Yokenella regensburgei, a novel pathogen in farmed American alligators.

Increased acute mortality of farmed American alligators (Alligator mississippiensis) was observed in various pens from 2 different farms in Louisiana over 2 years (2019-2021). A total of 14 alligators from multiple events of increased mortality were subjected to postmortem investigations. Except for one alligator with acute neurologic signs, no premonitory signs were observed. All animals had pneumonia (14/14), coelomitis (14/14), and intravascular short Gram-negative bacilli (14/14). Myocarditis (13/14) was common. Yokenella regensburgei was isolated from all alligators tested (13/13). These data suggest the respiratory tract may be a primary target system and could be involved in transmission, either through exhaled bacteria or through swallowing of contaminated respiratory fluids with passage through the feces. Available sensitivity data for Y. regensburgei in this study indicates in vitro sensitivity to aminoglycosides, fluoroquinolones, chloramphenicol, and trimethoprim/sulphamethoxazole antibiotics. Yokenella regensburgei should be included in the differential diagnosis of septicemia and acute death in alligators.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

H7N9 influenza A virus in turkeys in Minnesota.

Introductions of H7 influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAVs in Minnesota (MN) turkey farms during 2009 and 2011. The full genome was sequenced from eight isolates as well as the haemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum-likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAVs, estimated the time and geographical origin of the ancestral viruses, and determined the relatedness between the 2009 and 2011 turkey-origin H7N9 IAVs. Analyses supported that the 2009 and 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further supported that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAVs isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAVs could not be determined, our findings suggested that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi Flyway.

Detection of Tritrichomonas foetus by RT-rtPCR in pooled bovine preputial washings.

Trichomonosis is a venereal disease of cattle caused by the protozoan Tritrichomonas foetus. T. foetus infection in cattle herds can be economically costly for cattle producers; therefore, testing is important for detection of the agent. Given that bulls are considered to be subclinical carriers of T. foetus, it is important to detect T. foetus infection prior to movement and/or breeding season. We have described previously the development of an updated set of PCR primers and probes that offer increased sensitivity of T. foetus detection in preputial washings collected in PBS by utilizing reverse-transcription real-time PCR (RT-rtPCR) that targets the 5.8S ribosomal RNA of the T. foetus organism. Here, we report improvements in the updated RT-rtPCR reagents as well as the evaluation of testing of pooled preputial washings. We found that up to 5 preputial washings can be pooled, similar to routine testing practices (InPouch culture), without reducing the sensitivity of detection of T. foetus.

Deer mastadenovirus B pneumonia in a white-tailed deer fawn.

A 7-mo-old farmed white-tailed deer fawn (Odocoileus virginianus) died after several weeks of progressive deterioration associated with endoparasitism and respiratory signs. A field autopsy was performed, and lung tissue was submitted for histologic examination. The findings were consistent with necrosuppurative bronchointerstitial pneumonia with intranuclear viral inclusions. Immunofluorescence using fluorescently labeled polyclonal antibodies to bovine adenovirus 3 and 5 was positive. To rule out cross-reactivity with other adenoviruses, formalin-fixed, paraffin-embedded tissue sections were submitted for genome sequence analysis, which revealed a 99.6% match to Deer mastadenovirus B (formerly Odocoileus adenovirus 2, OdAdV2). To our knowledge, natural clinical disease associated with OdAdV2 has not been reported previously.

Development of a novel real-time PCR multiplex assay for detection of Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus.

Strangles is a contagious bacterial disease of horses caused by Streptococcus equi subspecies equi (SEE) that occurs globally. Rapid and accurate identification of infected horses is essential for controlling strangles. Because of limitations of existing PCR assays for SEE, we sought to identify novel primers and probes that enable simultaneous detection and differentiation of infection with SEE and S. equi subsp. zooepidemicus (SEZ). Comparative genomics of U.S. strains of SEE and SEZ (n = 50 each) identified SE00768 from SEE and comB from SEZ as target genes. Primers and probes for real-time PCR (rtPCR) were designed for these genes and then aligned in silico with the genomes of strains of SEE (n = 725) and SEZ (n = 343). Additionally, the sensitivity and specificity relative to microbiologic culture were compared between 85 samples submitted to an accredited veterinary medical diagnostic laboratory. The respective primer and probe sets aligned with 99.7 % (723/725) isolates of SEE and 97.1 % (333/343) of SEZ. Of 85 diagnostic samples, 20 of 21 (95.2 %) SEE and 22 of 23 SEZ (95.6 %) culture-positive samples were positive by rtPCR for SEE and SEZ, respectively. Both SEE (n = 2) and SEZ (n = 3) were identified by rtPCR among 32 culture-negative samples. Results were rtPCR-positive for both SEE and SEZ in 21 of 44 (47.7 %) samples that were culture-positive for SEE or SEZ. The primers and probe sets reported here reliably detect SEE and SEZ from Europe and the U.S., and permit detection of concurrent infection with both subspecies.

