in the mid-Atlantic region of the United States should consider C felis infection in cats that become ill with fever, icterus, and pancytopenia or bicytopenia, especially in the spring and summer months.}, number={4}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Birkenheuer, Adam J. and Le, Jaime A. and Valenzisi, Amy M. and Tucker, Melissa D. and Levy, Michael G. and Breitschwerdt, Edward B.}, year={2006}, month={Feb}, pages={568–571} }
 @article{birkenheuer_marr_alleman_levy_breitschwerdt_2006, title={Development and evaluation of a PCR assay for the detection of Cytauxzoon felis DNA in feline blood samples}, volume={137}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2005.12.007}, abstractNote={Cytauxzoonosis is an emerging tick borne infectious disease of domestic cats in the United States, caused by the organism Cytauxzoon felis (C. felis). In naturally infected domestic cats the disease is almost always fatal. Currently there are no commercially available molecular or serologic tests to facilitate the antemortem diagnosis of C. felis infection. Clinical and pathological diagnosis of cytauxzoonosis is based on microscopic identification of parasites in tissues or on blood smears. We have developed and evaluated the sensitivity and specificity of a polymerase chain reaction (PCR) based assay for the diagnosis of C. felis infections in feline blood samples. The assay is sensitive enough to detect one copy of a cloned fragment of the C. felis 18S rRNA gene. This PCR assay can be used for the rapid clinical diagnosis of cytauxzoonosis and for epidemiological studies that will better define the geographic distribution of C. felis infection in cats.}, number={1-2}, journal={VETERINARY PARASITOLOGY}, author={Birkenheuer, AJ and Marr, H and Alleman, AR and Levy, MG and Breitschwerdt, EB}, year={2006}, month={Apr}, pages={144–149} }
 @article{gookin_copple_papich_poore_stauffer_birkenheuer_twedt_levy_2006, title={Efficacy of ronidazole for treatment of feline Tritrichomonas foetus infection}, volume={20}, ISSN={["1939-1676"]}, DOI={10.1892/0891-6640(2006)20[536:EORFTO]2.0.CO;2}, abstractNote={To determine the efficacy of ronidazole (RDZ), tinidazole (TDZ), and metronidazole (MDZ) against Tritrichomonas foetus in vitro and of RDZ for treatment of feline naturally occurring or experimentally induced T. foetus infection.A cat naturally infected with T. foetus infection and diarrhea. Ten specific-pathogen-free (SPF) kittens.RDZ, TDZ, and MDZ were tested for activity against 3 different feline isolates of T. foetus in vitro. RDZ then was administered to a naturally infected cat at 10 mg/kg PO q24h for 10 days. SPF kittens were infected orogastrically with feline T. foetus and treated with either placebo or RDZ (10 mg/kg PO q12h for 14 days). Cats with relapsing infection or those receiving placebo were treated subsequently with RDZ (either 30 or 50 mg/kg PO q12h for 14 days). Feces were examined for T. foetus by direct microscopy, culture, and polymerase chain reaction (PCR) testing weekly.Both RDZ and TDZ killed T. foetus at concentrations >0.1 microg/mL in vitro. In the naturally infected cat, RDZ abolished diarrhea and T. foetus infection for 85 days after treatment, at which time infection and diarrhea relapsed. Retreatment with RDZ eradicated diarrhea and T. foetus infection for over 407 days. In experimentally induced infection, RDZ at 10 mg/kg caused initial improvement, but infection relapsed in all 5 cats 2 to 20 weeks after treatment. At 30 or 50 mg/kg, 10/10 cats were negative for T. foetus infection for follow-up durations of 21 to 30 weeks after treatment.Oral administration of RDZ at 30 to 50 mg/kg q12h for 14 days resolved diarrhea and eradicated infection (on the basis of polymerase chain reaction [PCR] testing) in 1 naturally infected cat and 10 experimentally inoculated cats receiving a different isolate of T. foetus.}, number={3}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Gookin, Jody L. and Copple, Christina N. and Papich, Mark G. and Poore, Matthew F. and Stauffer, Stephen H. and Birkenheuer, Adam J. and Twedt, David C. and Levy, Michael G.}, year={2006}, pages={536–543} }
