Moraxella oculi sp. nov., isolated from a cow with infectious bovine keratoconjunctivitis.

A novel species of the genus Moraxella was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was Moraxella bovis (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related Moraxella species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name Moraxella oculi sp. nov. for this new species. The type strain is Tifton1T and has been deposited into the American Type Culture Collection (TSD-373T) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942T).

SURVEILLANCE OF BATS IN THE UNITED STATES FOR SARS-COV-2 AND OTHER CORONAVIRUSES.

Bat coronaviruses (CoVs) are extremely prevalent throughout the globe and exhibit a wide range of genetic diversity. Currently, little is known about the susceptibility of New World bats to severe acute respiratory syndrome-2 (SARS-CoV-2), the causative agent of COVID-19. Also, there is limited information about the genetic diversity of other CoVs in the New World bats. The determination of genetic diversity of bat CoVs through continuous surveillance is essential to predict and mitigate the emergence of new CoVs and their impacts on the health of both humans and animals. In this study, 491 guano specimens collected from New World bats and 37 specimens collected from Old World bats during July 2020 to July 2021 were tested for SARS-COV-2 and other CoVs using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) panel and pan-coronavirus PCR that target a highly conserved region of CoVs. No evidence of SARS-CoV-2 was found in the tested specimens. An alpha CoV was detected in a single specimen from a big brown bat (Eptesicus fuscus). This information was used by wildlife agencies and rehabilitation facilities to permit the release of bats during the pandemic while mitigating the risk of spreading SARS-CoV-2 among North American bats and other wild animal populations.

Two stage, nested isothermal amplification in a single tube.

Sensitive, specific and rapid molecular diagnosis of respiratory diseases in animals and humans is critical to facilitate appropriate control measures and treatment. Conventional polymerase chain reaction (PCR)-based molecular diagnostics requires relatively expensive equipment and trained staff, restricting its use to centralized laboratories with significant delays between sample collection and test results. Herein, we report a highly sensitive, rapid, point-of-need, two-stage-molecular test that requires minimal instrumentation and training. Our test, dubbed Penn-RAMP, combines recombinase polymerase amplification (RPA, 38 °C) and loop-mediated isothermal amplification (LAMP, 63 °C) in one tube, enabling nested, two-stage isothermal amplification. We demonstrate Penn-RAMP's efficacy by testing for two common viral respiratory diseases of chickens: infectious laryngotracheitis (ILT) and infectious bronchitis (IB) that impose great economic burden worldwide. Test results of clinical samples with our closed-tube Penn-RAMP assays concord with the gold standard quantitative PCR (qPCR) assay; with 10-fold better limit of detection than LAMP and qPCR. Our closed-tube Penn-RAMP assays have the potential to greatly reduce false negatives while requiring minimal instrumentation and training.

Natural Canine Distemper Virus Infection in Linnaeus's 2-Toed Sloths (Choloepus didactylus).

An outbreak of canine distemper virus in a private zoo in eastern Tennessee in July 2016 led to fatal clinical disease in 5 adult, wild-caught Linnaeus's 2-toed sloths (Choloepus didactylus). Clinical signs included hyporexia, lethargy, mucopurulent nasal discharge, and oral and facial ulcers. At necropsy, affected animals had crusts and ulcers on the lips, nose, tongue, and oral cavity. Microscopically, all sloths had widespread, random, hepatic necrosis; lymphoid depletion; and bronchointerstitial pneumonia. The central nervous system did not contain gross or histopathologic lesions in any of the 5 sloths, although immunoreactivity for viral antigen was present within vessel walls. Epithelial cells and histiocytes within numerous organs contained intranuclear and intracytoplasmic inclusions and occasional syncytial cells. Canine distemper virus was confirmed with immunohistochemistry and virus isolation. Viral sequencing identified the novel American-4 strain prevalent in eastern Tennessee wildlife. This is the first pathologic characterization of canine distemper virus infection in sloths (family Choloepodidae, order Pilosa) and emphasizes the significant morbidity and mortality in this species.

GENETIC CHARACTERISTICS OF CANINE DISTEMPER VIRUSES CIRCULATING IN WILDLIFE IN THE UNITED STATES.

