Anil J. Thachil

Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies

Porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was first detected in North America in early 2014 and associated with enteric disease in pigs, resulting in an urgent need to further investigate the ecology of this virus. While assays detecting nucleic acids were implemented quickly, assays to detect anti-PDCoV antibodies have not been available. In this study, an indirect anti-PDCoV IgG enzyme-linked immunosorbent assay (ELISA) based on the putative S1 portion of the spike protein was developed and utilized to determine the prevalence of anti-PDCoV IgG in U.S. pigs. The diagnostic sensitivity of the PDCoV ELISA was 91% with a diagnostic specificity of 95%. A total of 968 serum samples were tested including samples with confirmed infection with PDCoV, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus or porcine respiratory coronavirus. There was no cross-reactivity with any of the other coronaviruses. Among 355 arbitrarily selected serum samples collected in 2014 and originating from 51 farms across 18 U.S. states, anti-PDCoV IgG antibodies were detected in 8.7% of the samples and in 25.5% of the farms whereas anti-PEDV IgG was detected in 22.8% of the samples and in 54.9% of the farms. In addition, anti-PDCoV IgG antibodies were detected in archived samples collected in 2010, perhaps indicating an earlier undetected introduction into the U.S. pig population. Overall, the obtained data suggest that PDCoV seroprevalence in U.S. pigs is lower compared to PEDV and PDCoV may have been introduced to the U.S. prior to PEDV.

Role of Clostridium perfringens and Clostridium septicum in Causing Turkey Cellulitis

Abstract The role of Clostridium perfringens and Clostridium septicum in the development of cellulitis and mortality in turkey poults was examined. Studies were done in turkeys of two age groups: 3-wk-old and 7-wk-old turkey poults. The effect of varying doses of C. perfringens and C. septicum in reproducing cellulitis lesions and mortality in turkeys was investigated. Both in vitro and in vivo assays were conducted to study their toxic and biologic activities. Clostridium septicum spore culture was found to be more potent than that of C. perfringens in both in vitro assays, such as the hemolysis test, and in vivo assays in mice and turkeys. Both C. perfringens and C. septicum spore cultures were found to be capable of inducing cellulitis lesions and mortality in turkey poults when inoculated by subcutaneous route. Histopathology examination of affected tissues revealed a “moth-eaten appearance,” with abundant growth of C. perfringens and C. septicum in the sarcomeres of muscle tissues and in the subcutaneous tissues. However, C. septicum was found to be more potent than C. perfringens in causing cellulitis lesions and mortality in turkeys. Three-week-old poults were found to be less susceptible than 7-wk-old poults in the development of cellulitis lesions and mortality after inoculation with either spore cultures of C. perfringens or C. septicum. The results of the current study suggest that although C. septicum is more potent in causing cellulitis lesions and mortality, infection with either C. septicum or C. perfringens can cause cellulitis lesions and mortality in turkeys.

Human Metapneumovirus in Turkey Poults

TOC summary: Human metapneumovirus causes clinical signs in turkey poults.

Temporal Genomic Phylogeny Reconstruction Indicates a Geospatial Transmission Path of Salmonella Cerro in the United States and a Clade-Specific Loss of Hydrogen Sulfide Production.

Salmonella Cerro has become one of the most prevalent Salmonella serotypes isolated from dairy cattle in several U.S. states, including New York where it represented 36% of all Salmonella isolates of bovine origin in 2015. This serotype is commonly isolated from dairy cattle with clinical signs of salmonellosis, including diarrhea and fever, although it has also been identified in herds without evidence of clinical disease or decreased production. To better understand the transmission patterns and drivers of its geographic spread, we have studied the genomic similarity and microevolution of S. Cerro isolates from the northeast U.S. and Texas. Eighty-three out of 86 isolates were confirmed as multilocus sequence type 367. We identified core genome SNPs in 57 upstate New York (NY), 2 Pennsylvania (PA), and 27 Texas S. Cerro isolates from dairy cattle, farm environments, raw milk, and one human clinical case and used them to construct a tip-dated phylogeny. S. Cerro isolates clustered in three distinct clades, including (i) clade I (n = 3; 2013) comprising isolates from northwest Texas (NTX), (ii) clade II (n = 14; 2009–2011, 2014) comprising isolates from NY, and (iii) clade III comprising isolates from NY, PA, and central Texas (CTX) in subclade IIIa (n = 45; 2008–2014), and only CTX isolates in subclade IIIb (n = 24; 2013). Temporal phylogenetic analysis estimated the divergence of these three clades from the most recent common ancestor in approximately 1980. The CTX clade IIIb was estimated to have evolved and diverged from the NY ancestor around 2004. Furthermore, gradual temporal loss of genes encoding a D-alanine transporter, involved in virulence, was observed. These genes were present in the isolates endemic to NTX clade I and were gradually lost in clades II and III. The virulence gene orgA, which is part of the Salmonella Pathogenicity Island 1, was lost in a subgroup of Texas isolates in clades I and IIIb. All S. Cerro isolates had an additional cytosine inserted in a cytosine-rich region of the virulence gene sopA, resulting in premature termination of translation likely responsible for loss of pathogenic capacity in humans. A group of closely related NY isolates was characterized by the loss of hydrogen sulfide production due to the truncation or complete loss of phsA. Our data suggest the ability of Salmonella to rapidly diverge and adapt to specific niches (e.g., bovine niche), and to modify virulence-related characteristics such as the ability to utilize tetrathionate as an alternative electron acceptor, which is commonly used to detect Salmonella. Overall, our results show that clinical outcome data and genetic data for S. Cerro isolates, such as truncations in virulence genes leading to novel pheno- and pathotypes, should be correlated to allow for accurate risk assessment.