Detection and characterization of a distinct bornavirus lineage from healthy Canada geese (Branta canadensis).

Avian bornaviruses (ABV), identified in 2008, infect captive parrots and macaws worldwide. The natural reservoirs of these viruses are unknown. Reverse transcription-PCR (RT-PCR) was used to screen oropharyngeal/cloacal swab and brain samples from wild Canada geese (Branta canadensis) for ABV. Approximately 2.9% of swab samples were positive for bornavirus sequences. Fifty-two percent of brain samples from 2 urban flocks also tested positive, and brain isolates were cultured in duck embryo fibroblasts. Phylogenetic analyses placed goose isolates in an independent cluster, and more notably, important regulatory sequences present in Borna disease virus but lacking in psittacine ABVs were present in goose isolates.

Avian influenza virus investigation in wild bobwhite quail from Texas.

The objective of this study was to determine the prevalence of avian influenza viruses (AIV) in bobwhite quail (Colinus virginianus) populations from the rolling plains of Texas, U. S. A. A total of 1320 swab samples (652 tracheal swabs and 668 cloacal swabs) and 44 serum samples were collected from wild-captured or hunter-harvested bobwhite quail from November 2009 to April 2011 at the Rolling Planes Quail Research Ranch, Fisher County, Texas, U. S. A. The presence of AIV in the swabs was determined by real-time reverse-transcription-PCR (rRT-PCR) and all samples positive or suspicious by rRT-PCR were further processed for virus isolation in embryonated chicken eggs. A total of 18 (1.4%) swab samples tested positive for AIV by rRT-PCR (cycle threshold [Ct] values < 35): 13 cloacal swabs (1.9%) and 5 tracheal swabs (0.8%). In addition, 100 (7.6%) swab samples were considered suspicious (Ct values 35.1-40): 69 cloacal swabs (10.3%) and 31 tracheal swabs (4.7%). No virus was isolated from any of the rRT-PCR-positive or suspicious samples tested. Additionally, 44 serum samples were screened for AIV antibodies and were negative. The results presented here indicate low prevalence of AIV in wild populations of bobwhite quail.

Avian influenza virus surveillance in hunter-harvested waterfowl, Texas coast, September 2009-January 2010.

Wild waterfowl are considered the natural reservoir of type A influenza viruses, and the migratory nature of many waterfowl species presents a possible vehicle for global dissemination of these infectious agents. In order to fully understand the ecology of influenza viruses, multiyear surveillance efforts are critical, particularly in understudied areas, such as waterfowl wintering areas. Herein we report results obtained during the fifth year ofa 5-yr avian influenza virus (AIV) surveillance project conducted on waterfowl wintering grounds of the Texas Coast. During year 5, the 2009-2010 hunting season (September, November-January), 655 cloacal swabs were collected from hunter-harvested waterfowl and screened for AIV by real-time RT-PCR (rRT-PCR) followed by virus isolation on all positive samples. Molecular methods were used for subtyping all AIV isolates. Sixty-five (9.5%) samples were positive for AIV by rRT-PCR, and 24 (3.7%) AIVs were isolated. Eight different hemagglutinin (H3, 4, 5, 6, 8, 9, 10, and 11) and seven different neuraminidase (N1, 2, 3, 4, 6, 8, and 9) subtypes were identified. This was the first year H8 and H9 were isolated throughout the 5-yr survey. Our results support the fact that continued multiyear surveillance of natural reservoirs, particularly in understudied areas, is needed in order to better understand the ecology of AIVs in nature.

Use of FTA sampling cards for molecular detection of avian influenza virus in wild birds.

Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n = 6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anasplatyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 x 10(4.7) median embryo infectious dose (EID50)/ml (range: 1 x 10(4.3) to 1 x 10(5.4) EID50/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.

Role of nonmigratory mottled ducks (Anas fulvigula) as sentinels for avian influenza surveillance.