Canine distemper virus (CDV) is a highly contagious disease of wild and domestic mammals. Maintenance of CDV among wildlife plays an important role in the disease epidemiology. Wild animals, including raccoons (Procyon lotor) and gray foxes (Urocyon cinereoargenteus), serve as reservoirs of CDV and hamper the control of the disease. Recently, we discovered that at least three different CDV lineages (America-3 [Edomex], America-4, and America-5] that are genetically different from the available vaccine strains are circulating in domestic dogs in the United States. Because wildlife serve as a reservoir for the virus, it is important to determine if wildlife play a role in the maintenance and spread of these lineages. To determine the genetic characteristics of circulating strains of CDV in wildlife in various geographic regions in the United States, we studied the nucleotide sequences of the hemagglutinin (H) gene of 25 CDV strains detected in nondomestic species. The species included were free-ranging wildlife: three fishers (Martes pennanti), six foxes, one skunk (Mephitis mephitis), 10 raccoons, two wolves (Canis lupus), and one mink (Neovison vison). Strains from two species in managed care, one sloth (Choloepus didactylus) and one red panda (Ailurus fulgens), were also evaluated. Phylogenetic analysis of the H genes indicated that in addition to America-3, America-4, and America-5 lineages, there are at least two other lineages circulating in US wildlife. One of these includes CDV nucleotide sequences that grouped with that of a single CDV isolate previously detected in a raccoon from Rhode Island in 2012. The other lineage is independent and genetically distinct from other CDV strains included in the analysis. Additional genetically variable strains were detected, mainly in raccoons, suggesting that this species may be the host responsible for the genetic variability of newly detected strains in the domestic dog population.

SAFETY OF AND HUMORAL IMMUNE RESPONSE TO THE MERIAL RECOMBITEK CANINE DISTEMPER VIRUS VACCINE IN MANED WOLVES (CHRYSOCYON BRACHYURUS).

This study evaluated the safety of and humoral response to the Merial Recombitek® recombinant canine distemper virus (rCDV) vaccine in maned wolves (n = 9, age 2-9 yr). All maned wolves had prior history of annual vaccination with the Merial Purevax® ferret rCDV vaccine. Serum neutralization (SN) to CDV was measured prior to initial vaccination with the rCDV Recombitek vaccine followed by a booster vaccination at 4-6 wk. Final SN titers were obtained at 13 wk post initial vaccination. The maned wolves developed no observable adverse side effects through the study. Pre-Recombitek vaccination SN titers ranged from negative to 1: 8. Postvaccination CDV titers ranged from negative to 1: 8, and were therefore below the range of that considered protective in domestic dogs.

TIGER (PANTHERA TIGRIS) AND DOMESTIC CAT (FELIS CATUS) IMMUNE RESPONSES TO CANARYPOX-VECTORED CANINE DISTEMPER VACCINATION.

Two methods for delivering a canarypox-vectored canine distemper vaccine to tigers (Panthera tigris) and domestic cats (Felis catus) were investigated. Eight tigers were divided randomly into two vaccination groups: subcutaneous injection or topical tonsillar application. Each tiger received 2 ml of canine distemper virus (CDV) vaccine (Merial Ferret Distemper Vaccine). Blood was collected from tigers on days 0, 21, 35 or 37, and 112 post-initial vaccination (PIV). Domestic cats were divided randomly into four treatment groups: saline injection (negative controls), low- and high-dose oral, and subcutaneous vaccinates. Blood was collected from domestic cats on days 0, 7, 21, and 28 and 165 or 208 PIV. Sera were tested for CDV antibodies by virus neutralization. All individuals were seronegative at the beginning of the study. One tiger vaccinated subcutaneously developed a titer of 32 by day 35, which reduced to 16 by day 112. Another tiger vaccinated by tonsillar application developed a titer of 8 on day 112. All other tigers remained seronegative. Cats that received saline injection or oral vaccination remained seronegative at each sampling time. Domestic cats vaccinated subcutaneously developed titers ranging from 4 to >128 by day 28, and those re-bled at day 166 had titers of 16 or 64. The disparity in response between domestic cats and tigers may be due to species differences or it may represent a dose-dependent effect. Subcutaneous vaccination with canarypox-vectored Purevax Ferret Distemper® is safe and elicits persistent antibody titers in domestic cats vaccinated parenterally.

RED PANDAS' (AILURUS FULGENS) SEROLOGICAL RESPONSE TO CANARYPOX-VECTORED CANINE DISTEMPER VACCINES.

