A Rapid Field-Deployable Reverse Transcription-Insulated Isothermal Polymerase Chain Reaction Assay for Sensitive and Specific Detection of Bluetongue Virus.

Bluetongue is a non-contagious, haemorrhagic, Culicoides-borne disease of ruminants. The causative agent, bluetongue virus (BTV), is a member of the Orbivirus genus of the Reoviridae family. So far, 26 BTV serotypes have been identified worldwide. The global distribution of bluetongue has been expanding, and rapid detection of BTV, preferably in the field, is critical for timely implementation of animal movement restrictions and vector control measures. To date, many laboratory-based, molecular assays for detection of BTV have been developed. These methods require the samples to be shipped to a central laboratory with sophisticated instruments and highly skilled technicians to perform the assays, conduct analyses and interpret the results. Here, we report the development and evaluation of a rapid, portable, user-friendly, pan-BTV reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay that can potentially be used in low-resource field conditions. The total length of the assay was <60 min, and at the end of the assay, the results were automatically displayed as '+' or '-' without the need for data interpretation. The RT-iiPCR assay detected 36 BTV isolates and two in vitro transcribed RNA samples representing all 26 BTV serotypes. The assay did not cross-react with other animal viruses tested, including two closely related orbiviruses. The analytical sensitivity of the assay was as low as nine copies of in vitro transcribed double-stranded BTV RNA. Analysis of BTV-infected whole blood samples showed that the BTV RT-iiPCR assay was as sensitive as real-time RT-PCR. The assay can potentially be used for rapid screening of animals for BTV in routine diagnostics and for monitoring bluetongue outbreaks both in ruminants and in Culicoides vectors in the field and in the laboratory.

Detection of bluetongue virus RNA in field-collected Culicoides spp. (Diptera: Ceratopogonidae) following the discovery of bluetongue virus serotype 1 in white-tailed deer and cattle in Louisiana.

In November 2004, bluetongue virus (family Reoviridae, genus Orbivirus, BTV) serotype 1 (BTV-1) was detected for the first time in the United States from a hunter-killed deer in St. Mary Parish, LA. In 2005, sera surveys were conducted on three cattle farms near the area where the deer was found, and BTV-1-seropositive cattle were found on two of the three farms; in 2006, sera surveys from the cattle on the three farms did not detect any BTV-1-positive animals. The purpose of this study was to survey ceratopogonid populations at the three farms and test field-collected specimens for the presence of BTV and epizootic hemorrhagic disease virus (family Reoviridae, genus Orbivirus, EHDV). Miniature CDC light traps and New Jersey traps were used to capture ceratopogonids on the three farms from January 2006 through November 2007. In total, 3,319 ceratopogonids were captured, including 1,790 specimens of 10 different species of Culicoides. IR-RT-polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in 264 pools representing 2,309 specimens collected at the farms. All positive samples were sequenced for serotype determination. Five pools of 275 (1.8%) were positive for BTV. Pools of four species of Culicoides were found to be positive: Culicoides crepuscularis (Malloch), Culicoides debilipalpis Lutz (two pools), Culicoides haematopotus Malloch, and Gulicoidesfurens (Poey). The amplicons of the positive specimens were sequenced and found to be identical to both BTV-17 and BTV-13. During our study, no BTV-1 transmission was detected in cattle, and no BTV-1 was detected in specimens of ceratopogonids.

Distribution of bluetongue in the United States of America, 1991-2002.

Bluetongue virus (BTV) distribution in the United States of America (USA) is limited by the range of the vector Culicoides spp. Regional differences exist with the north-eastern states being free of BTV, while the central and north-western states are seasonally free of virus. Activity of the virus can be observed throughout the year in the southern USA. Serological evidence defining the distribution of BTV in selected regions of the USA is gathered regularly through serological surveys conducted on samples from slaughter cattle. From 1991 to 2002, ten serological surveys were completed. Results from Alaska, Hawaii, Michigan, Minnesota, New York, Wisconsin and New England consistently demonstrated a seropositive rate of less than 2%, confirming BTV-free status. Antibody against BTV was sporadically detected in cattle originating from states contiguous to the BTV-free regions. Additional information on BTV distribution in the USA is obtained through identification of BTV or BTV RNA in diagnostic, surveillance and export specimens submitted to the National Veterinary Services Laboratories. Results confirm that BTV serotypes 2, 10, 11, 13 and 17 are present in the USA.

Bluetongue surveillance methods in the United States of America.

Historical surveillance for bluetongue virus (BTV) exposure in the United States of America (USA) has relied on periodical serological surveillance using samples collected from cattle at slaughter. Most of this surveillance has been focused on the north-eastern portion of the USA due to the lack of competent vectors of BTV in this region. For most of the states tested in this region, the prevalence of seropositive animals has been less than 2%. Recently, a study was conducted in north-central USA using sentinel cattle herds. Results of serological testing showed an increasing gradient of exposure from north to south. In addition, detection of Culicoides sonorensis showed a similar gradient with detection in the northern areas being relatively rare. The results of these studies indicate that cattle herds in the northern and north-eastern areas of the USA are likely to be free of BTV.

Equine West Nile encephalitis, United States.