The objective of this study was to evaluate the mottled duck (Anas fulvigula), a nonmigratory dabbling duck, as a sentinel species for avian influenza virus (AIV) surveillance. A total of 235 cloacal swabs from 147 live-captured and 88 hunter-harvested mottled ducks during summer (June-August 2007) and winter (November 2007 to January 2008), respectively, were collected along the upper Texas coast. Samples were screened for AIV using real-time reverse transcription polymerase chain reaction (rRT-PCR); all rRT-PCR-positive samples were processed for virus isolation. Three samples were positive for AIV by AIV-matrix rRT-PCR. One of these samples also was positive for H5 by rRT-PCR, and a low pathogenic H5N2 AIV was isolated. Although isolation of AIVs from mottled ducks during the winter has been reported previously, to the authors' knowledge, this is the first H5 isolate from mottled ducks. Interestingly, this isolation was made during the same season that other H5N2 viruses were obtained from migratory waterfowl on the Texas coast, which suggests AIV transmission among waterfowl on the wintering grounds and the potential role of mottled ducks as a naturally occurring sentinel species for AIV surveillance.

Clostridium piliforme and canine distemper virus coinfection in 2 domestic dog littermates and a gray fox kit.

Concurrent Clostridium piliforme and canine distemper virus (CDV) infection was diagnosed in 2 canine littermates and 1 gray fox kit from Texas, USA. In all 3 animals, intracytoplasmic, filamentous bacteria, consistent with C. piliforme, were present along the margins of foci of hepatic necrosis. Additional histologic findings included intracytoplasmic and intranuclear inclusion bodies in bile duct and bronchial epithelial cells of the fox kit, and mild intestinal necrosis in 1 puppy. PCR assays confirmed the presence of C. piliforme in all 3 animals, CDV in both puppies, and canine parvovirus in 1 puppy. Fluorescent antibody testing confirmed the presence of CDV in the fox kit. Concurrent canine distemper and Tyzzer disease in canine littermates and the gray fox has not been reported previously, to our knowledge.

Genetic Sequencing of Attwater's Prairie Chicken Avian Poxvirus and Evaluation of Its Potential Role in Reticuloendotheliosis Virus Outbreaks.

Efforts to breed Attwater's prairie chickens (APC; Tympanuchus cupido attwateri) in captivity to supplement wild populations of this endangered bird have been negatively affected by infections with Avipoxvirus and reticuloendotheliosis virus (REV). Because REV can be integrated into the genome of fowlpox virus (FPV) and may be transmitted in that manner, identifying the source of avipox disease in APC is important to mitigate the impact of this virus. Tissue samples from APC were collected from breeding programs in Texas from 2016 to 2020. These samples consisted of 11 skin lesions and three internal organs from a total of 14 different birds that died of unknown causes or were euthanized. Avipoxvirus was detected by PCR and isolation in embryonating chicken eggs in all skin lesion samples but was not detected in internal organs. Using sequence analysis of FPV polymerase and 4b genes, we determined that 10 out of 11 Avipoxvirus detections resided within the fowlpox clade and a single sample resided within the canarypox clade. REV sequences were detected in all FPV positive samples and in all internal organ tissues but were not detected in the sample matching the canarypox clade. Analysis of REV sequences and PCR detection showed the REV infecting APC was consistent with REV-A and had little variability on analysis of the U3 region of the long terminal repeat. The results of this study indicate control of REV in APC breeding colonies may benefit by a vaccination program targeting FPV and REV. However, a commercially available vaccine for REV is not available at this time.

Secuenciación genética de un virus de la viruela aviar de un gallo grande de las praderas Attwater y evaluación de su papel potencial en los brotes del virus de la reticuloendoteliosis. Los esfuerzos para criar gallos de las praderas grandes de Attwater (APC; Tympanuchus cupido attwateri) en cautiverio para complementar las poblaciones silvestres de esta ave en peligro de extinción se han visto afectados negativamente por infecciones con Avipoxvirus y con el virus de la reticuloendoteliosis (REV). Debido a que el virus de la reticuloendoteliosis puede integrarse en el genoma del virus de la viruela del pollo (FPV) y puede transmitirse de esa manera, identificar la fuente del virus pox en gallos de las praderas grandes es importante para mitigar el impacto de este virus. Se recolectaron muestras de tejido de gallos de las praderas grandes de programas de reproducción en Texas entre los años 2016 a 2020. Estas muestras consistieron en 11 lesiones cutáneas y tres órganos internos de un total de 14 aves diferentes que murieron por causas desconocidas o fueron sacrificadas. El Avipoxvirus se detectó mediante PCR y por aislamiento en huevos embrionados de pollo en todas las muestras de lesiones cutáneas, pero no se detectó en los órganos internos. Utilizando el análisis de secuencia de la polimerasa del virus de la viruela del pollo y de los genes 4b, se determinó que diez de las once detecciones de Avipoxvirus residían dentro del clado de la viruela aviar del pollo y una sola muestra residía dentro del clado de la viruela del canario. Se detectaron secuencias del virus de la reticuloendoteliosis en todas las muestras positivas para virus de la viruela de pollo y en todos los tejidos de órganos internos, pero no se detectaron en la muestra que coincidía con el clado de la viruela del canario. El análisis de las secuencias del virus de la reticuloendoteliosis y la detección por PCR mostró que los virus de reticuloendoteliosis que infectan a gallos de las praderas grandes eran compatible con virus de la reticuloendoteliosis A y tenía poca variabilidad en el análisis de la región U3 de la región repetida terminal larga. Los resultados de este estudio indican que el control del virus de la reticuloendoteliosis en colonias reproductoras de gallos de las praderas grandes puede beneficiarse de un programa de vacunación dirigido los virus de la viruela del pollo y de la reticuloendoteliosis. Sin embargo, una vacuna disponible comercialmente contra el virus de la reticuloendoteliosis no está disponible en este momento.