 @article{birkenheuer_whittington_neel_large_barger_levy_breitschwerdt_2006, title={Molecular characterization of a Babesia species identified in a North American raccoon}, volume={42}, ISSN={["1943-3700"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33748280658&partnerID=MN8TOARS}, DOI={10.7589/0090-3558-42.2.375}, abstractNote={Piroplasmosis was first described in raccoons (Procyon lotor) in 1926, and the official description of a small piroplasm as Babesia lotori was done in 1981. Babesia microti-like gene sequences have been characterized in raccoons in both North American and Japan. It is well documented that the microscopic appearance of piroplasms does not always accurately predict the genotype and phylogenetic classification. Discrepancies using phenotype to predict genotype have been reported most frequently when evaluating small piroplasms. We amplified and sequenced the full-length 18S rRNA gene from a small piroplasm identified in a raccoon and used this sequence for phylogenetic analyses. Based on these analyses, the organism was placed in the Babesia sensu stricto clade, confirming that it is a true Babesia sp. This documents that at least two Babesia spp. can infect raccoons. The data generated in this study can be used to design molecular diagnostic tests for detection of this Babesia sp., which will be useful for epidemiologic and comparative phylogenetic studies. As piroplasmosis has been documented with increased frequency in humans in recent years, the results of this study will aid in the recognition of zoonotic babesiosis.}, number={2}, journal={JOURNAL OF WILDLIFE DISEASES}, author={Birkenheuer, Adam J. and Whittington, Julia and Neel, Jennifer and Large, Edward and Barger, Anne and Levy, Michael G. and Breitschwerdt, Edward B.}, year={2006}, month={Apr}, pages={375–380} }
 @article{wardrop_reine_birkenheuer_hale_hohenhaus_crawford_lappin_2005, title={Canine and feline blood donor screening for infectious disease}, volume={19}, ISSN={["1939-1676"]}, DOI={10.1892/0891-6640(2005)19<135:CAFBDS>2.0.CO;2}, number={1}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Wardrop, KJ and Reine, N and Birkenheuer, A and Hale, A and Hohenhaus, A and Crawford, C and Lappin, MR}, year={2005}, pages={135–142} }
 @article{birkenheuer_correa_levy_breitschwerdt_2005, title={Geographic distribution of babesiosis among dogs in the United States and association with dog bites: 150 cases (2000-2003)}, volume={227}, ISSN={0003-1488}, url={http://dx.doi.org/10.2460/javma.2005.227.942}, DOI={10.2460/javma.2005.227.942}, abstractNote={Abstract Objective —To identify the geographic distribution of babesiosis among dogs in the United States and determine, for dogs other than American Pit Bull Terriers (APBTs), whether infection was associated with a recent dog bite. Design —Retrospective study. Animals —150 dogs. Procedure —Canine blood samples submitted to the North Carolina State University Vector-Borne Disease Diagnostic Laboratory between May 2000 and October 2003 for which results of a Babesia -specific polymerase chain reaction assay were positive were identified, and breed and geographic origin of dogs from which samples were obtained were recorded. History and hematologic abnormalities for dogs that were not APBTs were recorded, and possible associations with a recent dog bite were examined. Results —Dogs positive for Babesia DNA were located in 29 states and 1 Canadian province (Ontario). Babesia gibsoni was the most commonly detected species, with B gibsoni DNA detected in blood samples from 131 of 144 (91%) dogs. Of the 131 dogs positive for B gibsoni DNA, 122 (93%) were APBTs. Of the 10 dogs positive for Babesia canis vogeli DNA, 6 were Greyhounds. In dogs other than APBTs, there was an association between having recently been bitten by another dog, particularly an APBT, and infection with B gibsoni . Conclusions and Clinical Relevance —Results document an expansion of the known geographic range for babesiosis among dogs in the United States. Testing for babesiosis should be pursued in dogs with clinicopathologic abnormalities consistent with immunemediated hemolytic anemia or thrombocytopenia, particularly if there is a history of a recent dog bite. ( J Am Vet Med Assoc 2005;227:942–947)}, number={6}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Birkenheuer, Adam J. and Correa, Maria T. and Levy, Michael G. and Breitschwerdt, Edward B.}, year={2005}, month={Sep}, pages={942–947} }
 @article{haber_birkenheuer_2005, title={Icterus and pancytopenia in a cat}, volume={3}, ISBN={1542-4014}, number={7}, journal={NAVC Clinician's Brief}, author={Haber, M. and Birkenheuer, A.}, year={2005}, pages={21} }
