Molecular detection and characterization of transient bovine viral diarrhea virus (BVDV) infections in cattle commingled with ten BVDV persistently infected cattle.

Fifty-three cattle of unknown serologic status that were not persistently infected (PI) with bovine viral diarrhea virus (BVDV) were commingled with 10 cattle that were PI with different strains of BVDV, and were monitored for an extended commingle period using a reverse-transcription real-time PCR (RT-rtPCR) BVDV assay on various sample types. Transient infections with BVDV were also assessed by virus isolation, virus neutralization (VN) assays, and direct buffy coat 5'-UTR sequencing. Infections were demonstrated in all cattle by RT-rtPCR; however, the detection rate was dependent on the type of sample. Buffy coat samples demonstrated a significantly greater number of positive results ( p ≤ 0.05) than either serum or nasal swab samples. Presence of elevated BVDV VN titers at the onset inversely correlated with the number of test days positive that an individual would be identified by RT-rtPCR from buffy coat samples, and directly correlated with the average Ct values accumulated over all RT-rtPCR test days from buffy coat samples. Both single and mixed genotype/subgenotype/strain infections were detected in individual cattle by direct sample 5'-UTR sequencing. A BVDV-2a strain from a PI animal was found to be the predominant strain infecting 64% of all non-PI cattle; BVDV-1b strains originating from 3 PI cattle were never detected in non-PI cattle. Although direct sample 5'-UTR sequencing was capable of demonstrating mixed BVDV infections, identifying all strains suspected was not always efficient or possible.

Tissue localization, shedding, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus following inoculation of 4-week-old feeder pigs.

We determined tissue localization, shedding patterns, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus (PEDV) following inoculation of 4-week-old feeder pigs. Thirty-three pigs were randomly assigned to 1 of 3 groups for the 42-day study: inoculated (group A; n = 23), contact transmission (group B; n = 5), and aerosol transmission (group C; n = 5). Contact transmission occurred rapidly to group B pigs whereas productive aerosol transmission failed to occur to group C pigs. Emesis was the first clinical sign noted at 3 days postinoculation (dpi) followed by mild to moderate diarrhea lasting 5 more days. Real-time PCR detected PEDV in fecal and nasal swabs, oral fluids, serum, and gastrointestinal and lymphoid tissues. Shedding occurred primarily during the first 2 weeks postinoculation, peaking at 5-6 dpi; however, some pigs had PEDV nucleic acid detected in swabs collected at 21 and 28 dpi. Antibody titers were measurable between 14 and 42 dpi. Although feces and intestines collected at 42 dpi were PEDV negative by PCR and immunohistochemistry, respectively, small intestines from 70% of group A pigs were PCR positive. Although disease was relatively mild and transient in this age group, the results demonstrate that 4-week-old pigs are productively infected and can sustain virus replication for several weeks. Long-term shedding of PEDV in subclinically affected pigs should be considered an important source for PEDV transmission.

Diagnostic Tools for Bluetongue and Epizootic Hemorrhagic Disease Viruses Applicable to North American Veterinary Diagnosticians.

This review provides an overview of current and potential new diagnostic tests for bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses compiled from international participants of the Orbivirus Gap Analysis Workshop, Diagnostic Group. The emphasis of this review is on diagnostic tools available to North American veterinary diagnosticians. Standard diagnostic tests are readily available for BT/EHD viruses, and there are described tests that are published in the World Organization for Animal Health (OIE) Terrestrial Manual. There is however considerable variation in the diagnostic approach to these viruses. Serological assays are well established, and many laboratories are experienced in running these assays. Numerous nucleic acid amplification assays are also available for BT virus (BTV) and EHD virus (EHDV). Although there is considerable experience with BTV reverse-transcriptase PCR (RT-PCR), there are no standards or comparisons of the protocols used by various state and federal veterinary diagnostic laboratories. Methods for genotyping BTV and EHDV isolates are available and are valuable tools for monitoring and analyzing circulating viruses. These methods include RT-PCR panels or arrays, RT-PCR and sequencing of specific genome segments, or the use of next-generation sequencing. In addition to enabling virus characterization, use of advanced molecular detection methods, including DNA microarrays and next-generation sequencing, significantly enhance the ability to detect unique virus strains that may arise through genetic drift, recombination, or viral genome segment reassortment, as well as incursions of new virus strains from other geographical areas.

Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus-colonized bulls.

The objectives of the current study were 1) to compare sample preparation workflows and quantitative real-time polymerase chain reaction assays (qPCR) as currently used in veterinary diagnostic laboratories with a study protocol utilizing commercially available reagents for individual Tritrichomonas foetus testing, 2) to assess the accuracy of pooling cultured smegma samples followed by extraction and qPCR testing as used in the study laboratory, and 3) to assess the specificity of the currently used primers and probes by sequencing all positive and presumptive positive samples identified in the study laboratory in an attempt to capture any nucleotide variability between T. foetus isolates and to rule out false-positive results possibly due to Simplicimonas moskowitzi. Eight hundred three cultured smegma samples were collected from different regions of the United States with the collaboration of 5 veterinary testing laboratories. The samples were processed individually by the respective laboratories, and then sent to the study laboratory and retested using the study protocol. Comparison testing showed an overall agreement of 95.89% between the veterinary testing laboratories and the study laboratory. One hundred seventy-six positive or presumptive positive samples plus 625 negative qPCR samples were combined and retested using a pooling protocol. Pools consisted of 1 positive sample and 4 negative samples (1/5). These pools were processed using the same study laboratory protocols, and 96% of the positive samples were detected in these pools. Nested PCR followed by sequencing confirmed 175 of the 178 samples classified as positive or presumptive positive in the study laboratory as containing T. foetus-specific DNA.

Rapid detection of the pandemic 2009 H1N1 virus M gene by real-time and gel-based RT-PCR assays.

BACKGROUND: Since the first pandemic 2009 H1N1 (pH1N1) virus was isolated from humans, it has also been detected in other mammalian (pigs, cats, dogs, ferrets) and avian (turkey) species, most likely because of cross-species transmission from humans. The pH1N1 contains six genes derived from swine influenza viruses (SIVs) currently circulating in North America of human- (PB1), avian- (PB2, PA), and swine- (HA, NP, and NS) origin and two genes (NA and M) derived from Eurasian SIVs. The novel genetic composition of pH1N1 necessitates development of novel molecular and serological assays to differentiate the pH1N1 virus from circulating human, swine, turkey, canine, and feline influenza viruses. METHODS: To detect and discriminate the pH1N1 from currently circulating SIVs in North America, we developed and evaluated a TaqMan probe-based real-time and a gel-based RT-PCR assay, both targeting the pH1N1 matrix gene. RESULTS: The real-time and gel-based RT-PCR assays were able to specifically detect the pH1N1 M gene and differentiate it from SIVs circulating in North America, including the classical and novel human-like H1N1 influenza virus as well as H1, H2, and H3 subtype triple reassortant SIVs. Both assays were highly sensitive and specific for the pH1N1 virus. CONCLUSIONS: The newly developed pH1N1-specific real-time and gel-based RT-PCR assays can be used to detect and differentiate the pH1N1 virus from currently circulating SIVs in North America. We suggest a combinational diagnostic approach where the real-time RT-PCR is used for high-throughput detection of influenza positive or suspect samples and the gel-based RT-PCR for confirmation and sequencing of the M-gene.

Mycoplasma bovis outbreak in a herd of North American bison (Bison bison).