Presumptive Primary Cerebral T-Cell Lymphoma in a White-Tailed Deer (Odocoileus virginianus).

Primary central nervous system (CNS) lymphomas are rare in humans and even more uncommon in animals. We report the clinical, pathological and immunohistochemical features of a presumptive primary cerebral T-cell lymphoma (PCTCL) in an aged female white-tailed deer (Odocoileus virginianus) that had chronic progressive neurological disease characterized by ataxia, claudication and eventual circling. The animal was euthanized due to poor prognosis. Grossly, a 2.5 cm dark red, friable nodule effaced the cortical neuroparenchyma of the left anterior cingulate cortex (LACC). Microscopically, the meningeal vasculature and adjacent grey and white matter cortical neuroparenchyma of the LACC were infiltrated by a poorly demarcated, unencapsulated and densely cellular round cell neoplasm with a consistent angiocentric pattern. The neoplasm was associated with extensive regions of haemorrhage and liquefactive necrosis. Neoplastic cells immunolabelled for CD3 antigen and had high proliferative activity, as indicated by Ki-67 labelling. Based on the cytohistomorphological and immunohistochemical features and absence of metastasis, a diagnosis of PCTCL was determined. This case indicates that PCTCL should be considered in the differential diagnosis of neurological disease and intracranial, intra-axial CNS masses in deer.

Multiyear surveillance for avian influenza virus in waterfowl from wintering grounds, Texas coast, USA.

We studied the prevalence of influenza A virus in wintering waterfowl from the Central Flyway on the Gulf Coast of Texas. Of 5,363 hunter-harvested migratory and resident waterfowl and wetland-associated game birds sampled during 3 consecutive hunting seasons (September-January 2006-07, 2007-08, and 2008-09), real-time reverse transcription-PCR detected influenza A matrix sequences in 8.5% of samples, H5 in 0.7%, and H7 in 0.6%. Virus isolation yielded 134 influenza A viruses, including N1-N9, H1-H7, H10, and H11 subtypes. Low-pathogenicity H7 subtype was isolated during January, September, and November 2007 and January 2008; low-pathogenicity H5 subtype was isolated during November and December 2007.

Comparison of real-time reverse transcription-PCR and virus isolation for estimating prevalence of avian influenza virus in hunter-harvested wild birds at waterfowl wintering grounds along the Texas mid-Gulf Coast (2005-2006 through 2008-2009).

Historically, virus isolation has been the method of choice for conducting surveillance for avian influenza virus (AIV) in avian species. More recently, the primary screening method has become real-time reverse transcription-polymerase chain reaction (RRT-PCR). We wanted to determine how these two testing methods (virus isolation and RRT-PCR) affected AIV prevalence estimation, particularly in an understudied, low-prevalence region-the waterfowl wintering grounds along the Texas mid-Gulf Coast. Cloacal swabs were collected from hunter-harvested waterfowl and other wetland-associated game birds during four consecutive hunting seasons (2005-2006 through 2008-2009). Overall prevalence by RRT-PCR (5.9%, 6.5%, 11.2%, and 5.5%) was approximately an order of magnitude higher than prevalence by virus isolation (0.5%, 1.3%, 3.9%, and 0.7%) for the four hunting seasons, respectively. Apparent AIV prevalence by virus isolation conducted only on RRT-PCR-positive samples resulted in estimates nearly identical in magnitude to those derived from parallel testing (0.5% vs. 0.6%, 1.3% vs. 1.7%, and 3.9% vs. 4.0% for 2005-2006, 2006-2007, and 2007-2008, respectively). Unlike most reports of seasonal variation in AIV prevalence, we documented differences in prevalence estimates among months by RRT-PCR only during 2008-2009 and by virus isolation only during 2006-2007 and 2007-2008. Our data indicate that screening samples by RRT-PCR followed by virus isolation only on RRT-PCR-positive samples provides a reasonable means to generate prevalence estimates close to the true prevalence as determined by virus isolation. We also confirmed the low prevalence of AIV in waterfowl wintering grounds along the Texas mid-Gulf Coast and demonstrated little variation in prevalence among months during the four hunting seasons sampled.

