Retrospective study of laboratory-based surveillance of rabies in wild and domestic animals in the southern United States, 2010-2021.

We performed a retrospective study of all case submissions for the rabies virus (RABV) direct fluorescent antibody test (DFAT) requested of the Tifton Veterinary Diagnostic and Investigational Laboratory (Tifton, GA, USA) between July 2010 and June 2021. Submitted were 792 samples from 23 animal species from 89 counties in Georgia, and 4 neighboring counties in Florida, 1 in South Carolina, and 1 in Alabama. In 13 (1.6%) cases, the DFAT result was inconclusive; 779 (98.4%) cases had a conclusive (positive or negative) test result. Of these 779 cases, 79 (10.1%) tested positive across 10 species. The remaining 700 (89.9%) cases were negative. The main reason for submission for RABV testing was human exposure to a potentially rabid animal in 414 (52.3%) cases. Among the 79 positive cases, 74 (93.7%) involved wildlife; raccoons (51 cases; 68.9%) were the primary host confirmed with RABV infection, followed by skunk and fox (8 cases each; 10.8%), bobcat (5 cases; 6.8%), and bats (2 cases; 2.7%). Only 5 domestic animals (6.3% of the positive cases) tested positive during our study period; one from each of the bovine, canine, caprine, equine, and feline species. Hence, the sylvatic cycle plays the predominant role in circulating RABV infection in our study area.

Antimicrobial Metaphylaxis and its Impact on Health, Performance, Antimicrobial Resistance, and Contextual Antimicrobial Use in High-Risk Beef Stocker Calves.

The objective of this blinded, cluster-randomized, complete block trial was to evaluate the impact of metaphylaxis on health, performance, antimicrobial resistance, and contextual antimicrobial use (AMU) in high-risk beef stocker calves. Calves (n = 155) were randomly assigned to receive either saline or tulathromycin at the time of arrival processing. Deep nasopharyngeal swabs were collected from each calf at arrival and 14 d later. Calves were monitored for bovine respiratory disease (BRD) for 42 d. Body weights were obtained at arrival, days 14, 28, and 42. Contextual antimicrobial use (AMU) was calculated using dose and mass-based metrics. Calves given tulathromycin had a greater average daily gain (0.96 ±â€…0.07 kg vs. 0.82 ±â€…0.07 kg; P = 0.034) and lower prevalence of BRD than controls (17% vs. 40%; P = 0.008). Proportions of calves with BRD pathogens identified at arrival were similar between treatment groups [17%; P = 0.94]. Proportions of calves with BRD pathogens identified at day 14 were lower for calves receiving tulathromycin compared to controls (15% vs. 60%, P < 0.001). Overall, 81% of Pastuerella multocida isolates and 47% of Mannheimia haemolytica isolates were pansusceptible. When measured as regimens per head in, AMU in calves receiving tulathromycin was higher than calves receiving saline (P = 0.01). Under the conditions of this study, metaphylaxis had positive impacts on the health and performance of high-risk beef stocker calves, did not contribute to the selection of resistant bacterial isolates in the nasopharynx of treated cattle, and increased AMU.

In this study, we investigated the impact of metaphyactic antimicrobial administration on health, performance, and antimicrobial use in high-risk beef stocker calves. Our findings demonstrated that metaphylaxis improves performance and has positive effects on animal health and well-being but increases total antimicrobial use. Additionally, our study revealed that metaphylaxis alone does not contribute to the selection of antimicrobial-resistant pathogens in the upper airway of treated cattle.

Predominance of Canine Parainfluenza Virus and Mycoplasma in Canine Infectious Respiratory Disease Complex in Dogs.