Red pandas (Ailurus fulgens) are susceptible to canine distemper, with a number of reported vaccine-induced canine distemper cases. Canarypox-vectored recombinant canine distemper vaccines (PureVax Ferret Distemper [PFD] and Recombitek CDV [rCDV]) provide protection without inoculating a live distemper virus, but there are currently no published data regarding these vaccines' safety and efficacy in red pandas. One hundred twenty-two serum samples were collected from 50 captive red pandas and analyzed for antibodies to canine distemper. All naïve red pandas (n = 20) had negative titers. Naïve pandas receiving two PFD vaccinations had either negative or intermediate titers (n = 4). In contrast, naïve pandas receiving a series of two or three rCDV vaccinations (n = 14) had greater antibody responses. Red pandas vaccinated with PFD >12 mo since their last vaccination and a rCDV booster vaccination showed the highest titers observed. We recommend red pandas be administered a series of at least three recombinant vaccine (PDF or rDCV) vaccinations, followed by annual booster vaccinations.

Evaluation of Targeted Next-Generation Sequencing for Detection of Bovine Pathogens in Clinical Samples.

The laboratory diagnosis of infectious diseases, especially those caused by mixed infections, is challenging. Routinely, it requires submission of multiple samples to separate laboratories. Advances in next-generation sequencing (NGS) have provided the opportunity for development of a comprehensive method to identify infectious agents. This study describes the use of target-specific primers for PCR-mediated amplification with the NGS technology in which pathogen genomic regions of interest are enriched and selectively sequenced from clinical samples. In the study, 198 primers were designed to target 43 common bovine and small-ruminant bacterial, fungal, viral, and parasitic pathogens, and a bioinformatics tool was specifically constructed for the detection of targeted pathogens. The primers were confirmed to detect the intended pathogens by testing reference strains and isolates. The method was then validated using 60 clinical samples (including tissues, feces, and milk) that were also tested with other routine diagnostic techniques. The detection limits of the targeted NGS method were evaluated using 10 representative pathogens that were also tested by quantitative PCR (qPCR), and the NGS method was able to detect the organisms from samples with qPCR threshold cycle (CT ) values in the 30s. The method was successful for the detection of multiple pathogens in the clinical samples, including some additional pathogens missed by the routine techniques because the specific tests needed for the particular organisms were not performed. The results demonstrate the feasibility of the approach and indicate that it is possible to incorporate NGS as a diagnostic tool in a cost-effective manner into a veterinary diagnostic laboratory.

Phylogenetic analysis of the wild-type strains of canine distemper virus circulating in the United States.

BACKGROUND: Canine distemper (CD) is a highly contagious, systemic, viral disease of dogs seen worldwide. Despite intensive vaccination in developed countries, recent reports suggest both the re-emergence and increased activity of Canine distemper virus (CDV) worldwide, including the United States. CDV is an RNA virus of the genus Morbillivirus within the family Paramyxoviridae. Viral genomic RNA encodes six structural proteins. Of the six structural proteins, the hemagglutinin (H) gene has the greatest genetic variation and is therefore a suitable target for molecular epidemiological studies. The majority of neutralizing epitopes are found on the H protein, making this gene also important for evaluation of changes over time that may result in antigenic differences among strains. The aim of this study was to determine the phylogenetic relationship of CDV strains circulating in the US. METHODS: Fifty-nine positive canine distemper virus samples collected from dogs from different regions and states from 2014 to 2017 were sequenced with a targeted next-generation sequencing (NGS) method. The sequences of the H, F, and P genes and the matrix-fusion (M-F) intergenic region of the amplified CDVs were analyzed individually. RESULTS: Sequence analysis of the H gene revealed that there are at least 3 different lineages of CDV currently circulating in the US. These lineages include America-3 (Edomex), America-4, and a clade that was previously reported in association with an outbreak in Wyoming, which was linked to a domestic dog-breeding facility in Kansas in 2010. These lineages differ from the historically identified lineages in the US, including America-1, which contains the majority of the vaccine strains. Genetic differences may result in significant changes to the neutralizing epitopes that consequently may lead to vaccine failure. Phylogenetic analyses of the nucleotide sequences obtained in this study of the F and P genes and the M-F intergenic region with sequences from the GenBank database produced similar findings to the H gene analysis. CONCLUSIONS: The CDV lineages currently circulating in the US differ from the historically identified lineages America-1. Continuous surveillance is required for monitoring circulating CDV strains in the US, to prevent potential vaccine breakthrough events.

SEROLOGIC RESPONSE TO CANINE DISTEMPER VACCINATION IN CAPTIVE LINNAEUS'S TWO-TOED SLOTHS ( CHOLOEPUS DIDACTYLUS) AFTER A FATAL CANINE DISTEMPER VIRUS OUTBREAK.