Accurate Genomic Predictions for Chronic Wasting Disease in U.S. White-Tailed Deer.

The geographic expansion of chronic wasting disease (CWD) in U.S. white-tailed deer (Odocoileus virginianus) has been largely unabated by best management practices, diagnostic surveillance, and depopulation of positive herds. Using a custom Affymetrix Axiom single nucleotide polymorphism (SNP) array, we demonstrate that both differential susceptibility to CWD, and natural variation in disease progression, are moderately to highly heritable ([Formula: see text] among farmed U.S. white-tailed deer, and that loci other than PRNP are involved. Genome-wide association analyses using 123,987 quality filtered SNPs for a geographically diverse cohort of 807 farmed U.S. white-tailed deer (n = 284 CWD positive; n = 523 CWD non-detect) confirmed the prion gene (PRNP; G96S) as a large-effect risk locus (P-value < 6.3E-11), as evidenced by the estimated proportion of phenotypic variance explained (PVE ≥ 0.05), but also demonstrated that more phenotypic variance was collectively explained by loci other than PRNP Genomic best linear unbiased prediction (GBLUP; n = 123,987 SNPs) with k-fold cross validation (k = 3; k = 5) and random sampling (n = 50 iterations) for the same cohort of 807 farmed U.S. white-tailed deer produced mean genomic prediction accuracies ≥ 0.81; thereby providing the necessary foundation for exploring a genomically-estimated CWD eradication program.

Evaluation of a 384-well format for high-throughput real-time reverse transcription polymerase chain reaction for avian influenza testing.

As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384-well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96- and 384-well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96- and 384-well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96- and 384-well assays, respectively. The data presented herein supports the utility of the 384-well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.

Novel real-time PCR assays for genomic group identification of tick-borne relapsing fever species Borrelia hermsii.

Borrelia hermsii is a non-Lyme borreliosis pathogen that is responsible for causing tick-borne relapsing fever in humans and animals in the western United States. B. hermsii has been described to encompass two divergent genomic groups, GGI and GGII, which have been suggested to maintain a unique geographical distribution and potential range of pathogenicity. Though the genomic groups have been extensively documented in the literature, a real-time PCR tool for identifying these genomic groups is lacking. This study describes the development and validation of two flaB-based quantitative real-time PCR assays for differentiating between the two genomic groups of B. hermsii while also maintaining specificity against other closely related Borrelia species. The diagnostic specificity of the assays were evaluated using a large panel of various Borrelia species, including a collection of 22 B. hermsii culture isolates purified from various hosts. The high sensitivity and specificity of the assays provide a useful tool for supporting future studies aimed at evaluating the geographical distribution as well as potential intraspecies pathogenicity within arthropod vectors and mammalian hosts.

TickPath Layerplex: adaptation of a real-time PCR methodology for the simultaneous detection and molecular surveillance of tick-borne pathogens.

Tick-borne diseases (TBD) are common across the United States and can result in critical and chronic diseases in a variety of veterinary patients. Moreover, borreliosis, anaplasmosis, rickettsiosis, ehrlichiosis, and babesiosis are zoonotic and have been cited as the most common TBDs. Molecular diagnostic methodologies utilized for screening domestic dogs for these causative agents include real-time PCR (qPCR) assays in both singleplex and multiplex formats. However, current limitations of qPCR instruments restrict the number of fluorogenic labels that can be differentiated by the instrument for a given reaction. This study describes the development of the TickPath Layerplex, a diagnostic assay based on qPCR methodology that was adapted for the simultaneous detection and characterization of 11 pathogens responsible for causing 5 common TBDs in domestic dogs. The analytical and diagnostic performance of the layerplex assay was evaluated and shown to be compatible with common instruments utilized in molecular diagnostic laboratories. Test results revealed no inhibition or reduction in sensitivity during validation of the layerplex assay, and the limit of detection was determined to be near 16 genome copy equivalents per microliter. Overall, the high sensitivity, specificity, and screening capability of the assay demonstrate its utility for broadly screening dogs for common TBDs.