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BackgroundRecent surveillance studies for equine respiratory viruses have shown that equine influenza virus (EIV) continues to be a prevalent respiratory virus of equids throughout the United States and Europe.ObjectivesTo gain a better understanding of the prevalence and epidemiology of EIV shed by horses, mules and donkeys in the United States from March 2010 to November 2013.Animals2,605 equids.MethodsNasal secretions from index cases with acute onset of respiratory disease were tested by qPCR for EIV. Multilevel logistic regression was used to model the association between EIV status and prevalence factors. Furthermore, observations from EIV-positive study horses were compared to previous data from March 2008 to February 2010.ResultsA total of 230 (9.7%) index cases tested qPCR positive for EIV. A higher-than-expected proportion of EIV qPCR-positive horses occurred in the 1-5, 6-10, and 11-15 age groups when compared to the

The purpose of this study was to describe clinical, hematological and fecal PCR results from 161 horses involved in outbreaks associated with ECoV. The outbreaks happened at four separate boarding facilities between November 2011 and April 2012 in the States of CA, TX, WI and MA. Following the molecular detection of ECoV in the feces from the initial index cases, the remaining herdmates were closely observed for the development of clinical signs. Fecal samples were collected from sick and healthy horses for the PCR detection of ECoV. All four outbreaks involved primarily adult horses. Fifty-nine horses developed clinical signs with 12-16 sick horses per outbreak. The main clinical signs reported were anorexia, lethargy and fever. Four horses from 3 different outbreaks were euthanized or died due to rapid progression of clinical signs. The cause of death could not be determined with necropsy evaluation in 2 horses, while septicemia secondary to gastrointestinal translocation was suspected in 2 horses. Blood work was available from 10 horses with clinical disease and common hematological abnormalities were leucopenia due to neutropenia and/or lymphopenia. Feces were available for ECoV testing by real-time PCR from 44 and 96 sick and healthy horses, respectively. 38/44 (86%) horses with abnormal clinical signs tested PCR positive for ECoV, while 89/96 (93%) healthy horses tested PCR negative for ECoV. The overall agreement between clinical status and PCR detection of ECoV was 91%. The study results suggest that ECoV is associated with self-limiting clinical and hematological abnormalities in adult horses.

Food contaminated with bacterial pathogens is a great threat to human health and food spoilage, having an impact on public health and the food industry. Research in food safety seeks to develop a practical, rapid, and sensitive detection technique for food-borne pathogens. In the past few decades, real-time quantitative polymerase chain reaction (qPCR) has been developed, and multiplex qPCR is a preferred feature. Multiplex qPCR enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. In this study, we have developed and evaluated a hydrolysis (TaqMan) probe-based system for simultaneous detection of eight of the most common food-borne pathogens in a single-step procedure by multiplex qPCR. A multicolor combinational probe coding (MCPC) strategy was utilized that allows multiple fluorophores to label different probes in combinatorial manner. This strategy enabled simultaneous detection, identification, and quantification of targeted genes. The efficiency of the individual qPCR reactions for each target gene had values comparable to those established for multiplex qPCR, with detection limits of approximately < 10 copies of DNA per reaction. Pathogen load helps to predict bacteriological quality status in food products and serves to validate the efficiency of procedures to minimize or eliminate their presence, so newly developed multiplex qPCR was quantitative for each pathogen. During sample preparation, a step to concentrate the target organism from a relatively large sample size, remove all potential PCR inhibitors, and yield samples in a volume suitable for qPCR was incorporated.