 @article{gookin_birkenheuer_st john_spector_levy_2005, title={Molecular characterization of trichomonads from feces of dogs with diarrhea}, volume={91}, ISSN={["1937-2345"]}, DOI={10.1645/ge-474r.1}, abstractNote={Trichomonads are occasionally observed in the feces of dogs with diarrhea. On the basis of superficial morphological appearance, these infections have been attributed to opportunistic overgrowth of the commensal, Pentatrichomonas hominis. However, molecular characterization of canine trichomonads has never been reported. This study was performed to determine, by means of rRNA gene sequence analysis, the identity of trichomonads observed in feces from dogs with diarrhea. Total DNA was isolated from fecal samples obtained from a 3-mo-old mixed breed dog and litter of German Shepherd puppies having profuse liquid diarrhea containing numerous trichomonads. Total DNA was subject to PCR amplification of partial 18S rRNA gene or 5.8S, ITS1, ITS2, and partial 18S and 28S rRNA genes using species-specific and universal primers, respectively. Products of 642 and 1864 base-pair length were amplified and cloned. On the basis of rRNA gene sequence, the trichomonads observed in the single dog and the litter of puppies shared 100% identity with Tritrichomonas foetus and P. hominis, respectively. The present study is the first to establish the molecular identity of trichomonads infecting dogs with diarrhea. These studies validate the longstanding assumption that canine trichomoniasis may be attributed to P. hominis. Importantly, these studies additionally recognize that canine trichomoniasis may also be caused by infection with T. foetus.}, number={4}, journal={JOURNAL OF PARASITOLOGY}, author={Gookin, JL and Birkenheuer, AJ and St John, V and Spector, M and Levy, MG}, year={2005}, month={Aug}, pages={939–943} }
 @article{birkenheuer_neel_ruslander_levy_breitschwerdt_2004, title={Detection and molecular characterization of a novel large Babesia species in a dog}, volume={124}, ISSN={0304-4017}, url={http://dx.doi.org/10.1016/j.vetpar.2004.07.008}, DOI={10.1016/j.vetpar.2004.07.008}, abstractNote={Babesia canis has generally been considered the only large Babesia to infect dogs. Here we describe the molecular characterization of a large Babesia species that was detected in the blood and bone marrow of a dog with clinical and hematological abnormalities consistent with babesiosis. Analysis of the 18S rRNA genes revealed a unique sequence that shared 93.9% sequence identity with B. bigemina and 93.5% sequence identity with B. caballi, compared to 91.2-91.6% identity with B. canis canis, B. c. vogeli, and B. c. rossi. Cross-reactive antibodies against B. canis, B. gibsoni (Asian genotype), or B. gibsoni (California genotype) antigens were not detected in acute or convalescent serum samples. The dog was treated with imidocarb diproprionate, which resulted in the resolution of clinical signs, and subsequently Babesia DNA was not detectable by PCR in post-treatment samples. The organism described in this report represents a genetically unique large Babesia sp. and is the eighth genetically distinct piroplasm capable of infecting the domestic dog.}, number={3-4}, journal={Veterinary Parasitology}, publisher={Elsevier BV}, author={Birkenheuer, A.J. and Neel, J. and Ruslander, D. and Levy, M.G. and Breitschwerdt, E.B.}, year={2004}, month={Oct}, pages={151–160} }
 @article{birkenheuer_levy_breitschwerdt_2004, title={Efficacy of Combined Atovaquone and Azithromycin for Therapy of Chronic Babesia gibsoni (Asian Genotype) Infections in Dogs}, DOI={10.1111/j.1939-1676.2004.tb02573.x}, abstractNote={Babesiosis caused by Babesia gibsoni (Asian genotype) is an emerging disease in dogs in the United States. To date, no drugs have been shown to eliminate B gibsoni (Asian genotype) infections from dogs. Twenty-two dogs that remained persistently infected with B gibsoni (Asian genotype) after either imidocarb diproprionate and or diminazine aceturate therapy were identified and randomly and evenly distributed into 2 groups. One group was treated with atovaquone and azithromycin combination therapy, and the other group received a placebo. Eight of 10 dogs in the treatment group had no detectable B gibsoni (Asian genotype) DNA, as determined by a sensitive and specific polymerase chain reaction (PCR) assay, in any of their posttreatment samples. In contrast, B gibsoni (Asian genotype) DNA was detectable by PCR in the posttreatment samples from 11 of 11 of the placebo-treated dogs. One dog in the treatment group was excluded from the treatment outcome analysis. This dog had 2 consecutive negative PCR assay results and was euthanized because of ongoing degenerative joint disease prior to completion of the study. No adverse effects of treatment were reported in any dog during the study period. A combination of atovaquone and azithromycin is the 1st described treatment that will either eliminate B gibsoni (Asian genotype) infections or suppress the parasitemia below the limit of detection in the majority of treated dogs.