Canine infectious respiratory disease complex (CIRDC) is caused by different viruses and bacteria. Viruses associated with CIRDC include canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine herpesvirus type 1 (CHV-1), canine respiratory coronavirus (CRCoV), and canine parainfluenza virus (CPIV). Bacteria associated with CIRDC include Bordetella bronchiseptica, Streptococcus equi subspecies zooepidemicus (S. zooepidemicus), and Mycoplasma spp. The present study examined the prevalence of CIRDC pathogens in specimens received by a Veterinary Diagnostic Laboratory in Georgia, USA., from 2018 to 2022. Out of 459 cases, viral agents were detected in 34% of cases and bacterial agents were detected in 58% of cases. A single pathogen was detected in 31% of cases, while two or more pathogens were identified in 24% of cases. The percentages of viral agents identified were CAV-2 (4%), CDV (3%), CPIV (16%), CRCoV (7%), and CIV (2%). The percentages of bacterial agents were B. bronchiseptica (10%), Mycoplasma canis (24%), Mycoplasma cynos (21%), and S. zooepidemicus (2%). Over the five-year period, the positive cases ranged from 2-4% for CAV-2, 1-7% for CDV, 1-4% for CHV-1, 9-22% for CPIV, 4-13% for CRCoV, and 1-4% for CIV. Overall, the most prevalent pathogens associated with CIRDC were CPIV, M. canis, and M. cynos.

Rapid Detection of SARS-CoV-2 RNA in Human Nasopharyngeal Specimens Using Surface-Enhanced Raman Spectroscopy and Deep Learning Algorithms.

A rapid and cost-effective method to detect the infection of SARS-CoV-2 is fundamental to mitigating the current COVID-19 pandemic. Herein, a surface-enhanced Raman spectroscopy (SERS) sensor with a deep learning algorithm has been developed for the rapid detection of SARS-CoV-2 RNA in human nasopharyngeal swab (HNS) specimens. The SERS sensor was prepared using a silver nanorod array (AgNR) substrate by assembling DNA probes to capture SARS-CoV-2 RNA. The SERS spectra of HNS specimens were collected after RNA hybridization, and the corresponding SERS peaks were identified. The RNA detection range was determined to be 103-109 copies/mL in saline sodium citrate buffer. A recurrent neural network (RNN)-based deep learning model was developed to classify 40 positive and 120 negative specimens with an overall accuracy of 98.9%. For the blind test of 72 specimens, the RNN model gave a 97.2% accuracy prediction for positive specimens and a 100% accuracy for negative specimens. All the detections were performed in 25 min. These results suggest that the DNA-functionalized AgNR array SERS sensor combined with a deep learning algorithm could serve as a potential rapid point-of-care COVID-19 diagnostic platform.

Point-of-care testing in companion and food animal disease diagnostics.

Laboratory diagnoses of animal diseases has advanced tremendously in recent decades with the advent of cutting-edge technologies such as real-time polymerase chain reaction, next generation sequencing (NGS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and others However, most of these technologies need sophisticated equipment, laboratory space and highly skilled workforce. Therefore, there is an increasing market demand for point-of-care testing (POCT) in animal health and disease diagnostics. A wide variety of assays based on antibodies, antigens, nucleic acid, and nanopore sequencing are currently available. Each one of these tests have their own advantages and disadvantages. However, a number of research and developmental activities are underway in both academia and industry to improve the existing tests and develop newer and better tests in terms of sensitivity, specificity, turnaround time and affordability. In both companion and food animal disease diagnostics, POCT has an increasing role to play, especially in resource-limited settings. It plays a critical role in improving animal health and wellbeing in rural communities in low- and middle-income countries. At the same time, ensuring high standard of quality through proper validation, quality assurance and regulation of these assays are very important for accurate diagnosis, surveillance, control and management of animal diseases. This review addresses the different types of POCTs currently available for companion and food animal disease diagnostics, tests in the pipeline and their advantages and disadvantages.

Isothermal amplification using sequence-specific fluorescence detection of SARS coronavirus 2 and variants in nasal swabs.

Coronavirus disease 2019 is a public health challenge requiring rapid testing for the detection of infections and transmission. Nucleic acid amplification tests targeting SARS coronavirus 2 (CoV2) are used to detect CoV2 in clinical samples. Real-time reverse transcription quantitative PCR is the standard nucleic acid amplification test for CoV2, although reverse transcription loop-mediated isothermal amplification is used in diagnostics. The authors demonstrate a sequence-specific reverse transcription loop-mediated isothermal amplification-based nucleic acid amplification assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay using labeled primers and a quencher oligonucleotide. This assay can achieve rapid (30 min) and sensitive (1000 plaque-forming units/ml) fluorescence detection of CoV2 (WA1/2020), B.1.1.7 (Alpha) and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples.