Application of Polymerase Chain Reaction Fingerprinting to Differentiate Ornithobacterium Rhinotracheale Isolates

Ornithobacterium rhinotracheale (ORT) is an infectious respiratory pathogen of chickens, turkeys, and wild birds. There are 18 serotypes of ORT reported worldwide. In this study, enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction and random amplified polymorphic DNA assay with Universal M13 primer-based fingerprinting techniques were investigated for their ability to differentiate ORT isolates. The authors examined 50 field isolates and 8 reference strains of ORT for their genetic differences. The fingerprint patterns were compared with serotyping results of ORT by the agar gel precipitation test. M13 fingerprinting revealed different patterns for 6 reference serotypes of ORT that were tested, namely, C, D, E, I, J, and K. Ornithobacterium rhinotracheale reference serotypes A and F yielded indistinguishable fingerprints with M13 fingerprinting. The ERIC 1R technique discerned only 5 of the 8 reference serotypes of ORT. Distinct fingerprints were also found within the ORT serotypes with both techniques. From 58 isolates of ORT that were fingerprinted belonging to 8 ORT serotypes, 10 different fingerprints were obtained with M13 fingerprinting and 6 different fingerprints were obtained with ERIC 1R fingerprinting. M13 fingerprinting technique was found to be more discriminative in differentiating ORT isolates than the ERIC 1R fingerprinting technique. These results suggest that fingerprinting techniques may be a more discerning tool for characterizing ORT isolates than the serological test using the agar gel precipitation test. This fingerprinting technique could potentially be a valuable tool in identifying an isolate from a clinical outbreak of ORT infection for development of an autogenous vaccine.

