Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

Necrotizing hepatitis caused by Clostridium novyi type B in a dog with no predisposing liver lesions: a case report.

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.

Giraffe skin disease: Clinicopathologic characterization of cutaneous filariasis in the critically endangered Nubian giraffe (Giraffa camelopardalis camelopardalis).

Giraffe skin disease (GSD) is an emerging disease of free-ranging giraffe recognized in the last 25 years in several species, including the critically endangered Nubian giraffe (Giraffa camelopardalis camelopardalis) of Uganda. Identifying the cause of GSD and understanding its impact on health were deemed paramount to supporting these vulnerable populations. Sixty-four giraffes were immobilized in Murchison Falls National Park, Uganda, from 2017 to 2019, and GSD lesions were opportunistically biopsied. Fifty-five giraffes (86%) had GSD lesions on the neck, axilla, chest, and cranial trunk. Lesions were categorized into early, intermediary, and dormant stages based on gross and histological characteristics. Early lesions were smaller, crusted nodules with eosinophilic and pyogranulomatous dermatitis and furunculosis. Intermediary lesions were thick plaques of proliferative and fissured hyperkeratosis and acanthosis with dense dermal granulation tissue and severe eosinophilic and granulomatous dermatitis. Lesions appeared to resolve to dormancy, with dormant lesions consisting of hairless plaques of hyperkeratosis with dermal scarring and residual inflammation. The periphery of early and intermediary lesions included follicular granulomas containing adult filarid nematodes, with myriad encysted microfilariae in the superficial dermis. Stage L3 larvae were common in early and intermediary lesions, and dormant lesions had remnant encysted microfilariae with no adult or stage L3 larvae. Nematodes were morphologically and genetically novel with close identity to Stephanofilaria spp. and Brugia malayi, which cause infectious filariasis. Identification of potential insect vectors, long-term monitoring of GSD lesions, and evaluating response to therapy is ongoing in the efforts to help conserve the Nubian giraffe.

Cholesterol Granuloma of the Cranial Mediastinum in a Dog.

A 10 yr old female spayed Pomeranian presented with a history of dyspnea and coughing and was diagnosed with a cranial mediastinal mass presumed to be a thymoma. Surgical removal was elected and occurred without intraoperative complications. Histopathology revealed the lesion to be a cholesterol granuloma. The patient developed a brief period of increased respiratory difficulty 3 days postoperatively. Thoracic radiographs showed mild pleural effusion and the patient improved with supportive care. Five months postoperatively, repeat thoracic radiographs revealed no evidence of recurrence or respiratory pathology. This case report describes a cholesterol granuloma in a unique location and reviews the pathogenesis/pathophysiology of this type of mass.

Theileria orientalis Ikeda Genotype in Cattle, Virginia, USA.

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.

Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing.

BACKGROUND: Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. METHODS: An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow. RESULTS: For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq. CONCLUSION: The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.

Multistate Infestation with the Exotic Disease-Vector Tick Haemaphysalis longicornis - United States, August 2017-September 2018.

Haemaphysalis longicornis is a tick indigenous to eastern Asia and an important vector of human and animal disease agents, resulting in such outcomes as human hemorrhagic fever and reduction of production in dairy cattle by 25%. H. longicornis was discovered on a sheep in New Jersey in August 2017 (1). This was the first detection in the United States outside of quarantine. In the spring of 2018, the tick was again detected at the index site, and later, in other counties in New Jersey, in seven other states in the eastern United States, and in Arkansas. The hosts included six species of domestic animals, six species of wildlife, and humans. To forestall adverse consequences in humans, pets, livestock, and wildlife, several critical actions are indicated, including expanded surveillance to determine the evolving distribution of H. longicornis, detection of pathogens that H. longicornis currently harbors, determination of the capacity of H. longicornis to serve as a vector for a range of potential pathogens, and evaluation of effective agents and methods for the control of H. longicornis.

Frontal and caudal maxillary sinus lipoma in a horse.

An adult horse was diagnosed with a frontal and caudal maxillary sinus lipoma, which was surgically removed. This is the first known report of a sinus lipoma in a horse. Lipomas should be considered in the differential diagnoses of equine sinus masses; complete surgical excision appears to be curative.