Determining the role of natural SARS-CoV-2 infection in the death of domestic pets: 10 cases (2020-2021).

OBJECTIVE: To establish a pathoepidemiological model to evaluate the role of SARS-CoV-2 infection in the first 10 companion animals that died while infected with SARS-CoV-2 in the US. ANIMALS: 10 cats and dogs that tested positive for SARS-CoV-2 and died or were euthanized in the US between March 2020 and January 2021. PROCEDURES: A standardized algorithm was developed to direct case investigations, determine the necessity of certain diagnostic procedures, and evaluate the role, if any, that SARS-CoV-2 infection played in the animals' course of disease and death. Using clinical and diagnostic information collected by state animal health officials, state public health veterinarians, and other state and local partners, this algorithm was applied to each animal case. RESULTS: SARS-CoV-2 was an incidental finding in 8 animals, was suspected to have contributed to the severity of clinical signs leading to euthanasia in 1 dog, and was the primary reason for death for 1 cat. CONCLUSIONS AND CLINICAL RELEVANCE: This report provides the global community with a standardized process for directing case investigations, determining the necessity of certain diagnostic procedures, and determining the clinical significance of SARS-CoV-2 infections in animals with fatal outcomes and provides evidence that SARS-CoV-2 can, in rare circumstances, cause or contribute to death in pets.

Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs.

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

Complete Genome Sequences of Six Staphylococcus pseudintermedius Strains from Dogs with Superficial Pyoderma in Georgia, USA.

Staphylococcus pseudintermedius is a pathogen of veterinary importance, as it is the major causative agent of superficial pyoderma in dogs. We present the complete genome sequences of six strains of S. pseudintermedius derived from dogs affected with epidermal collarettes and superficial bacterial folliculitis, which are two variants of superficial pyoderma.

A milk-line sampling system to detect foodborne pathogens: A field case investigation from the United States and Argentina.

The objective of this short communication was to discuss two field case investigations to determine the usefulness of a milk-line sampling device to detect bacteria either coming from a group of cows suffering from mastitis or from the milking line potentially contaminated with environmental bacteria. In Case 1, the in-line sampling device was able to detect certain segments of the milk-line contaminated with environmental bacteria, but not coming from the cows. In Case 2, 19 out of 25 pooled in-line samples were in agreement with at least one of the individual sampled cows shedding either Staphylococcus or Streptococcus spp. or both, which accounted for 76% accordance between both methods. The in-line system, although not perfect, provided a reliable method to detect individual cows shedding mastitis-causing organisms. In conclusion, the milk-line sampling device system was able to help identify foodborne pathogens. Regular monitoring of the microbial quality of milk through a milk-line sampling device is recommended for groups of cows within the dairy herd to detect potential mastitis-causing microorganisms. Furthermore, the sampling device was an effective tool to screen the efficacy of cleaning and disinfecting mechanisms of the milk lines to identify and control potential foodborne pathogens that are collected in the bulk tank.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

An outbreak of Neospora caninum abortion in a dairy herd from the State of Georgia, United States.