Canine distemper virus (CDV) affects many wild and captive, nondomestic species worldwide but has not been previously reported in Xenarthra. Paucity of information on vaccination safety and efficacy presents challenges for disease prevention in captive collections. CDV infections and subsequent mortalities in five captive Linnaeus's two-toed sloths ( Choloepus didactylus) in eastern Tennessee are reported. Clinical signs included oculonasal discharge, oral ulcerations, and diarrhea, and the diagnosis was confirmed by necropsy, histopathology, immunohistochemistry, virus isolation, and polymerase chain reaction. Viral sequencing identified the strain to be consistent with a new CDV lineage currently affecting domestic dogs and wildlife in Tennessee. Seven sloths were examined and vaccinated using a recombinant CDV vaccine on days 0 and 21. Subsequent blood samples showed increased titers in 3/4 sloths. Based on the outbreak and serologic findings postvaccination without adverse effects, the authors recommend recombinant CDV vaccination in sloths exposed to known carriers of CDV.

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs.

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

Metagenomics for Pathogen Detection During a Mass Mortality Event in Songbirds.

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria forming the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes, including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events.

Investigation of the pathogens contributing to naturally occurring outbreaks of infectious bovine keratoconjunctivitis (pinkeye) using Next Generation Sequencing.

Infectious bovine keratoconjunctivitis (IBK), commonly known as pinkeye, has a marked negative impact on the economy of the cattle industry. Moraxella species, including Mor. bovis and Mor. bovoculi, which have been associated with this disease, colonize clinically healthy eyes as well, suggesting that there are intrinsic changes that may occur to the ocular microbiota or the involvement of additional unrecognized organisms that contribute to IBK. To evaluate this, 104 ocular swabs collected from eyes with IBK or clinically healthy eyes from 16 different cattle herds were subjected to 16 S rRNA gene PCR and next generation sequencing (NGS) analysis. Organisms detected were similar across the herds and there was no difference in the total number of bacterial groups detected among IBK cases and controls. However, the percentages of the different organisms detected varied between the two groups, including Moraxella spp., with more Moraxella spp. in eyes with IBK than controls. Further, using culture and whole genome NGS, a new species of Moraxella (suggested name Mor. oculobovii) was detected from the eyes of cattle from two farms. This strain is non-hemolytic on blood agar, is missing the RTX operon, and is likely a non-pathogenic strain of the bovine ocular microbiome. Alteration of the ocular microbiota composition may have a predisposing role, enhancing bacterial infection and the occurrence of clinical IBK. Future studies are required to evaluate if these changes are permanent or if there is a shift in the microbiome following recovery from the infection and how antibiotics might affect the microbiome.

Transfer of naturally acquired specific passive immunity against Anaplasma phagocytophilum in foals in Southeastern Pennsylvania and Northern Maryland.

BACKGROUND: Equine granulocytic anaplasmosis (EGA) is a common disease in adult horses, but clinical disease in foals is rarely reported. The relationship between equine maternal and neonatal antibodies to Anaplasma phagocytophilum is unclear. HYPOTHESIS/OBJECTIVES: That mares in an endemic region would be seropositive for A. phagocytophilum and that mare and foal serum IgG concentrations for A. phagocytophilum would correlate. Additionally, we hypothesized that foal IgG concentrations for A. phagocytophilum acquired by passive immunity would decline by 6 months of age. ANIMALS: Twenty-two healthy mare-foal pairs. METHODS: This prospective observational study investigated serum IgG concentrations specific for A. phagocytophilum in mares and foals using an immunofluorescent antibody test (IFA). The association between foal titer (as a binary variable) and age in months was assessed using a mixed-effects logistic regression. RESULTS: A positive correlation between newborn foal antibody titers and mare titers was identified at both the pre-foaling (τa = 0.38, τb = 0.50, P = .009) and foaling timepoints (τa = 0.36, τb = 0.47, P = .01). In A. phagocytophilum seropositive neonates, it was unlikely that a positive titer would be detected by 3 months of age (OR = 0.002, P = .02, 95% CI: 0.00001-0.38). Three out of 20 foals seroconverted between 3 and 6 months of age. CONCLUSIONS AND CLINICAL IMPORTANCE: Transfer of specific passive immunity to A. phagocytophilum occurred in 80% of foals born to seropositive mares and declined by 3 months of age. A. phagocytophilum infection should be considered in foals displaying clinical signs consistent with EGA.

Bat Red Blood Cells Express Nucleic Acid-Sensing Receptors and Bind RNA and DNA.