}, journal={Journal of Veterinary Internal Medicine}, author={Birkenheuer, Adam J. and Levy, Michael G. and Breitschwerdt, Edward B.}, year={2004} }
 @article{birkenheuer_levy_breitschwerdt_2004, title={Efficacy of combined atovaquone and azithromycin for therapy of chronic Babesia gibsoni (Asian genotype) infections in dogs}, volume={18}, ISSN={["1939-1676"]}, DOI={10.1892/0891-6640(2004)18<494:EOCAAA>2.0.CO;2}, abstractNote={Babesiosis caused by Babesia gibsoni (Asian genotype) is an emerging disease in dogs in the United States. To date, no drugs have been shown to eliminate B. gibsoni (Asian genotype) infections from dogs. Twenty-two dogs that remained persistently infected with B. gibsoni (Asian genotype) after either imidocarb diproprionate and or diminazine aceturate therapy were identified and randomly and evenly distributed into 2 groups. One group was treated with atovaquone and azithromycin combination therapy, and the other group received a placebo. Eight of 10 dogs in the treatment group had no detectable B. gibsoni (Asian genotype) DNA, as determined by a sensitive and specific polymerase chain reaction (PCR) assay, in any of their posttreatment samples. In contrast, B. gibsoni (Asian genotype) DNA was detectable by PCR in the posttreatment samples from 11 of 11 of the placebo-treated dogs. One dog in the treatment group was excluded from the treatment outcome analysis. This dog had 2 consecutive negative PCR assay results and was euthanized because of ongoing degenerative joint disease prior to completion of the study. No adverse effects of treatment were reported in any dog during the study period. A combination of atovaquone and azithromycin is the 1st described treatment that will either eliminate B. gibsoni (Asian genotype) infections or suppress the parasitemia below the limit of detection in the majority of treated dogs.}, number={4}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Birkenheuer, AJ and Levy, MG and Breitschwerdt, EB}, year={2004}, pages={494–498} }
 @article{tuttle_birkenheuer_juopperi_levy_breitschwerdt_2003, title={Concurrent bartonellosis and babesiosis in a dog with persistent thrombocytopenia}, volume={223}, ISSN={0003-1488}, url={http://dx.doi.org/10.2460/javma.2003.223.1306}, DOI={10.2460/javma.2003.223.1306}, abstractNote={A 12-year-old castrated male West Highland White Terrier was referred because of recurrent episodes of collapsing. The dog was mildly anemic and severely thrombocytopenic and had high serum alanine aminotransferase activity. Infection with Bartonella vinsonii (berkhoffii) was initially diagnosed on the basis of serologic testing. Despite treatment with a series of antimicrobials and prolonged use of immunosuppressive drugs, thrombocytopenia persisted. After 5 months of treatment, Babesia canis organisms were seen during examination of a direct blood smear. The dog was treated with imidocarb dipropionate for babesiosis, after which thrombocytopenia resolved, and administration of immunosuppressive drugs was discontinued. Retrospective review of blood smears failed to identify organisms; however, polymerase chain reaction (PCR) analysis of multiple stored blood samples obtained during the 5-month period of persistent thrombocytopenia identified DNA of B. canis vogeli. Babesiosis may cause persistent, unexplained thrombocytopenia in dogs that are not anemic. A PCR assay can facilitate a diagnosis of babesiosis when organisms are not evident or when serologic testing fails to detect Babesia-specific antibodies.}, number={9}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Tuttle, Allison D. and Birkenheuer, Adam J. and Juopperi, Tarja and Levy, Michael G. and Breitschwerdt, Edward B.}, year={2003}, month={Nov}, pages={1306–1310} }