Lipome du sinus maxillaire frontal et caudal chez un cheval. Un cheval adulte a été diagnostiqué avec un lipome du sinus maxillaire frontal et caudal qui a été enlevé par chirurgie. Il s'agit du premier rapport d'un lipome de sinus chez un cheval. Les lipomes devraient être considérés dans les diagnostics différentiels des masses des sinus chez les équidés; l'excision chirurgicale complète semble être curative.(Traduit par Isabelle Vallières).

Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum.

BACKGROUND: Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259-93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin selection. RESULTS: Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259-93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259-93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02-90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n = 174) and without rifampicin (n = 126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains. CONCLUSION: Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).

Widespread Circulation of Tick-Borne Viruses in Virginia-Evidence of Exposure to Heartland, Bourbon, and Powassan Viruses in Wildlife and Livestock.

Emerging tick-borne viruses such as Powassan virus (POWV), Bourbon virus (BRBV), and Heartland virus (HRTV), whilst rare, can cause severe health problems in humans. While limited clinical cases have been reported thus far in Virginia, the presence of tick-borne viruses poses a serious health threat, and the extent of their prevalence in Virginia is unknown. Here, we sought evidence of POWV, BRBV, and HRTV exposure in Virginia via a serological assessment of wildlife and livestock. Wildlife in Virginia were found to be seropositive against POWV (18%), BRBV (8%), and HRTV (5%), with western and northern regions of the state having a higher prevalence. Multiple wildlife species were shown to have been exposed to each virus examined. To a lesser extent, cattle also showed exposure to tick-borne viruses, with seroprevalences of 1%, 1.2%, and 8% detected in cattle against POWV, BRBV, and HRTV, respectively. Cross-reactivity against other known circulating mosquito-borne flaviviruses was ruled out. In conclusion, there is widespread exposure to tick-borne viruses in western and northern Virginia, with exposure to a diverse range of animal populations. Our study provides the first confirmation that HRTV is circulating in the Commonwealth. These findings strengthen the existing evidence of emerging tick-borne viruses in Virginia and highlight the need for public health vigilance to avoid tick bites.

Prevalence of Latent Equid Herpesvirus Type 1 in Submandibular Lymph Nodes of Horses in Virginia.

Equine Herpesvirus type 1 (EHV-1) typically causes mild respiratory disease, but it can also cause late-term abortion, neonatal foal death and neurologic disease. Once a horse is infected, the virus concentrates to local lymphoid tissue, where it becomes latent. The virus can be reactivated during times of stress, which can lead to the initiation of devastating outbreaks. Understanding the carriage rate of latent EHV-1 in different geographic regions is essential for managing the disease. The objective of the current study was to estimate the prevalence of latent EHV-1 and compare the frequency of each variant in the submandibular lymph nodes of horses in Virginia. Sixty-three submandibular lymph nodes were collected post-partem from horses submitted to regional labs for necropsy, and qPCR was performed. All samples were negative for the gB gene of EHV-1. The results demonstrated a low apparent prevalence of latent EHV-1 DNA in submandibular lymph nodes in this population of horses in Virginia. Despite this, the mainstay for outbreak prevention and mitigation continues to focus on minimizing risks and using appropriate and diligent biosecurity.

Case report: Clitoral adenocarcinoma in a mixed-breed female dog.

A 9-year-old, spayed female, mixed-breed dog was initially presented for evaluation of chronic dermatitis on the nasal planum, where a clitoral mass was discovered as an incidental finding during the exam. No further investigation of the clitoral mass was undertaken due to other significant dermal lesions and the lack of clinical significance of the mass at the time. However, ~1 month later, the dog was presented to the Emergency Service for bleeding from the vulva. The clitoral mass was found to have prolapsed; the mass was manually reduced back into a position within the vulvar folds and maintained with a purse-string suture. The dog was referred to the Theriogenology Service for further investigation and removal. On follow-up evaluation, the mass was noted to be multi-lobulated, ulcerated, cystic, and involving the clitoris but not the urethra. The urethra was easily catheterized, and no urinary abnormalities were found. No evidence of lymph node metastasis or hypercalcemia was noted prior to surgery. Ultrasonographic evaluation of the anal sacs was normal. The mass was removed, and histopathologic evaluation revealed a primary clitoral adenocarcinoma. On recheck evaluation, after 1 month, no evidence of metastasis or local recurrence was observed. Clitoral adenocarcinoma is a rarely reported neoplasm of the canine genital tract that shares many clinical, histopathological, and immunohistochemical features with canine apocrine gland anal sac adenocarcinoma. This case adds to the available knowledge on the condition, specifically regarding the frequency of complications such as hypercalcemia and metastasis, as previous reports suggest that these are present at least 50% of the time.