A disease outbreak of high morbidity and high mortality in bison (Bison bison) was investigated. Clinical signs included lameness, swollen joints, respiratory distress, and lethargy. Fifty-three of 194 animals died. Cows between 5 and 10 years of age were the most affected group, in which 40 of 88 animals died. Necropsies were performed on several animals. There were abscesses in the lung and liver, as well as fibrinosuppurative pleuritis, polyarthritis, and disseminated microabscesses in various organs. No significant bacteria were isolated by routine aerobic cultures of lung and liver from 2 representative cases. However, Mycoplasma cultures were positive. Polymerase chain reaction tests on the isolated bacteria were positive for Mycoplasma bovis. Histologically, the abscesses were characterized by areas of necrosis with variable mineralization rimmed by granulomatous inflammation and fibrous tissue. No new animals had been introduced into the herd, but a cattle herd was present adjacent to the affected bison herd. Two restriction fragment length polymorphism techniques were used to compare the bison isolate and another bison isolate from an outbreak in North Dakota with a field isolate of M. bovis from cattle and with a laboratory control strain of M. bovis; the isolates and control strain were found to be similar. The isolates and the control were sequenced and compared with sequences in GenBank. Bison isolates were more than 99% homologous to M. bovis sequences in GenBank. It was concluded that M. bovis in bison can cause disseminated infection with a high morbidity and mortality and that bison isolates are similar to bovine M. bovis isolates.

A field evaluation of mortality rate and growth performance in pigs vaccinated against porcine circovirus type 2.

OBJECTIVE: To evaluate, under field conditions, the effects of a commercial porcine circovirus type 2 (PCV2) vaccine on mortality rate and growth performance in a herd infected with PCV2 that had a history of porcine circovirus disease. DESIGN: Randomized controlled clinical trial. ANIMALS: 485 commercial, cross-bred, growing pigs. PROCEDURES: Prior to weaning, pigs were randomly assigned within litter to a vaccination or unvaccinated control group. Pigs in the vaccination group were given a commercial PCV2 vaccine at weaning and 3 weeks later. Mortality rate was recorded, and pigs were weighed prior to vaccination, when moved from the nursery, and prior to marketing. Infection status was assessed by serologic testing and detection of viral DNA in serum. RESULTS: Compared with control pigs, pigs vaccinated against PCV2 had a significantly lower mortality rate during the finishing phase, significantly higher average daily gain during the finishing phase, and significantly lower likelihood of being lightweight at the time of marketing. For vaccinated pigs, overall mortality rate was reduced by 50% and average daily gain during the finishing period was increased by 9.3%. At the time of marketing, vaccinated pigs weighed an average of 8.8 kg (19.4 lb) more than control pigs, without any difference in days to marketing. Serum PCV2 antibody titers increased in control pigs, and PCV2 DNA was detected, indicating active PCV2 infection. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that vaccination against PCV2 was effective at reducing mortality rate and improving growth performance among pigs in a herd infected with PCV2.

Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen.

To detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in semen, various RNA extraction techniques have been utilized for RT-PCR, but rarely compared, to determine an optimized extraction protocol. Due to the viscosity, non-homogeneity, high cellularity and large volume of boar semen produced, difficulties can be encountered in obtaining RNA from the seminal cell fraction. This study compared six RNA extractions, five which used a commercially available kit (RNeasy, Qiagen Inc.) for use on highly cellular samples and a traditional phenol/chloroform procedure. All extractions were compared on serially diluted PRRSV "spiked" seminal cell fractions. The two methods resulting in recovery of the highest amount of RNA, which included a Qiashredder (Qiagen Inc.) (protocol 1) or cell lysis/centrifugation technique (protocol 3) preceding the RNeasy procedure were then compared using naturally infected semen samples from experimentally infected boars. Both protocols detected similar amounts of virus in "spiked" samples, but protocol 1 detected eight additional PRRSV-positive semen samples in naturally infected semen. This study demonstrated that semen "spiked" with PRRSV (cell-free virus) may not be representative of naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen.

Detection of Escherichia coli O157:H7 in cattle feces using a polymerase chain reaction-based fluorogenic 5' nuclease (TaqMan) detection assay after secondary enrichment.