An outbreak of 92 abortions out of 1,700 pregnant cows (5.41%) in a period of 3 weeks (19 May to 05 June 2019) occurred in a Georgia Dairy, USA, in cattle that were between 3 and 7 months of gestation. Two sets of samples (aborted fetuses' organs, placental tissues, aborted cows blood) were submitted for laboratory investigations at the Tifton Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia (TVDIL, Tifton, GA, USA). An abortion panel testing for the major abortion-causing agents [e.g. Bovine Viral Diarrhoea Virus (BVDV), Infectious Bovine Rhinotracheitis Virus/ Bovine Herpes Virus-I (IBR/ BHV-I), Brucella spp., Leptospira spp.] was conducted on several of the samples. On the first set of samples, microbial cultures, serology and PCR tests for the common abortifacient agents revealed the presence of Neospora caninum (N. caninum) DNA, which was positive by PCR on the placenta and fetal tissues. The second set of diagnostic investigations also identified two out of three submitted freshly aborted fetuses to be positive for N. caninum by PCR and immunohistochemistry. Moreover, all three dams were also sero-positive for N. caninum. The entire herd was being fed on grass silage harvested from a pasture where feral pigs were hunted previously and carcasses were left behind. As a consequence of this action a large population of wild coyotes were attracted to these carcasses, and likely contaminated the pasture with potential N. caninum-infected feces. After the abortion outbreak was resolved, it was recommended that the farmers should avoid disposal of cadavers of hunted animals in the wild, as it could attract carnivorous and omnivorous animals that may potentially spread the disease to the cattle and other wildlife.

Serosurvey of arthropod-borne diseases among shelter dogs in the Cumberland Gap Region of the United States.

BACKGROUND: The Cumberland Gap Region (CGR) of the United States is a natural corridor between the southeastern, northeastern, and midwestern regions of the country. CGR has also many species of ticks and mosquitos that serve as competent vectors for important animal and human pathogens. In this study, we tested dogs from six different animal shelters in the CGR for Rocky Mountain spotted fever (RMSF), anaplasmosis, Lyme disease, canine ehrlichiosis and canine heartworm disease. RESULTS: Sera from 157 shelter dogs were tested for antibodies to RMSF agent, Rickettsia rickettsii, using an indirect immunofluorescence assay. Sixty-six dogs (42.0%) were positive for either IgM or IgG, or both IgM and IgG antibodies to R. rickettsii. Moreover, the same set of sera (n = 157) plus an and additional sera (n = 75) from resident dogs at the same shelters were tested using the SNAP 4Dx Plus. Of 232 dogs tested, two (0.9%) were positive for antibodies to Anaplasma phagocytophilum/A. platys, nine (3.9%) were positive for antibodies to Borrelia burgdorferi, 23 (9.9%) for positive for antibodies to Ehrlichia canis/E. ewingii, and 13 (5.6%) were positive for Dirofilaria immitis antigen. Co-infection with two or more etiologic agents was detected in five animals. Three dogs had antibodies to both B. burgdorferi and E. canis/E. ewingii, and two dogs were positive for D. immitis antigen and antibodies to B. burgdorferi and E. canis/E. ewingii. CONCLUSIONS: Shelter dogs in the CGR are exposed to a number of important vector-borne pathogens. Further studies are required to ascertain the roles these animals play in maintenance and transmission of these pathogens.

Leptospiral shedding and seropositivity in shelter dogs in the Cumberland Gap Region of Southeastern Appalachia.

BACKGROUND: Leptospirosis, caused by pathogenic Leptospira spp., is a zoonotic infection that affects humans, dogs and many other mammalian species. Virtually any mammalian species can act as asymptomatic reservoir, characterized by chronic renal carriage and shedding of a host-adapted leptospiral serovar. Environmental contamination by chronic shedders results in acquisition of infection by humans and susceptible animals. METHODS: In this study, we investigated if clinically normal shelter dogs and cats harbor leptospires in their kidneys by screening urine samples for the presence of leptospiral DNA by a TaqMan based-quantitative PCR (qPCR) that targets pathogen-associated lipl32 gene. To identify the infecting leptospiral species, a fragment of leptospiral rpoB gene was PCR amplified and sequenced. Additionally, we measured Leptospira-specific serum antibodies using the microscopic agglutination test (MAT), a gold standard in leptospiral serology. RESULTS: A total of 269 shelter animals (219 dogs and 50 cats) from seven shelters located in the tri-state area of western Virginia, eastern Tennessee, and southeastern Kentucky were included in this study. All cats tested negative by both qPCR and MAT. Of the 219 dogs tested in the study, 26/198 (13.1%, 95% CI: 8.4-17.8%) were positive for leptospiral DNA in urine by qPCR and 38/211 (18.0%, 95% CI: 12.8-23.2%) were seropositive by MAT. Twelve dogs were positive for both qPCR and MAT. Fourteen dogs were positive by qPCR but not by MAT. Additionally, leptospiral rpoB gene sequencing from a sub-set of qPCR-positive urine samples (n = 21) revealed L. interrogans to be the leptospiral species shed by dogs. CONCLUSIONS: These findings have significant implications regarding animal and public health in the Cumberland Gap Region and possibly outside where these animals may be adopted.