Contemporary data on equine herpesvirus-1 (EHV-1) genotype (non-neuropathogenic or N 752 , neuropathogenic or D 752 and new variant or H 752 ) in clinically diseased equids is important in order to determine the frequency of these genotypes and their association with disease expression. A total of 297 EHV-1 qPCR-positive swabs collected from 2019 to 2022 from horses with respiratory disease (EHV-1), neurological disease (equine herpesvirus-1 myeloencephalopathy [EHM]) and abortion were tested for the three different EHV-1 genotypes (N 752 , D 752 and H 752 ) using qPCR allelic discrimination assays. All submissions originated from the United States and included 257 EHV-1 cases, 35 EHM cases and 5 cases of abortion. EHV-1 qPCR-positive cases were predominantly seen during winter and spring. N 752 was the predominant genotype detected in EHV-1 cases (87.5%), EHM cases (74.3%) and abortions (80%). D 752 was detected less frequently in EHV-1 cases (9.3%) and EHM cases (25.7%), while H 752 was only detected in EHV-1 cases (3.1%). While the N 752 genotype has remained the predominant genotype affecting horses with respiratory disease and abortion, it has also become a leading genotype in cases of EHM, when compared to historical data. The new H 752 genotype, first reported in the United States in 2021, has remained confined to a cluster of geographically and temporally related outbreaks and the data showed no emerging spread of H 752 since it was first reported. While the monitoring of EHV-1 genotypes is important from a diagnostic and epidemiological standpoint, it may also help establish medical interventions and preventive protocols to reduce the risk of severe complications associated with EHV-1 infection., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Actively shedding healthy horses have been indicated as a possible source of respiratory pathogen outbreak, transmission, and spread. Using nasal swabs from clinically healthy sport horses submitted for qPCR testing after an outbreak of equine herpesvirus-1 (EHV-1) myeloencephalopathy (EHM) in the spring of 2022, this study aimed to identify the rate of clinically healthy horses shedding common and less characterized respiratory pathogens within the sport horse population to better understand their role in outbreaks. Swabs were collected during a required quarantine and testing period, according to the United States Equestrian Federation (USEF), and showed return-to-competition requirements. Common respiratory pathogens, such as equine influenza virus (EIV), EHV-4, and equine rhinitis B virus (ERBV), were found at low but stable frequencies within previously reported ranges, whereas EHV-1 and Streptococcus equi subspecies equi ( S. equi ) were found at or above previously reported frequencies. Less characterized respiratory pathogens, such as EHV-2, EHV-5, and S. equi subspecies zooepidemicus ( S. zooepidemicus ), were found within previously reported ranges. Common respiratory pathogens, especially EHV-1 following the multiple EHM outbreaks, were found to be circulating in clinically healthy sport horse populations, reflecting their silent transmission. The strategy of quarantine and EHV-1 qPCR testing of clinically healthy horses was successful at eliminating additional EHM outbreaks and facilitating safe return to competition with no reported respiratory disease outbreaks following the subsequent shows in California.

In this study, we examined the total bacterial community associated with ureolytic biomineralization from urine drainage systems. Biomineral samples were obtained from 11 California Department of Transportation public restrooms fitted with waterless, low-flow, or conventional urinals in 2019. Following high throughput 16S rRNA Illumina sequences processed using the DADA2 pipeline, the microbial diversity assessment of 169 biomineral and urine samples resulted in 3,869 reference sequences aggregated as 598 operational taxonomic units (OTUs). Using PERMANOVA testing, we found strong, significant differences between biomineral samples grouped by intrasystem sampling location and urinal type. Biomineral microbial community profiles and alpha diversities differed significantly when controlling for sampling season. Observational statistics revealed that biomineral samples obtained from waterless urinals contained the largest ureC/16S gene copy ratios and were the least diverse urinal type in terms of Shannon indices. Waterless urinal biomineral samples were largely dominated by the Bacilli class (86.1%) compared to low-flow (41.3%) and conventional samples (20.5%), and had the fewest genera that account for less than 2.5% relative abundance per OTU. Our findings are useful for future microbial ecology studies of urine source-separation technologies, as we have established a comparative basis using a large sample size and study area., Competing Interests: The authors have declared that no competing interests exist.