RBCs demonstrate immunomodulatory capabilities through the expression of nucleic acid sensors. However, little is known about bat RBCs, and no studies have examined the immune function of bat erythrocytes. In this study, we show that bat RBCs express the nucleic acid-sensing TLRs TLR7 and TLR9 and bind the nucleic acid ligands, ssRNA, and CpG DNA. Collectively, these data suggest that, like human RBCs, bat erythrocytes possess immune function and may be reservoirs for nucleic acids. These findings provide unique insight into bat immunity and may uncover potential mechanisms by which virulent pathogens of humans are concealed in bats.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

Detection of human papillomavirus DNA in feline premalignant and invasive squamous cell carcinoma.

Squamous cell carcinoma (SCC) is the most common malignant cutaneous and oral neoplasm of cats. Papillomavirus (PV) DNA has been identified in a proportion of feline Bowenoid in situ carcinomas (BISCs), cutaneous SCCs and a single oral SCC, but its exact role in the pathogenesis remains unknown. In humans, it has been suggested that ultraviolet (UV) light and human PV (HPV) may act as cofactors in cutaneous SCC carcinogenesis. Little is known about the influence of UV light on PV prevalence in feline cutaneous lesions, including actinic keratosis (AK). Additionally, PV prevalence in noncutaneous feline lesions, including oral SCC, is largely not known. This study aimed to determine the presence of PV in 84 cats with premalignant and invasive SCC from cutaneous and noncutaneous sites using polymerase chain reaction and to investigate an association with UV light. Papillomaviral DNA was amplified from two of 12 cases of AK, seven of 22 BISCs, nine of 39 cutaneous SCCs and two of 35 non-cutaneous SCCs. Of the PV DNA sequenced, 50% was most similar to HPV of the genus Betapapillomavirus, while the other 50% was most similar to Felis domesticus PV type 2. Exposure to UV was not associated with an increase in PV for cutaneous SCC. The results of this study suggest that in the cat, HPV DNA may be detectible within a higher percentage of squamous lesions than previously demonstrated, UV exposure may not be a confounder for PV presence, and noncutaneous lesions may have a low prevalence of PV.

Performance of commercial PCR assays to detect toxigenic Clostridioides difficile in the feces of puppies.

Clostridioides difficile is an important enteric pathogen that causes significant morbidity and mortality in humans. With community-acquired infections on the rise, it is important to identify reservoirs of the pathogen. Companion animals can be asymptomatic carriers of C. difficile and may therefore represent a reservoir, but epidemiological studies of C. difficile within the pet-owner unit are needed, along with validated methods to detect C. difficile in both people and animals. The goal of this study was to assess the performance of commercial qPCR assays and a multiplex PCR for C. difficile compared to toxigenic culture. These assays were tested on up to 103 fecal samples from puppies, a population in which the prevalence of C. difficile is the highest. The sensitivities, specificities, positive predictive values and negative predictive values were respectively 84.2%, 87.7%, 61.5%, and 95.9% for the Cepheid GeneXpert; 66.7%, 66.7%, 29.6%, and 90.9% for the DiaSorin Simplexa; and 94.4%, 85.0%, 65.4%, and 98.1%, for the multiplex qPCR. The agreement was highest between the GeneXpert and the multiplex PCR (90.1% agreement, with a kappa statistic of 0.77). For diagnostic purposes, the positive predictive values of the assays were low. However, the high sensitivities of the assays could render them useful for epidemiologic purposes.

Molecular Detection of Infectious Laryngotracheitis Virus in Chickens with a Microfluidic Chip.

Infectious laryngotracheitis (ILT) is a viral disease of chickens' respiratory system that imposes considerable financial burdens on the chicken industry. Rapid, simple, and specific detection of this virus is crucial to enable proper control measures. Polymerase chain reaction (PCR)-based molecular tests require relatively expensive instruments and skilled personnel, confining their application to centralized laboratories. To enable chicken farms to take timely action and contain the spread of infection, we describe a rapid, simple, semi-quantitative benchtop isothermal amplification (LAMP) assay, and a field-deployable microfluidic device for the diagnosis of ILTV infection in chickens. Our assay performance was compared and favorably agreed with quantitative PCR (qPCR). The sensitivity of our real-time LAMP test is 250 genomic copies/reaction. Clinical performance of our microfluidic device using samples from diseased chickens showed 100% specificity and 100% sensitivity in comparison with benchtop LAMP assay and the gold-standard qPCR. Our method facilitates simple, specific, and rapid molecular ILTV detection in low-resource veterinary diagnostic laboratories and can be used for field molecular diagnosis of suspected ILT cases.