ACVIM consensus statement on the diagnosis of immune thrombocytopenia in dogs and cats.

Immune thrombocytopenia (ITP) is the most common acquired primary hemostatic disorder in dogs. Immune thrombocytopenia less commonly affects cats but is an important cause of mortality and treatment-associated morbidity in both species. Immune thrombocytopenia remains a diagnosis of exclusion for which diagnostic guidelines are lacking. Primary, or non-associative, ITP refers to autoimmune platelet destruction. Secondary, or associative, ITP arises in response to an underlying disease trigger. However, evidence for which comorbidities serve as ITP triggers has not been systematically evaluated. To identify key diagnostic steps for ITP and important comorbidities associated with secondary ITP, we developed 12 Population Evaluation/Exposure Comparison Outcome (PECO) format questions. These questions were addressed by evidence evaluators utilizing a literature pool of 287 articles identified by the panelists using a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations that then were integrated by diagnosis and comorbidity domain chairs. The revised PECO responses underwent a Delphi survey process to reach consensus on final guidelines. A combination of panel expertise and PECO responses were employed to develop algorithms for diagnosis of ITP in dogs and cats, which also underwent 4 iterations of Delphi review. Comorbidity evidence evaluators employed an integrated measure of evidence (IME) tool to determine evidence quality for each comorbidity; IME values combined with evidence summaries for each comorbidity were integrated to develop ITP screening recommendations, which also were subjected to Delphi review. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The final consensus statement provides clinical guidelines for the diagnosis of, and underlying disease screening for, ITP in dogs and cats. The systematic consensus process identified numerous knowledge gaps that should guide future studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Treatment of Immune Thrombocytopenia.

Transmission of Cytauxzoon felis by injection of Amblyomma americanum salivary glands.

BACKGROUND: Cytauxzoonosis is a life-threatening disease of cats, caused by the tick-borne piroplasmid hemoparasite, Cytauxzoon felis. Current experimental models for cytauxzoonosis rely on either tick transmission or direct injection of infected cat tissues. These models require researchers to directly work with infected ticks or use cats with acute cytauxzoonosis. To improve the feasibility and accessibility, there is a need to establish sharable resources among researchers. In related piroplasmid parasites, sporozoite-based inoculums are routinely produced from tick salivary glands, cryopreserved and distributed to other investigators and facilities. For these parasites, sporozoites have been the basis for vaccine development and in vitro cultivation, both of which remain lacking for C. felis research. If infectious sporozoites can be similarly isolated for C. felis, it would significantly broaden our capabilities to study this parasite. Aims of this study was to determine if C. felis sporozoites inoculums collected from the salivary glands of Amblyomma americanum ticks were capable of inducing cytauxzoonosis in naïve cats. MATERIALS AND METHODS: A. americanum nymphs were acquisition-fed on a donor cat chronically infected with C. felis and allowed to molt to adults. Four groups of adult ticks (n = 50/group) were either stimulation-fed for 4 days on naïve cats or were heated at 37 °C for 4 days. After these treatments, salivary glands (SG) of each group of ticks were collected to create inoculums. Infectivity of these inoculums was then tested by subcutaneous injection into naïve cats. RESULTS: The two naïve cats used for stimulation feeding and as controls both developed cytauxzoonosis, indicating these groups of ticks were capable of producing infectious sporozoites. Of the 2 cats that were injected with SGs from the stimulation-fed ticks, one cat developed cytauxzoonosis and C. felis infection was confirmed by both light microscopy and PCR. The other cat did not develop cytauxzoonosis and only had equivocal evidence of infection. Neither cat injected with SGs from the heated ticks developed cytauxzoonosis. One of these cats had equivocal evidence of infection and one had no evidence of infection. CONCLUSION: This study validates the feasibility of collecting infectious sporozoites from C. felis-infected ticks that can be used to infect naïve cats. While this model requires further optimization, it has the potential to expand resources to study C. felis and further advance research in this field.

A practical protocol to prepare paraffin-embedded whole tick histology sections.

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.

Cytauxzoon felis in salivary glands of Amblyomma americanum.

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.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Prevalence and genetic characterization of a Babesia microti-like species in the North American river otter (Lontra canadensis).

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.

DETECTION OF VECTOR-BORNE INFECTIONS IN LIONS AND TIGERS AT TWO ZOOS IN TENNESSEE AND OKLAHOMA, USA.

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.

Direct injection of Amblyomma americanum ticks with Cytauxzoon felis.

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.

A novel missense mutation of the NAT10 gene in a juvenile Schnauzer dog with chronic respiratory tract infections.

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.

Pathognomonic thoracic radiographic findings are lacking in cats with acute Cytauxzoonosis.

Cytauxzoon felis is a tick-borne haemoprotozoan parasite that often causes fatal disease in domestic cats. Histological studies have described substantial pulmonary pathology due to cytauxzoonosis. Published reports were not found describing the thoracic radiographic signs associated with acute cytauxzoonosis in cats. The purpose of this retrospective descriptive study was to describe thoracic radiographic findings in a group of felines with confirmed acute cytauxzoonosis. A total of 37 cats with confirmed cytauxzoonosis and with available thoracic radiographs were included. A subset of 7 cats in this sample also had histopathologic evaluation of their lung parenchyma. Thoracic radiographs were retrieved and reviewed. A bronchial pulmonary pattern was identified as the most common finding (n = 27/37; 73%). Other radiographic findings included cardiomegaly (n = 19/37; 51%), interstitial pattern (n = 17/37; 46%), pleural effusion (n = 12/37; 32%), arterial vascular distension (n = 10/37; 27%), arterial and venous distension (n = 10/37; 27%), and venous distension (n = 1/37; 3%). The primary histological features present in 7 cats with additional histopathologic evaluation, similar to previously published studies, were vascular occlusion. Our study suggests that, despite severe histologic evidence of disease, there are no pathognomonic thoracic radiographic findings in cats with acute cytauxzoonosis.

Global distribution of canine Babesia species identified by a commercial diagnostic laboratory.

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.

Cytauxzoon felis cytochrome b gene mutation associated with atovaquone and azithromycin treatment.

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.

Second illness due to subsequent Cytauxzoon felis infection in a domestic cat.

CASE SUMMARY: 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. RELEVANCE AND NOVEL INFORMATION: 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.

Perceptions and attitudes of Small Animal Internal Medicine specialists toward the publication requirement for board certification.

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.

Serum procalcitonin concentrations in dogs with induced endotoxemia.

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.

Prevalence of Babesia spp. and clinical characteristics of Babesia vulpes infections in North American dogs.

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.

Endothelial alterations in a canine model of immune thrombocytopenia.

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.

Clinical, morphological, and molecular characterization of an undetermined Babesia species in a maned wolf (Chrysocyon brachyurus).

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.

Genetic conservation of Cytauxzoon felis antigens and mRNA expression in the schizont life-stage.

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.

Evaluation of cell culture-grown Bartonella antigens in immunofluorescent antibody assays for the serological diagnosis of bartonellosis in dogs.

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.