Differential virulence of clinical and bovine-biased enterohemorrhagic Escherichia coli O157:H7 genotypes in piglet and Dutch belted rabbit models.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) is an important cause of food and waterborne illness in the developed countries. Cattle are a reservoir host of EHEC O157 and a major source of human exposure through contaminated meat products. Shiga toxins (Stxs) are an important pathogenicity trait of EHEC O157. The insertion sites of the Stx-encoding bacteriophages differentiate EHEC O157 isolates into genogroups commonly isolated from cattle but rarely from sick humans (bovine-biased genotypes [BBG]) and those commonly isolated from both cattle and human patients (clinical genotypes [CG]). Since BBG and CG share the cardinal virulence factors of EHEC O157 and are carried by cattle at similar prevalences, the infrequent occurrence of BBG among human disease isolates suggests that they may be less virulent than CG. We compared the virulence potentials of human and bovine isolates of CG and BBG in newborn conventional pig and weaned Dutch Belted rabbit models. CG-challenged piglets experienced severe disease accompanied by early and high mortality compared to BBG-challenged piglets. Similarly, CG-challenged rabbits were likely to develop lesions in kidney and intestine compared with the BBG-challenged rabbits. The CG strains used in this study carried stx2 and produced significantly higher amounts of Stx, whereas the BBG strains carried the stx2c gene variant only. These results suggest that BBG are less virulent than CG and that this difference in virulence potential is associated with the Stx2 subtype(s) carried and/or the amount of Stx produced.

Evaluation of Tumor Grade and Proliferation Indices before and after Short-Course Anti-Inflammatory Prednisone Therapy in Canine Cutaneous Mast Cell Tumors: A Pilot Study.

Glucocorticoid administration is a common clinical practice that attempts to decrease the inflammation associated with and improve the resectability of canine mast cell tumors (MCTs). However, the impact of neoadjuvant glucocorticoids on the histological features and proliferation indices of canine MCTs is unknown. The objective of this study was to evaluate changes in tumor grade, mitotic count, Ki67, AgNOR, and AgNORxKi67 scores following short-course anti-inflammatory neoadjuvant prednisone in canine patients with MCTs. This was a prospective single-arm pilot study. Client-owned dogs with treatment-naïve cytologically confirmed MCTs were enrolled. Patients underwent an initial incisional biopsy followed by a 10-14-day course of anti-inflammatory prednisone and surgical resection. All histological samples were randomized, masked, and evaluated by a single pathologist. Unstained paired pre- and post-treatment samples were submitted to a commercial laboratory for Ki67 and AgNOR immunohistochemical analysis. There were 11 dogs enrolled with 11 tumors. There were no statistical differences between the pre- and post-treatment histological parameters of mitotic index, Ki67, AgNOR, or Ki67xAgNOR. There were no clinically significant alterations between pre-treatment and post-treatment in the assignment of tumor grades. A short course of anti-inflammatory prednisone does not appear to alter the histological parameters that affect grade determination or significantly alter the proliferation indices in canine MCTs.

Optimizing Pooled Testing for Estimating the Prevalence of Multiple Diseases.

Pooled testing can enhance the efficiency of diagnosing individuals with diseases of low prevalence. Often, pooling is implemented using standard groupings (2, 5, 10, etc.). On the other hand, optimization theory can provide specific guidelines in finding the ideal pool size and pooling strategy. This article focuses on optimizing the precision of disease prevalence estimators calculated from multiplex pooled testing data. In the context of a surveillance application of animal diseases, we study the estimation efficiency (i.e., precision) and cost efficiency of the estimators with adjustments for the number of expended tests. This enables us to determine the pooling strategies that offer the highest benefits when jointly estimating the prevalence of multiple diseases, such as theileriosis and anaplasmosis. The outcomes of our work can be used in designing pooled testing protocols, not only in simple pooling scenarios but also in more complex scenarios where individual retesting is performed in order to identify positive cases. A software application using the shiny package in R is provided with this article to facilitate implementation of our methods. Supplementary materials accompanying this paper appear online. Supplementary materials for this article are available at 10.1007/s13253-022-00511-4.