Effects of Dexamethasone Immunosuppression on Turkey Clostridial Dermatitis

SUMMARY Clostridia represents a group of anaerobic spore-forming bacteria ubiquitous in the poultry environment. They are widely distributed in soil and survive for many years as highly resistant, inactive spores. They enter the body through wounds and contaminated feed as active bacteria or spores. Multiplication of clostridial bacteria occurs only in the absence of oxygen or in environments with very low concentrations of oxygen. During active multiplication, the clostridial organisms produce several toxins that are responsible for most of the clinical signs seen in clostridial diseases. Immunosuppression is a problem for the poultry industry. In modern, intensive poultry-rearing conditions, stress due to high population densities pose a considerable challenge for the immune system, and infectious agents can exploit this situation to cause disease. Immunosuppression may predispose turkeys to clostridial infection, resulting in clostridial dermatitis and mortality. The purpose of this study was to determine whether immunosuppression predisposes turkeys to clostridial infection and causes clostridial dermatitis. We immunosuppressed 10-wk-old turkey poults with dexamethasone. The birds immunosuppressed and not immunosuppressed were then challenged with Clostridium perfringens, Clostridium septicum, or both and examined for the development of clostridial dermatitis. The dexamethasone-treated birds were found to be more susceptible to C. perfringens/C. septicum challenge and developed clostridial dermatitis than the no-dexamethasone-treated birds through the subcutaneous route. However, oral inoculation of the same agents did not cause any dermatitis lesions in either of the groups. RESUMEN Efectos de la inmunodepresión por dexametasona en la dermatitis clostridial de los pavos. Las bacterias del género Clostridium representan un grupo de bacterias anaerobias que forman esporas y que son ubicuas en los ambientes avícolas. Están ampliamente distribuidas en el suelo y sobreviven durante muchos años como esporas inactivas, altamente resistentes. Entran en el cuerpo a través de heridas y alimentos contaminados en forma de bacterias activas o esporas. La multiplicación de las bacterias clostridiales sólo se produce en ausencia de oxígeno o en ambientes con concentraciones muy bajas del mismo. Durante la multiplicación activa, los organismos clostridiales producen varias toxinas que son responsables de la mayoría de los signos clínicos observados en las enfermedades clostridiales. La inmunosupresión es un problema para la industria avícola. Bajo las condiciones de crianza intensiva modernas, el estrés debido a la alta densidad de población plantea un reto considerable para el sistema inmune y los agentes infecciosos pueden aprovechar esta situación para causar enfermedades. La inmunodepresión puede predisponer a los pavos a la infección por clostridios y causar dermatitis clostridial y mortalidad. El propósito de este estudio fue determinar si la inmunosupresión predispone a los pavos a la infección por clostridios y si es causa dermatitis por clostridios. Se inmunodeprimieron pavipollos de 10 semanas de edad con dexametasona. Posteriormente, aves inmunodeprimidas y normales fueron expuestas a Clostridium perfringens, a Clostridium septicum, o a ambos y se examinaron para el desarrollo de la dermatitis clostridial. Se encontró que las aves tratadas con dexametasona eran más susceptibles al desafío por C. perfringens/C. septicum y desarrollaron dermatitis clostridial a diferencia de las aves que no fueron tratadas con dexametasona por vía subcutánea. Sin embargo, la inoculación oral de los mismos agentes no causó lesiones de dermatitis en ninguno de los grupos.

Vaccination of Turkeys with Clostridium septicum Bacterin-Toxoid: Evaluation of Protection Against Clostridial Dermatitis

SUMMARY Clostridial dermatitis is an acute disease causing high mortality in turkeys. Both Clostridium septicum and Clostridium perfringens have been isolated from these cases; however, reports from several diagnostic laboratories indicate an increased isolation rate of C. septicum compared with C. perfringens from cases of clostridial dermatitis in recent years. Previous studies suggested C. septicum was more potent than C. perfringens in causing clostridial dermatitis in turkeys. The objective of this study was to develop and evaluate the use of a C. septicum bacterin-toxoid to control clostridial dermatitis in turkeys. A C. septicum bacterin-toxoid was prepared and was initially tested in 6-wk-old commercial turkeys under laboratory conditions for its safety and efficacy. Subsequently, the bacterin-toxoid was evaluated for use in commercial turkey farms with a consistent history of clostridial dermatitis. Birds in the field were vaccinated subcutaneously once at 6 wk of age with C. septicum bacterin-toxoid, and then mortality in both vaccinated and unvaccinated groups was recorded and compared. Blood samples from birds in both groups were examined using ELISA to detect antibody response to the C. septicum toxoid. The C. septicum bacterin-toxoid was found to be safe and to elicit antibodies against the toxoid. In vaccinated commercial turkeys, control of clostridial dermatitis was achieved via antibiotic use and clostridial dermatitis mortality was significantly reduced compared with that of birds in the unvaccinated group. The C. septicum bacterin-toxoid seems to be a valuable tool for the turkey industry to reduce losses due to clostridial dermatitis. RESUMEN Vacunación de pavos con una bacterina-toxoide de Clostridium septicum: Evaluación de la protección contra la dermatitis clostridial. La dermatitis clostridial es una enfermedad aguda que causa una alta mortalidad en pavos. Tanto Clostridium septicum como Clostridium perfringens se han aislado de estos casos. Sin embargo en los últimos años, informes de varios laboratorios de diagnóstico indican un aumento de la tasa de aislamiento de C. septicum en comparación con C. perfringens a partir de casos de dermatitis clostridial. Los estudios previos han sugerido que C. septicum era más potente que C. perfringens para causar dermatitis clostridial en pavos. El objetivo de este estudio fue desarrollar y evaluar el uso de una bacterina-toxoide de C septicum para controlar la dermatitis clostridial en los pavos. La bacterina-toxoide contra C. septicum se preparó y fue probada inicialmente en pavos comerciales de seis semanas de edad, bajo condiciones de laboratorio, para determinar su seguridad y eficacia. Posteriormente, la bacterina-toxoide se evaluó para su uso en granjas de pavos comerciales con un historial de dermatitis clostridial constante. Las aves en el campo fueron vacunadas por vía subcutánea una vez a las seis semanas de edad con la bacterina-toxoide de C. septicum bacterina y se registró y comparó la mortalidad en los grupos vacunados y no vacunados. Las muestras de sangre de aves en ambos grupos se examinaron usando una prueba de ELISA para detectar la respuesta de anticuerpos contra el toxoide de C. septicum. Se determinó que la bacterina-toxoide de C. septicum era segura y que inducía anticuerpos contra el toxoide. En pavos comerciales vacunados, el control de la dermatitis clostridial se logró mediante el uso de antibióticos y se redujo significativamente la mortalidad por dermatitis clostridial en comparación con las aves del grupo no vacunado. La bacterina-toxoide contra C. septicum parece ser una herramienta valiosa para la industria de los pavos para reducir las pérdidas debidas a la dermatitis clostridial.