 @article{birkenheuer_levy_breitschwerdt_2003, title={Development and Evaluation of a Seminested PCR for Detection and Differentiation of Babesia gibsoni (Asian Genotype) and B. canis DNA in Canine Blood Samples}, volume={41}, ISSN={0095-1137}, url={http://dx.doi.org/10.1128/jcm.41.9.4172-4177.2003}, DOI={10.1128/JCM.41.9.4172-4177.2003}, abstractNote={ABSTRACT Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asian genotype), B. canis subsp. vogeli , B. canis subsp. canis , and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify an ∼340-bp fragment of the 18S rRNA genes from B. gibsoni (Asian genotype), B. canis subsp. vogeli , B. canis subsp. rossi , and B. canis subsp. canis but not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an ∼370-bp fragment of the B. gibsoni (USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected with Theileria annae at a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis of B. gibsoni (USA/California genotype) or T. annae . The PCR test did not amplify Toxoplasma gondii , Neospora caninum , Leishmania infantum , Cryptosporidium parvum , or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminate B. gibsoni (Asian genotype), B. canis subsp. vogeli , B. canis subsp. canis , and B. canis subsp. rossi DNA in blood samples from infected dogs.}, number={9}, journal={Journal of Clinical Microbiology}, publisher={American Society for Microbiology}, author={Birkenheuer, A. J. and Levy, M. G. and Breitschwerdt, E. B.}, year={2003}, month={Sep}, pages={4172–4177} }
 @article{birkenheuer_levy_stebbins_poore_breitschwerdt_2003, title={Serosurvey of antiBabesia antibodies in stray dogs and American pit bull terriers and American Staffordshire terriers from North Carolina}, volume={39}, ISSN={["0587-2871"]}, DOI={10.5326/0390551}, abstractNote={Stray dogs (n=359) and kennel dogs (n=149) from North Carolina were tested for evidence of antiBabesia antibodies. AntiBabesia antibodies were detected in 21/359 and 22/149 of the stray and kennel dogs, respectively. A total of 57 dogs from both groups were tested for babesiasis by light microscopy and polymerase chain reaction (PCR). Babesia deoxyribonucleic acid (DNA) was detected in 3/28 of the stray dogs and 14/29 of the kennel dogs. When Babesia DNA was detected by PCR, the species-specific PCR results differed from the Babesia species antibody titer results in 6/17 of the PCR-positive dogs. There was no association between antiBabesia antibodies and the presence of ticks. There are currently Babesia gibsoni epizootics affecting American pit bull terrier kennels.}, number={6}, journal={JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION}, author={Birkenheuer, AJ and Levy, MG and Stebbins, M and Poore, M and Breitschwerdt, E}, year={2003}, pages={551–557} }
 @article{stegeman_birkenheuer_kruger_breitschwerdt_2003, title={Transfusion-associated Babesia gibsoni infection in a dog}, volume={222}, ISSN={0003-1488}, url={http://dx.doi.org/10.2460/javma.2003.222.959}, DOI={10.2460/javma.2003.222.959}, abstractNote={A 2.5-year-old spayed female German Shepherd Dog was referred for evaluation of progressive anemia, lethargy, and weight loss. Seventeen days earlier, the dog had received a whole blood transfusion to manage hemorrhage after ovariohysterectomy. Mild fever, splenomegaly, and thrombocytopenia were also identified. Von Willebrand disease and Babesia gibsoni infection were diagnosed. Because of the serologic cross-reactivity of B gibsoni and B canis in the immunofluorescent antibody assay for IgG antibodies against these organisms, polymerase chain reaction amplification of parasite DNA was required to identify the infecting Babesia sp. The source of the B gibsoni infection was traced to an apparently healthy American Pit Bull Terrier blood donor. Despite resolution of clinical signs in the dog of this report, a series of antiparasitic treatments failed to eliminate the B gibsoni infection. Screening of potential blood donor dogs for Babesia spp is becoming increasingly important in the United States.}, number={7}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Stegeman, Julie R. and Birkenheuer, Adam J. and Kruger, John M. and Breitschwerdt, Edward B.}, year={2003}, month={Apr}, pages={959–963} }