Equine respiratory viruses remain a leading cause of equine morbidity and mortality, with the resurgence of certain infections, an increasing population of elderly, more susceptible horses, the growth of international equine commerce, and an expansion in geographic distribution of pathogens. The focus of rapid diagnosis of infectious diseases has also shifted recently, with the appearance and increasing importance of nucleic acid amplification-based techniques, primarily polymerase chain reaction (PCR), at the expense of traditional methods such as clinical microbiology. While PCR is fast, reliable, cost-effective, and more sensitive than conventional detection methods, careful interpretation of diagnostic test results is required, taking into account the clinical status of the patient, sample type, assay used and biological relevance of the detected viruses. The interpretation of common equine respiratory viruses such as influenza virus (EIV), alpha herpesviruses (EHV-1, EHV-4), arteritis virus (EAV) and rhinoviruses (ERAV, ERBV) is straight forward as causality can generally be established. However, the testing of less-characterized viruses, such as the gamma herpesviruses (EHV-2, EHV-5), may be confusing, considering their well-established host relationship and frequent detection in both diseased and healthy horses. For selected viruses, absolute quantitation (EHV-1 and EHV-4) and genotyping (EIV and EHV-1) has allowed additional information to be gained regarding viral state and virulence, respectively. This information is relevant when managing outbreaks so that adequate biosecurity measures can be instituted and medical interventions can be considered. The goal of this review is to help the equine practitioner navigate through the rapidly expanding field of molecular diagnostics for respiratory viruses and facilitate the interpretation of results., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Equine rhinitis B virus (ERBV) has been given little attention by practitioners compared to other respiratory viruses, mainly because of the lack of diagnostic modalities and association with clinical disease. The objective of the study was to determine the frequency of detection of ERBV in nasal secretions from 6568 horses with acute onset of respiratory signs., Methods: ERBV-positive qPCR results from nasal secretions submitted to a molecular diagnostic laboratory from 2013 to 2019 were reviewed., Results: A total of 333 ERBV qPCR-positive samples (5.1%) were detected with increasing yearly frequency since the introduction of the assay in 2013. In comparison, only three of 356 (0.8%) healthy horses tested qPCR-positive for ERBV. Median age for ERBV qPCR-positive horses was 3 years of age, and fever, coughing and nasal discharge were the most common signs reported. Further, co-infections with other respiratory pathogens were reported in 73 (21.9%) of ERBV qPCR-positive samples., Conclusion: ERBV is a commonly detected respiratory virus from nasal secretions of young horses presenting with fever, nasal discharge and coughing., (© 2021 British Veterinary Association.)

The best diagnostic test for cutaneous adverse food reactions (CAFR) in companion animals is an elimination diet and subsequent provocation trials. Many commercial diets contain novel protein ingredients used in elimination diets, and selection is based on label ingredients. Raw meat-based diets (RMBD) have become increasingly commercially available, gaining popularity despite potential health risks. Reliability of RMBD based on label ingredients has not been investigated. Using quantitative polymerase chain reaction (qPCR), 9 canine and 9 feline commercial RMBD were assessed for reliability of species-specific animal DNA. Two separate batches of each diet were assessed for content consistency. The DNA of 1 or more unlisted animal species was identified in > 60% of diets, as was discrepancy between batches. The unlisted DNA most frequently detected was lamb in canine diets and turkey in feline diets. Based on these findings, use of commercially available RMBD cannot be recommended as an elimination diet in clinical diagnosis of CAFR., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Given limited resources for managing invasive species, traditional survey methods may not be feasible to implement at a regional scale. Environmental DNA (eDNA) sampling has proven to be an effective method for detecting some invasive species, but comparisons between the detection probability of eDNA and traditional survey methods using modern occupancy modeling methods are rare. We developed a qPCR assay to detect two species of watersnake (Nerodia fasciata and Nerodia sipedon) introduced to California, USA, and we compared the efficacy of eDNA and aquatic trapping. We tested 3-9 water samples each from 30 sites near the known range of N. fasciata, and 61 sites near the known range of N. sipedon. We also deployed aquatic funnel traps at a subset of sites for each species. We detected N. fasciata eDNA in three of nine water samples from just one site, but captured N. fasciata in traps at three of ten sites. We detected N. sipedon eDNA in five of six water samples from one site, which was also the only site of nine at which this species was captured in traps. Traditional trapping surveys had a higher probability of detecting watersnakes than eDNA surveys, and both survey methods had higher detection probability for N. sipedon than N. fasciata. Occupancy models that integrated both trapping and eDNA surveys estimated that 5 sites (95% Credible Interval: 4-10) of 91 were occupied by watersnakes (both species combined), although snakes were only detected at four sites (three for N. fasciata, one for N. sipedon). Our study shows that despite the many successes of eDNA surveys, traditional sampling methods can have higher detection probability for some species. We recommend those tasked with managing species invasions explicitly compare eDNA and traditional survey methods in an occupancy framework to inform their choice of the best method for detecting nascent populations., Competing Interests: Two co-authors, JSW and SW, were employed by a commercial entity, Pisces Molecular, LLC, during their work on this study. The commercial affiliation of these two co-authors does not alter our adherence to PLOS ONE policies on sharing data and materials.