Complete Genome Sequence of Curtobacterium sp. Isolated from Surface-Sterilized Germinating Alfalfa Seeds.

We reported the complete genome sequence of a member of the pathogenic Curtobacterium genus. The sample includes a circular 3,955-kb chromosome, a 164-kb megaplasmid and a 42-kb plasmid. This strain was isolated from surface-sterilized alfalfa seeds.

Alginate microencapsulation of an attenuated O-antigen mutant of Francisella tularensis LVS as a model for a vaccine delivery vehicle.

Francisella tularensis is the etiologic agent of tularemia and a Tier I Select Agent. Subspecies tularensis (Type A) is the most virulent of the four subspecies and inhalation of as few as 10 cells can cause severe disease in humans. Due to its niche as a facultative intracellular pathogen, a successful tularemia vaccine must induce a robust cellular immune response, which is best achieved by a live, attenuated strain. F. tularensis strains lacking lipopolysaccharide (LPS) O-antigen are highly attenuated, but do not persist in the host long enough to induce protective immunity. Increasing the persistence of an O-antigen mutant may help stimulate protective immunity. Alginate encapsulation is frequently used with probiotics to increase persistence of bacteria within the gastrointestinal system, and was used to encapsulate the highly attenuated LVS O-antigen mutant WbtIG191V. Encapsulation with alginate followed by a poly-L-lysine/alginate coating increased survival of WbtIG191V in complement-active serum. In addition, BALB/c mice immunized intraperitoneally with encapsulated WbtIG191V combined with purified LPS survived longer than mock-immunized mice following intranasal challenge. Alginate encapsulation of the bacteria also increased antibody titers compared to non-encapsulated bacteria. These data suggest that alginate encapsulation provides a slow-release vehicle for bacterial deposits, as evidenced by the increased antibody titer and increased persistence in serum compared to freely suspended cells. Survival of mice against high-dose intranasal challenge with the LVS wildtype was similar between mice immunized within alginate capsules or with LVS, possibly due to the low number of animals used, but bacterial loads in the liver and spleen were the lowest in mice immunized with WbtIG191V and LPS in beads. However, an analysis of the immune response of surviving mice indicated that those vaccinated with the alginate vehicle upregulated cell-mediated immune pathways to a lesser extent than LVS-vaccinated mice. In summary, this vehicle, as formulated, may be more effective for pathogens that require predominately antibody-mediated immunity.

Retroperitoneal myxosarcoma in a cat.

This report details a retroperitoneal myxosarcoma in a cat that exhibited extremely aggressive biological behavior. An exploratory midline celiotomy revealed a left-sided retroperitoneal mass firmly adhered to the hypaxial musculature. Histopathological evaluation identified the mass as a myxosarcoma. Following surgical excision, the mass rapidly recurred within 6 weeks after surgery.

Coinfection of cattle in Virginia with Theileria orientalis Ikeda genotype and Anaplasma marginale.

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.

Distribution of Theileria orientalis in Virginia Market Cattle, 2018-2020.

Theileria orientalis, genotype Ikeda, was recently detected in North America. Determining the emerging distribution of this pathogen is critical for understanding spread and developing management strategies. Whole blood samples were collected from cattle at Virginia livestock markets from September 2018 through December 2020. Animals were tested for T. orientalis using a universal and then genotype specific real-time PCR based on the MPSP gene. Prevalence for each genotype was analyzed for temporal trends and mapped by county. Spatial patterns were compared between genotypes and assessed for associations with habitat features, cattle movements through cattle markets and county proximity. Overall, 212 of 1980 samples tested positive for T. orientalis with an overall prevalence of 8.7% (172/1980) for genotype Ikeda, 1.8% (36/1980) for genotype Chitose, 0.2% (3/1980) for genotype Buffeli. The Ikeda genotype increased over time in northern and southwestern Virginia markets. The Ikeda and Chitose genotypes occurred in different regions, with little overlap, but for each genotype, spatial distribution was associated with a combination of cattle movements and environmental factors. Genotype specific qPCR testing and surveillance of cattle from across a wide area of Virginia are providing information on temporal, spatial, and other patterns for this emerging disease.