Systemic humoral immunity in beef bulls following therapeutic vaccination against Tritrichomonas foetus.

The utility of therapeutic vaccination of bulls against Tritrichomonas foetus has been advocated in previous studies, but anecdotal reports suggest this practice does not clear infections and may additionally confound diagnostic testing by reducing parasite burdens below detectable limits. The objective of this study was to characterize the systemic humoral immune response to therapeutic vaccination in T. foetus-infected bulls over a period of four months using an indirect ELISA and to compare the dynamics of this response to culture and PCR results to establish the existence of a relationship (or lack thereof) between immunization and infection status. A study population of 4- to 6-year-old T. foetus-infected beef bulls (n = 20) was divided equally into a treatment group and a control group. The treatment group received two doses of commercially prepared whole cell killed vaccine 2 weeks apart while the control group received injections of vaccine diluent. Blood samples were collected at each injection and at 4 subsequent dates every 4 weeks thereafter (i.e. 0, 2, 6, 10, 14, and 18 wks) to measure IgG1 and IgG2 antibody subisotype response via an indirect ELISA. Preputial smegma samples were collected at the four monthly intervals following vaccination for diagnosis of infection via InPouch™ culture, Modified Diamond's Medium (MDM) culture, and PCR. Humoral response for both IgG isotypes from week 2 through week 18 were significantly increased in vaccinates compared to controls. No significant decrease in infection prevalence was detected in the treatment group for any of the diagnostic methods used. The apparent lack of pathogen clearance during a stimulated immune response suggests that therapeutic vaccination may not be a useful T. foetus management practice.

Development and Evaluation of a Novel Taqman Real-Time PCR Assay for Rapid Detection of Mycoplasma bovis: Comparison of Assay Performance with a Conventional PCR Assay and Another Taqman Real-Time PCR Assay.

The objective of this study was to develop and validate a Taqman real-time PCR assay for the detection of Mycoplasma bovis (M. bovis). Unique primers targeting the highly conserved house-keeping gene (uvrC) were designed and the probe sequence was derived from a previously published microarray study. There was 100% agreement in the outcome between our assay and the other two published assays for M. bovis detection. The analytical limit of detection of our assay is 83 copies of the uvrC gene. This assay was validated on a total of 214 bovine clinical specimens that were submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL), Texas, USA. The specificity of the assay was assessed to be 100% since no cross-reactivity occurred with 22 other bacterial and other Mycoplasma species. We conclude that the uvrC gene serves as a good and reliable diagnostic marker for the accurate and rapid detection of M. bovis from a wider variety of specimen matrices.

Campylobacter jejuni ferric-enterobactin receptor CfrA is TonB3 dependent and mediates iron acquisition from structurally different catechol siderophores.

Campylobacter jejuni NCTC11168 does not produce any endogenous siderophores of its own yet requires the CfrA enterobactin transporter for in vivo colonization. In addition, the genome of C. jejuni NCTC11168 contains three distinct TonB energy transduction systems, named TonB1, TonB2, and TonB3, that have not been tested for their role in siderophore uptake or their functional redundancy. We demonstrate that C. jejuni NCTC11168 transports ferric-enterobactin in an energy dependent manner that requires TonB3 for full activity with TonB1 showing partial functional redundancy. Moreover C. jejuni NCTC11168 can utilize a wide variety of structurally different catechol siderophores as sole iron sources during growth. This growth is solely dependent on the CfrA enterobactin transporter and highlights the wide range of substrates that this transporter can recognize. TonB3 is also required for growth on most catechol siderophores. Furthermore, either TonB1 or TonB3 is sufficient for growth on hemin or hemoglobin as a sole iron source demonstrating functional redundancy between TonB1 and TonB3. In vivo colonization assays with isogenic deletion mutants revealed that both TonB1 and TonB3 are required for chick colonization with TonB2 dispensable in this model. These results further highlight the importance of iron transport for efficient C. jejuni colonization.

