Theileria orientalis Ikeda genotype is a parasite that causes a disease in cattle that results in major economic issues in Asia, New Zealand, and Australia. The parasite is transmitted by Haemaphysalis longicornis ticks, which have recently been reported in numerous states throughout the eastern United States. Concurrently, cattle in Virginia showed clinical signs consistent with a hemoprotozoan infection. We used amplicons specific for the major piroplasm surface protein and small subunit rDNA of piroplasms to test blood samples from the cattle by PCR. Bidirectional Sanger sequencing showed sequences with 100% identity with T. orientalis Ikeda genotype 2 sequences. We detected the parasite in 3 unrelated herds and from various animals sampled at 2 time points. Although other benign T. orientalis genotypes are endemic to the United States, detection of T. orientalis Ikeda genotype might represent a risk for the cattle industry in Virginia.

Theileria orientalis Ikeda is a newly identified agent of bovine infectious anemia in the United States. Although T. orientalis Ikeda is transmitted by ticks other than the tick that transmits Anaplasma marginale—a bacterial etiology of bovine infectious anemia—the geographic distributions of these 2 infectious organisms overlap, with coinfection reported in some cattle. Only anaplasmosis has an approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex real-time PCR (rtPCR) for A. marginale and T. orientalis. With a cutoff of 38 cycles, the duplex assay has a sensitivity of 97.0% and a specificity of 100% for A. marginale; with a cutoff of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for T. orientalis, compared to existing tests. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, our rtPCR facilitates the study of coinfection of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for T. orientalis, 125 were positive for A. marginale, and 12 samples were positive for both T. orientalis and A. marginale. Hence, coinfection by these 2 agents of bovine infectious anemia does occur within Virginia. It is likely that this pattern of infection will be seen in other regions where T. orientalis and A. marginale infections are endemic, despite the difference in tick vectors.

MRL/lpr mice have been extensively used as a murine model of lupus. Disease progression in MRL/lpr mice can differ among animal facilities, suggesting a role for environmental factors. We noted a phenotypic drift of our in-house colony, which was the progeny of mice obtained from The Jackson Laboratory (JAX; stocking number 000485), that involved attenuated glomerulonephritis, increased splenomegaly, and reduced lymphadenopathy. To validate our in-house mice as a model of lupus, we compared these mice with those newly obtained from JAX, which were confirmed to be genetically identical to our in-house mice. Surprisingly, the new JAX mice exhibited a similar phenotypic drift, most notably the attenuation of glomerulonephritis. Interestingly, our in-house colony differed from JAX mice in body weight and kidney size (both sexes), as well as in splenic size, germinal center formation, and level of anti-dsDNA auto-IgG in the circulation (male only). In addition, we noted differential expression of microRNA (miR)-21 and miR-183 that might explain the splenic differences in males. Furthermore, the composition of gut microbiota was different between in-house and new JAX mice at early time points, which might explain some of the renal differences (e.g., kidney size). However, we could not identify the reason for attenuated glomerulonephritis, a shared phenotypic drift between the two colonies. It is likely that this was due to certain changes of environmental factors present in both JAX and our facilities. Taken together, these results suggest a significant phenotypic drift in MRL/lpr mice in both colonies that may require strain recovery from cryopreservation. ImmunoHorizons, 2022, 6: 36-46.

Theileria orientalis ikeda is a newly identified agent of bovine infectious anemia in the United States. Although it is transmitted by separate tick hosts than Anaplasma marginale - a bacterial etiology of bovine infectious anemia -the geographic distributions of these two infectious organisms overlap, with co-infection reported in some cattle. Only anaplasmosis has approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex qPCR for A. marginale and T. orientalis. With a cut-off of 38 cycles, the duplex assay has a sensitivity of 96.97% and a specificity of 100% for A. marginale; with a cut-off of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for T. orientalis. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, this qPCR facilitates the study of co-infection rate of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for the presence of T. orientalis, 125 were positive for the presence of A. marginale, and 12 samples were positive for both T. orientalis and A. marginale. This indicated that co-infection of both of these etiologies of bovine infectious anemia does occur within the state of Virginia. It is likely that this pattern of infection will be seen in regions where T. orientalis and A. marginale are endemic, despite the difference in tick vectors.

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Background Infectious necrotic hepatitis (INH) is typically a disease of ruminants caused by Clostridium novyi type B. Growth of the causative agent is supported by development of an anaerobic environment within the liver. In dogs, C. novyi is rare and has only been previously reported as a post-mortem diagnosis. In one case, infection was secondary to metastatic pancreatic adenocarcinoma and the other was presumptively diagnosed on histopathology of a hepatic lesion in a dog initially presented for acute collapse. Case presentation An 8-year-old spayed, female mixed breed dog was presented for acute onset of hyporexia and vomiting. Serum biochemistry revealed elevated hepatocellular injury and cholestatic liver enzymes. Ultrasound revealed peritoneal fluid accumulation and multiple hepatic masses. Cytologic examination of liver aspirates and peritoneal fluid revealed frequent 4 × 1 μm bacilli with a terminal endospore. Anaerobic bacterial growth isolated from the fluid sample could not be identified using typical laboratory identification techniques. Long-read, whole genome sequencing was performed, and the organism was identified as Clostridium novyi type B. Antimicrobial and hepatic support treatment were initiated. The patient re-presented 27 days later, and the follow up liver aspirate with cytology revealed no appreciable bacteria and anaerobic culture was negative. The patient was presented four months later and a large hepatic mass and peritoneal fluid were again identified on abdominal ultrasound. Cytologic examination of the peritoneal fluid revealed bacilli similar to those identified on initial presentation. The patient was euthanized. The most significant finding on necropsy was necrotizing hepatitis with intralesional endospore-forming bacilli compatible with recurrence of Clostridium novyi type B. There was no identifiable cause of an anaerobic insult to the liver. Conclusions This case demonstrates the diagnostic utility of using cytology as part of the initial diagnostic work up for infectious hepatitis. The cytologic findings coupled with whole genome sequencing and anaerobic culture were crucial for the identification and classification of the organism identified on fine needle aspirate. Clostridium novyi type B should be considered when bacilli organisms containing a terminal endospore are identified on liver aspirates collected from canine patients.

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease. The cause of SLE is not only genetic; in fact, environmental factors may play a more important role in disease development. The MRL/lpr mouse model lupus-like symptoms due to multiple SLE susceptible loci in the MRL background, and offers an accelerated model compared to the MRL parent strain due to the Faslpr mutation. Recently, our laboratory witnessed a loss of disease phenotype in our in-house colony of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX; Stock Number 000485) to our in-house mice. The single-nucleotide polymorphism (SNP) analysis showed no genetic drift, suggesting that environmental factors could be triggering a phenotypic drift. Surprisingly, the newly purchased JAX mice also had attenuation of glomerulonephritis. Even though JAX mice manifested similar attenuation of lupus nephritis, our in-house colony showed differences in organ weights. Furthermore, males showed a significantly higher level of anti-double stranded DNA auto-IgG consistent with germinal center maturation. In addition, in-house males had significantly higher levels of microRNA-21 and microRNA-183 explaining spleen size difference. Moreover, the composition of gut microbiota was different between in-house and new JAX mice at early age, with many groups of bacteria differing at later time points, which might explain some of the phenotypic differences. These results suggest that microRNAs and gut microbiota might be responsible for the phenotypic differences of MRL/lpr mice in JAX and our colonies as they were genetically identical. On the other hand, the attenuation of nephritis in both groups requires further investigation. Supported by R01AR073240