Currently, methods for recovering and identifying Escherichia coli O157:H7 from cattle feces are inconsistent and hindered by their inability to specifically and rapidly detect small numbers of organisms from this complex and highly variable matrix. A standard approach for isolating and characterizing E. coli O157:H7 from cattle feces was compared with a polymerase chain reaction (PCR)-based 5' nuclease assay specific for E. coli O157:H7 that included a secondary enrichment step. The PCR-based method proved a better indicator of the presence of the organism than the culture procedure. Retests indicated that the inclusion of a secondary enrichment step and the subsequent analysis by the 5' nuclease assay were reproducible and specific. Escherichia coli O157:H7 could be detected in fecal samples that were otherwise negative after a primary enrichment step, immunomagnetic separation, and plating onto sorbitol MacConkey agar plates containing cefixime and tellurite (CT-SMAC). In samples that were initially identified as culture positive but PCR negative, retesting of the culture isolates on CT-SMAC indicated that the sorbitol fermentation interpretations could frequently not be repeated in retests, whereas retesting using the 5' nuclease assay on the original samples demonstrated a high level of agreement with the initial PCR conclusions. These results indicate the necessity of confirmatory evaluation of isolates culturally recovered by standard cultural methods that involve the interpretation of CT-SMAC. The high level of disagreement between initial culture results and retests, and the high level of agreement between initial PCR results and retests, indicates the advantages of a gene-based detection system for identifying E. coli O157:H7 in cattle feces. Screening large numbers of fecal samples for E. coli O157:H7 would appear to be feasible by integrating the use of enrichment media in serial rounds of incubation with a PCR-based fluorogenic detection procedure in high throughput detection systems that had automated liquid-handling capabilities.

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots.

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.

Detection of Escherichia coli Shiga toxin (stx) and enterotoxin (estA and elt) genes in fecal samples from non-diarrheic and diarrheic greyhounds.

Virulence factors responsible for acute diarrhea in greyhounds have not been well established. The objective of this study was to determine if a correlation exists between disease and the presence of the Escherichia coli toxin genes in non-diarrheic and diarrheic greyhound feces. DNA extracted from broth cultures was evaluated for the presence of Shiga toxin and enterotoxin genes and broth samples were evaluated for Shiga toxin and heat-labile enterotoxin. Shiga toxin (stx1 and stx2) and enterotoxin (et and estA) genes were identified in both non-diarrheic and diarrheic samples after in vitro cultured of swabs at 37 degrees C for 16-24h. The stx1 gene was present in 3% of non-diarrheic and 15% diarrheic samples and the stx2 gene was identified in 36 and 23%, non-diarrheic and diarrheic samples, respectively. Shiga toxin was present in 48% diarrheic and 25% of the non-diarrheic in vitro cultured samples. The elt gene was detected in vitro cultured swabs in 12% of the non-diarrheic and 7% of the diarrheic samples. Labile toxin was present in the feces of small numbers of both groups of dogs. A significant correlation existed between the presence of both stx1 genes and Shiga toxin in feces, and lack of disease in non-diarrheic (P=0.01) and presence of disease in diarrheic (P=0.024) greyhounds. Correlation between production of Shiga toxin and detection of stx1 or stx2 was significant in both the diarrheic and non-diarrheic feces (P=0.03); however, only the presence of stx1 correlated with diarrhea in both groups of samples (P<0.008). The incidence of toxigenic E. coli in both non-diarrheic and diarrheic greyhounds indicates a zoonotic potential from dogs to humans and requires further study.

Diversity, frequency, and persistence of Escherichia coli O157 strains from range cattle environments.