Adenovirus and cryptosporidium co-infection in a corn snake (Elaphae guttata guttata).

A 1-yr-old albino male corn snake (Elaphae guttata guttata), which was part of a large breeding stock, was presented to the University of Florida, College of Veterinary Medicine, Zoo and Exotic Animal Clinic with a history of anorexia for 2 wk and progressively declining body condition. The animal was euthanized due to a poor prognosis. Histopathology, electron microscopy, and polymerase chain reaction analysis on tissues revealed concurrent infection with adenovirus and Cryptosporidium. Primary infection with adenovirus could have caused immunodeficiency in the snake, thus predisposing it to secondary infection with Cryptosporidium. To the authors' knowledge, this is the first report of co-infection of adenovirus and Cryptosporidium in a Colubrid species of snake.

Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow.

Calf diarrhea (scours) is a primary cause of illness and death in young calves. Significant economic losses associated with this disease include morbidity, mortality, and direct cost of treatment. Multiple pathogens are responsible for infectious diarrhea, including, but not limited to, Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp. Identification and isolation of carrier calves are essential for disease management. Texas Veterinary Medical Diagnostic Laboratory current methods for calf diarrhea pathogen identification include electron microscopy (EM) for BCV and BRV and a direct fluorescent antibody test (DFAT) for organism detection of Cryptosporidium spp. A workflow was developed consisting of an optimized fecal nucleic acid purification and multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) for single tube concurrent detection of BCV, BRV, and Cryptosporidium spp., and an internal control to monitor nucleic acid purification efficacy and PCR reagent functionality. In "spike-in" experiments using serial dilutions of each pathogen, the analytical sensitivity was determined to be <10 TCID(50)/ml for BCV and BRV, and <20 oocysts for Cryptosporidium spp. Analytical specificity was confirmed using Canine and Feline coronavirus, Giardia spp., and noninfected bovine purified nucleic acid. Diagnostic sensitivity was ≥98% for all pathogens when compared with respective traditional methods. The results demonstrate that the newly developed assay can purify and subsequently detect BCV, BRV, and Cryptosporidium spp. concurrently in a single PCR, enabling simplified and streamlined calf diarrhea pathogen identification.

Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni.

BACKGROUND: During gut colonization, the enteric pathogen Campylobacter jejuni must surmount the toxic effects of reactive oxygen species produced by its own metabolism, the host immune system, and intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. RESULTS: The mechanisms of oxidative stress defense in C. jejuni were characterized by transcriptional profiling and phenotypic analysis of wild-type and mutant strains. To define the regulon of the peroxide-sensing regulator, PerR, we constructed an isogenic DeltaperR mutant and compared its transcriptome profile with that of the wild-type strain. Transcriptome profiling identified 104 genes that belonged to the PerR regulon. PerR appears to regulate gene expression in a manner that both depends on and is independent of the presence of iron and/or H2O2. Mutation of perR significantly reduced motility. A phenotypic analysis using the chick colonization model showed that the DeltaperR mutant exhibited attenuated colonization behavior. An analysis of changes in the transcriptome induced by exposure to H2O2, cumene hydroperoxide, or menadione revealed differential expression of genes belonging to a variety of biological pathways, including classical oxidative stress defense systems, heat shock response, DNA repair and metabolism, fatty acid biosynthesis, and multidrug efflux pumps. Mutagenic and phenotypic studies of the superoxide dismutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed a role for these proteins in oxidative stress defense and chick gut colonization. CONCLUSION: This study reveals an interplay between PerR, Fur, iron metabolism and oxidative stress defense, and highlights the role of these elements in C. jejuni colonization of the chick cecum and/or subsequent survival.