 @article{levy_gookin_poore_birkenheuer_dykstra_litaker_2003, title={Tritrichomonas foetus and not Pentatrichomonas hominis is the etiologic agent of feline trichomonal diarrhea}, volume={89}, ISSN={["1937-2345"]}, DOI={10.1645/0022-3395(2003)089[0099:TFANPH]2.0.CO;2}, abstractNote={Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, a n organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).}, number={1}, journal={JOURNAL OF PARASITOLOGY}, author={Levy, MG and Gookin, JL and Poore, M and Birkenheuer, AJ and Dykstra, MJ and Litaker, RW}, year={2003}, month={Feb}, pages={99–104} }
 @article{birkenheuer_breitschwerdt_alleman_pitulle_2002, title={Differentiation of Haemobartonella canis and Mycoplasma haemofelis on the basis of comparative analysis of gene sequences}, volume={63}, ISSN={0002-9645}, url={http://dx.doi.org/10.2460/ajvr.2002.63.1385}, DOI={10.2460/ajvr.2002.63.1385}, abstractNote={To determine whether Haemobartonella canis and Mycoplasma haemofelis (formerly known as H felis [large form]) can be differentiated by use of comparative analysis of gene sequences.Blood samples obtained from 3 dogs infected with H canis and 2 cats infected with M haemofelis.The partial 16S rDNA and ribonuclease P RNA (RNase P) genes were amplified, cloned, and sequenced in blood samples obtained from H canis-infected dogs and M haemofelis-infected cats. The DNA sequences were subjected to comparative analysis.The 16S rDNA sequences of H canis and M haemofelis were nearly identical (homology of 99.3 to 99.7%). In contrast, RNase P gene sequences had a lower degree of sequence homology between the 2 organisms (94.3 to 95.5%).Haemobartonella canis and M haemofelis are not identical organisms. Molecular differentiation of H canis and M haemofelis is more clearly evident by use of comparative analysis of RNase P gene sequences than by comparative analysis of 16S rDNA gene sequences.}, number={10}, journal={American Journal of Veterinary Research}, publisher={American Veterinary Medical Association (AVMA)}, author={Birkenheuer, Adam J. and Breitschwerdt, Edward B. and Alleman, A. Rick and Pitulle, Christian}, year={2002}, month={Oct}, pages={1385–1388} }
 @article{rotstein_taylor_birkenhauer_roelke-parker_homer_2002, title={Retrospective study of proliferative papillary vulvitis in Florida panthers}, volume={38}, ISSN={["0090-3558"]}, DOI={10.7589/0090-3558-38.1.115}, abstractNote={Proliferative, papillary vulvitis was identified in 16 of 34 (47%) free-ranging and captive female Florida panthers (Puma concolor coryi) monitored over a period from 1983-98. Gross lesions were characterized by extensive papilliferous proliferation in the mucosa of the vestibulum vaginae. Within lesions, the mean length and width of vestibular papillae were 1.07 +/- 0.39 mm (CV = 36%) and 0.55 +/- 0.11 mm (CV = 20%) respectively. Histologically, three to 12 layers of non-cornified stratified squamous epithelium with various degrees of basal cell spongiosis and rete ridge formation covered fibrous papillae. Mixed leukocytic mucosal inflammation also was observed. Infectious organisms were not observed, and immunohistochemical testing for the presence of papillomavirus antigens in specimens from seven panthers was negative. Lesions in nearly all of the panthers were first observed during a six-year period (1986-92), with one each in 1983, 1996 and 1998. There were no significant differences between the number of females having litters, the number of litters between age-matched and interval-matched females, and the interval between litters among lesions positive and lesion negative females over the 15 yr period. The severity of lesions did not appear to differ between parous and nulliparous free-ranging lesion-positive females. The cause of proliferative vulvitis remains unknown. However, the lesion did not appear to have a significant effect on reproduction.}, number={1}, journal={JOURNAL OF WILDLIFE DISEASES}, author={Rotstein, DS and Taylor, SK and Birkenhauer, A and Roelke-Parker, M and Homer, BL}, year={2002}, month={Jan}, pages={115–123} }