Discrimination of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry:

Accurate and timely identification of infectious etiologies is of great significance in veterinary microbiology, especially for critical diseases such as strangles, a highly contagious disease of horses caused by Streptococcus equi subsp. equi. We evaluated a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform for use in species- and subspecies-level identification of S. equi isolates from horses and compared it with an automated biochemical system. We used 25 clinical isolates each of S. equi subsp. equi and S. equi subsp. zooepidemicus. Using the MALDI-TOF MS platform, it was possible to correctly identify all 50 isolates to the species level. Unique mass peaks were identified in the bacterial peptide mass spectra generated by MALDI-TOF MS, which can be used for accurate subspecies-level identification of S. equi. Mass peaks (mass/charge, m/z) 6,751.9 ± 1.4 (mean ± standard deviation) and 5,958.1 ± 1.3 were found to be unique to S. equi subsp. equi and S. equi subsp. zooepidemicus, respectively. The automated biochemical system correctly identified 47 of 50 of the isolates to the species level as S. equi, whereas at the subspecies level, 24 of 25 S. equi subsp. equi isolates and 22 of 25 S. equi subsp. zooepidemicus isolates were correctly identified. Our results indicate that MALDI-TOF MS can be used for accurate species- and subspecies-level identification of S. equi.

Detection of Salmonella spp. in veterinary samples by combining selective enrichment and real-time PCR

Rapid screening for enteric bacterial pathogens in clinical environments is essential for biosecurity. Salmonella found in veterinary hospitals, particularly Salmonella enterica serovar Dublin, can pose unique challenges for culture and testing because of its poor growth. Multiple Salmonella serovars including Dublin are emerging threats to public health given increasing prevalence and antimicrobial resistance. We adapted an automated food testing method to veterinary samples and evaluated the performance of the method in a variety of matrices including environmental samples (n = 81), tissues (n = 52), feces (n = 148), and feed (n = 29). A commercial kit was chosen as the basis for this approach in view of extensive performance characterizations published by multiple independent organizations. A workflow was established for efficiently and accurately testing veterinary matrices and environmental samples by use of real-time PCR after selective enrichment in Rappaport–Vassiliadis soya (RVS) medium. Using this method, the detection limit for S. Dublin improved by 100-fold over subculture on selective agars (eosin–methylene blue, brilliant green, and xylose–lysine–deoxycholate). Overall, the procedure was effective in detecting Salmonella spp. and provided next-day results.

Leptospirosis in Urban and Suburban American Black Bears in Western North Carolina

Abstract: American black bear (Ursus americanus) populations in North Carolina, US have recovered significantly in recent decades and now occupy much of western North Carolina, including urbansuburban areas. We used the black bear as a potential sentinel for leptospirosis, a bacterial zoonotic disease caused by Leptospira spp., which is maintained by domestic and wild mammals. We determined whether Leptospira spp. were present across a gradient of housing densities in the urban and suburban black bear population in and around Asheville, North Carolina using serologic and molecular surveys. We collected blood from captured black bears (n=94) and kidneys and bladders from carcasses (n=19). We tested a total of 96 (47 females, 47 males, and 2 unknown) serum samples by microscopic agglutination test (MAT) and had positive results (titer .1:100) for L. kirschneri serovar Grippotyphosa (L. Grippotyphosa) in 4 females (8%) and 5 males (10%). No other serovars showed elevated titers in MAT. We tested a total of 125 samples using PCR (n=96 serum, n=20 kidney, and n=9 bladders) and obtained positive results from one serum (1%), one kidney (5%), and one bladder (11%). The presence of Leptospira spp. in black bears occupying an urban and suburban landscape may indicate a more extensive occurrence of the bacteria among animals in the study region because black bears are the top carnivore in that ecosystem. Potential threats of widespread contamination during natural events such as flood or drought must be considered.