Genetic diversity, isolation frequency, and persistence were determined for Escherichia coli O157 strains from range cattle production environments. Over the 11-month study, analysis of 9,122 cattle fecal samples, 4,083 water source samples, and 521 wildlife fecal samples resulted in 263 isolates from 107 samples presumptively considered E. coli O157 as determined by culture and latex agglutination. Most isolates (90.1%) were confirmed to be E. coli O157 by PCR detection of intimin and Shiga toxin genes. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested preparations revealed 79 unique patterns (XbaI-PFGE subtypes) from 235 typeable isolates confirmed to be E. coli O157. By analyzing up to three isolates per positive sample, we detected an average of 1.80 XbaI-PFGE subtypes per sample. Most XbaI-PFGE subtypes (54 subtypes) were identified only once, yet the seven most frequently isolated subtypes represented over one-half of the E. coli O157 isolates (124 of 235 isolates). Recurring XbaI-PFGE subtypes were recovered from samples on up to 10 sampling occasions and up to 10 months apart. Seven XbaI-PFGE subtypes were isolated from both cattle feces and water sources, and one of these also was isolated from the feces of a wild opossum (Didelphis sp.). The number of XbaI-PFGE subtypes, the variable frequency and persistence of subtypes, and the presence of identical subtypes in cattle feces, free-flowing water sources, and wildlife feces indicate that the complex molecular epidemiology of E. coli O157 previously described for confined cattle operations is also evident in extensively managed range cattle environments.

Immunogenicity of chi4127 phoP- Salmonella enterica serovar Typhimurium in dogs.

Salmonellae are commonly isolated from dogs. The number of dogs infected with Salmonella spp. is surprisingly high and greater than the incidence of clinical disease would suggest. Salmonellosis is common in greyhound kennels. Morbidity can approach 100% in puppies and the mortality ranges to nearly 40%. To date, there has been little effort to evaluate the feasibility of a vaccine for control of this disease in dogs. In the studies described here, an attenuated strain of Salmonella enterica serovar Typhimurium (Se Typhimurium), chi4127, was capable of establishing a limited infection in dogs. The chi4127-attenuated salmonellae efficiently stimulated protective immune responses in serotype homologous, direct, oral challenge experiments. Morbidity in the wild-type-challenged dogs was 8.3% in immunized dogs but 100% in the non-vaccinated controls. In (9/12) control dogs, the disease involved both gastrointestinal and respiratory tracts with high fever (>40.2 degrees C) that persisted through 5 days after challenge. Serum IgG response against S. typhimurium lipopolysaccharide (LPS) significantly increased (P<0.01) in vaccinated dogs and in non-vaccinated dogs after challenge. The non-vaccinated dogs had 3 to 4 logs higher numbers of Se Typhimurium in splenic and hepatic tissue than did the vaccinated dogs. This particular attenuated strain has potential for use as a vaccine for canine salmonellosis.

Rol de los virus de hepatitis B y Delta en la etiología del hepatocarcinoma y otras crónicas / Etiological rol of B and Delta virus in hepatocarcinoma and other chronic liver disorders

Se estudiaron de forma prospectiva 23 pacientes con cuadros hepáticos crónicos hospitalizados en los Servicios de Medicina en un hospital de referencia de zonas de alta endemicidad de Hepatitis B y Delta ( Puerto maldonado, Abancay y La Convención) como es el Hospital Regional MINSA del Cusco, durante el período de un año. El diagnóstico de los cuadros hepáticos crónicos fue histopatológico por biopsia hepática en 16 de los pacientes, en el resto clínico-laboratorial, debido a que la biopsia hepática estaba contraindicada; la determinación de la infección viral se realizó mediante el dosaje de los siguientes marcadores serológicos: antígeno de superficie (AgHBs), anticuerpo anti-antígeno core IgG (Anti-HBc IgG) y antidelta.Seencontró que el 34.8 por ciento fueron antígeno de superficie positivos y el 65.2 por ciento anticuerpo anti-antígeno core IgG positivos; el 20 por ciento de estos últimos fueron además positivos a anticuerpo anti-antígeno delta