 @article{gookin_birkenheuer_breitschwerdt_levy_2002, title={Single-Tube Nested PCR for Detection of Tritrichomonasfoetus in Feline Feces}, volume={40}, ISSN={0095-1137}, url={http://dx.doi.org/10.1128/jcm.40.11.4126-4130.2002}, DOI={10.1128/JCM.40.11.4126-4130.2002}, abstractNote={ABSTRACT Tritrichomonas foetus , a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea. Recognition of the infection in cats has been mired by unfamiliarity with T . foetus in cats as well as misdiagnosis of the organisms as Pentatrichomonas hominis or Giardia sp. when visualized by light microscopy. The diagnosis of T . foetus presently depends on the demonstration of live organisms by direct microscopic examination of fresh feces or by fecal culturing. As T . foetus organisms are fastidious and fragile, routine flotation techniques and delayed examination and refrigeration of feces are anticipated to preclude the diagnosis in numerous cases. The objective of this study was to develop a sensitive and specific PCR test for the diagnosis of feline T . foetus infection. A single-tube nested PCR was designed and optimized for the detection of T . foetus in feline feces by using a combination of novel (TFITS-F and TFITS-R) and previously described (TFR3 and TFR4) primers. The PCR is based on the amplification of a conserved portion of the T . foetus internal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene. The absolute detection limit of the single-tube nested PCR was 1 organism, while the practical detection limit was 10 organisms per 200 mg of feces. Specificity was examined by using P . hominis , Giardia lamblia, and feline genomic DNA. Our results demonstrate that the single-tube nested PCR is ideally suited for (i) diagnostic testing of feline fecal samples that are found negative by direct microscopy and culturing and (ii) definitive identification of microscopically observable or cultivated organisms.}, number={11}, journal={Journal of Clinical Microbiology}, publisher={American Society for Microbiology}, author={Gookin, J. L. and Birkenheuer, A. J. and Breitschwerdt, E. B. and Levy, M. G.}, year={2002}, month={Nov}, pages={4126–4130} }
 @article{gaskin_schantz_jackson_birkenheuer_tomlinson_gramiccia_levy_steurer_kollmar_hegarty_et al._2002, title={Visceral leishmaniasis in a New York foxhound kennel}, volume={16}, ISSN={["0891-6640"]}, DOI={10.1892/0891-6640(2002)016<0034:VLIANY>2.3.CO;2}, abstractNote={Although endemic throughout much of the world, autochthonous visceral leishmaniasis has been reported on only 3 previous occasions in North America. After diagnosis of visceral leishmaniasis in 4 foxhounds from a kennel in Dutchess County, New York (index kennel), serum and ethylenediamine-tetraacetic acid (EDTA)-anticoagulated blood were collected from the remaining 108 American or cross-bred foxhounds in the index kennel and from 30 Beagles and Basset Hounds that were periodically housed in the index kennel. Samples were analyzed for antibodies to or DNA of tickborne disease pathogens and Leishmania spp. Most dogs had antibodies to Rickettsia spp., Ehrlichia spp., Babesia spp., or some combination of these pathogens but not to Bartonella vinsonii (berkhoffi). However, DNA of rickettsial, ehrlichial, or babesial agents was detected in only 9 dogs. Visceral leishmaniasis was diagnosed in 46 of 112 (41%) foxhounds from the index kennel but was not diagnosed in any of the Beagles and Basset Hounds. A positive Leishmania status was defined by 1 or more of the following criteria: a Leishmania antibody titer > or = 1:64, positive Leishmania polymerase chain reaction (PCR), positive Leishmania culture, or identification of Leishmania amastigotes by cytology or histopathology. The species and zymodeme of Leishmania that infected the foxhounds was determined to be Leishmania infantum MON-1 by isoenzyme electrophoresis. Foxhounds that were > 18 months of age or that had traveled to the southeastern United States were more likely to be diagnosed with visceral leishmaniasis. Transmission of Leishmania spp. in kennel outbreaks may involve exposure to an insect vector, direct transmission, or vertical transmission.}, number={1}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Gaskin, AA and Schantz, P and Jackson, J and Birkenheuer, A and Tomlinson, L and Gramiccia, M and Levy, M and Steurer, F and Kollmar, E and Hegarty, BC and et al.}, year={2002}, pages={34–44} }
 @article{gaskin_schantz_jackson_birkenheuer_tomlinson_gramiccia_levy_steurer_kollmar_hegarty_et al._2002, title={Visceral leishmaniasis in a New York foxhound kennel}, DOI={10.1111/j.1939-1676.2002.tb01604.x}, journal={Journal of Veterinary Internal Medicine}, author={Gaskin, Amanda A. and Schantz, Peter and Jackson, Joan and Birkenheuer, Adam and Tomlinson, Lindsay and Gramiccia, Marina and Levy, Michael and Steurer, Frank and Kollmar, Eleanor and Hegarty, Barbara C. and et al.}, year={2002} }
 @article{gasser_birkenheuer_breitschwerdt_2001, title={Canine Rocky Mountain spotted fever: A retrospective study of 30 cases}, volume={37}, ISSN={["0587-2871"]}, DOI={10.5326/15473317-37-1-41}, abstractNote={Rocky Mountain spotted fever (RMSF) was diagnosed in 30 dogs examined at North Carolina State University, Veterinary Teaching Hospital between 1984 and 1997. Historical, physical examination, and laboratory abnormalities were reviewed. Diagnostic criteria included a four-fold rise in antibody titer to Rickettsia rickettsii (R. rickettsii) (n=15) or a single R. rickettsii antibody titer of 1:1,024 or greater (n=15; when this initial titer was determined one week or more after the onset of clinical signs). Fifteen (50%) dogs were greater than seven years of age, and 13 (43%) dogs were between two and seven years of age. There was no sex predilection. Only five (17%) dogs had a history of known tick exposure. Presumably due to delayed diagnosis, dogs with antibody titers of 1:1,024 or greater at the time of presentation had a higher incidence of more severe neurological dysfunction (e.g., ataxia, hyperesthesia, vestibular disease, and seizures) and cutaneous lesions (e.g., hyperemia, edema, petechiae, ecchymoses, and necrosis). Laboratory findings included anemia, leukocytosis accompanied by toxic granulation of neutrophils, hypoalbuminemia, and coagulation abnormalities; signs were generally more severe in the 15 dogs with R. rickettsii antibody titers of 1:1,024 or greater at the time of presentation. Twelve (40%) dogs in this study were severely thrombocytopenic (less than 75 x10(3) platelets/microl; reference range, 200 to 450 x 10(3)/microl), without clinical evidence of fulminant disseminated intravascular coagulation. In this study, the survival rate following R. rickettsii infection was 100%.}, number={1}, journal={JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION}, author={Gasser, AM and Birkenheuer, AJ and Breitschwerdt, EB}, year={2001}, pages={41–48} }
 @article{kjemtrup_kocan_whitworth_meinkoth_birkenheuer_cummings_boudreaux_stockham_irizarry-rovira_conrad_2000, title={There are at least three genetically distinct small piroplasms from dogs}, volume={30}, ISSN={["0020-7519"]}, DOI={10.1016/S0020-7519(00)00120-X}, abstractNote={The 18S nuclear subunit ribosomal RNA (18S rRNA) gene of small piroplasms isolated from dogs from Okinawa (Japan), Oklahoma, North Carolina, Indiana, Missouri, and Alabama, was isolated and sequenced. Phylogenetic analysis of these sequences and comparisons with sequences from other Babesia, Cytauxzoon, and Theileria species revealed that all canine small babesial isolates, with the exception of isolates from California and Spain, were placed in a group containing the Babesia spp. sensu stricto. Within the Babesia spp. sensu stricto, there was support for separating the small canine piroplasms from the large canine piroplasm, Babesia canis. The isolate from California was in a distinct phylogenetic clade, closely related to babesial isolates from wildlife and humans from the Western US. The canine isolate from Spain was closely related to Babesia microti. These results suggest that there are multiple small piroplasm species in dogs. The isolates from the Midwestern and Eastern US and the one from Japan probably represent a single species with wide geographic distribution.}, number={14}, journal={INTERNATIONAL JOURNAL FOR PARASITOLOGY}, author={Kjemtrup, AM and Kocan, AA and Whitworth, L and Meinkoth, J and Birkenheuer, AJ and Cummings, J and Boudreaux, MK and Stockham, SL and Irizarry-Rovira, A and Conrad, PA}, year={2000}, month={Dec}, pages={1501–1505} }
 @article{birkenheuer_levy_savary_gager_breitschwerdt_1999, title={Babesia gibsoni infections in dogs from North Carolina}, volume={35}, ISSN={["0587-2871"]}, DOI={10.5326/15473317-35-2-125}, abstractNote={The recognition of canine babesiosis in North Carolina caused by Babesia gibsoni documents the expansion of the previously reported endemic area of this disease. Clinical signs ranged from severe hemolytic anemia and thrombocytopenia to subclinical infections. No infected dogs had traveled to endemic areas. Antibabesial treatment failed to eradicate the organism from infected dogs.}, number={2}, journal={JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION}, author={Birkenheuer, AJ and Levy, MG and Savary, KCM and Gager, RB and Breitschwerdt, EB}, year={1999}, pages={125–128} }