After the 1999 outbreak of West Nile (WN) encephalitis in New York horses, a case definition was developed that specified the clinical signs, coupled with laboratory test results, required to classify cases of WN encephalitis in equines as either probable or confirmed. In 2000, 60 horses from seven states met the criteria for a confirmed case. The cumulative experience from clinical observations and diagnostic testing during the 1999 and 2000 outbreaks of WN encephalitis in horses will contribute to further refinement of diagnostic criteria.

Detection of North American West Nile virus in animal tissue by a reverse transcription-nested polymerase chain reaction assay.

A traditional single-stage reverse transcription-polymerase chain reaction (RT-PCR) procedure is effective in determining West Nile (WN) virus in avian tissue and infected cell cultures. However, the procedure lacks the sensitivity to detect WN virus in equine tissue. We describe an RT-nested PCR (RT-nPCR) procedure that identifies the North American strain of WN virus directly in equine and avian tissues.

West Nile virus outbreak among horses in New York State, 1999 and 2000.

West Nile (WN) virus was identified in the Western Hemisphere in 1999. Along with human encephalitis cases, 20 equine cases of WN virus were detected in 1999 and 23 equine cases in 2000 in New York. During both years, the equine cases occurred after human cases in New York had been identified.

Foxtail-induced ulcerative stomatitis outbreak in a Missouri stable.

Twenty of 25 horses in a well-managed Missouri boarding stable were diagnosed with gingivitis/stomatitis. Gross examination of the affected horses revealed varying degrees of gingivitis ranging from mild periodontal swelling to marked swelling and erythema with ulceration and hemorrhage. Fine hair-like material was embedded within the intensely affected areas. Gingival biopsies from 4 affected horses contained pyogranulomatous inflammation with, in some cases, numerous eosinophils and several grass awns in cross and longitudinal section. Numerous foxtail seed heads were identified in hay samples. Examination of the records revealed that all of the affected horses had been fed the suspect hay, with the exception of 1 horse. Although not deliberately fed the suspect hay, this horse did have access to the hay when turned out into the exercise paddock. The lesions resolved following a change in hay source.

West Nile encephalitis.

WNV encephalitis in horses, previously reported in Africa, Asia, and Europe, occurred for the first time in the Western Hemisphere in 1999. The causative agent, WNV, is a flavivirus maintained in nature by a bird-mosquito cycle. The disease in horses is manifested primarily by ataxia of variable severity. Outbreaks of encephalitis may have a case fatality rate in excess of 40%, although this virus infection is inapparent in some horses. Early evidence indicates that WNV has overwintered in the northeastern United States and poses a threat for future disease occurrences in horses. No vaccine is available to protect against WNV infection in horses; disease control is predicated on mosquito abatement.

Equid herpesvirus-induced immunosuppression is associated with lymphoid cells and not soluble circulating factors.

A paresis isolate of equid herpesvirus 1 (EHV1, Ab4/8) and a plaque-purified virus derived from it (EHV1, Ab4/13), induced long-term suppression of both mitogenic and antigen-specific lymphocyte proliferations in adult outbred ponies. Peripheral blood mononuclear cells (PBMC) taken from a pony after EHV1 infection suppressed the in vitro function of normal cells but serum did not. This showed that the observed immune suppression was associated with circulating PBMC and/or their products rather than circulating soluble factors such as antigen or immune complexes. The results suggested that productive infection of lymphocytes by EHV1 was unlikely to result in the observed in vitro effects. Moreover, prostaglandin release from monocytes was not likely to have caused the observed suppression, because lymphocyte responsiveness was not restored in the presence of indomethacin.

Effect of vaccine-induced immune activation on the performance of early-weaned pigs.

Two groups of 96 pigs were studied to determine the influence of weaning age, nursery site and a challenge to their immune system on their performance. The weaning ages were 11 to 16 days and 16 to 21 days. One nursery was on-site and the second nursery was off-site. Immune activation was stimulated by the administration of infectious bovine rhinotracheitis virus (IBR) vaccine to half of the pigs at each site. Serum virus neutralisation titres to IBR and total immunoglobulins were monitored in some of the pigs in each group. Performance was measured in terms of feed intake, average daily gain in weight, and feed conversion ratio (FCR). The mean serum immunoglobulin concentrations of all the groups of pigs tended to decrease in the first two weeks after weaning and then increase. Twelve of 20 pigs vaccinated with IBR had neutralisation titres to the virus. The site of the nursery did not significantly affect average daily gain in weight, feed intake or FCR. Pigs weaned at 16 to 21 days of age had a significantly better daily gain in weight than the pigs weaned at 11 to 16 days of age. Immune stimulation of the older weaned pigs did not influence their performance, but it had a significantly (P < 0.016) negative effect on the performance of the younger weaned pigs.

The equine herpesviruses.

Two viruses, EHV-1 and EHV-4, are now known to be responsible for disease conditions formerly considered caused by "equine rhinopneumonitis virus." Although these viruses share several laboratory and clinical features, they differ in epidemiology and pathogenic potential. EHV-4 is primarily associated with clinical respiratory disease, whereas EHV-1 is more frequently isolated from aborted fetuses, sickly foals, and neurologic cases. Both viruses frequently establish latent infections, but the relevance of latency to clinical disease is unclear. Diagnosis based on identification of the pathogen is generally superior to serologic methods. Vaccines containing each virus are available, and vaccination in concert with careful management limits the number of clinical cases. Immunity following vaccination or disease is not absolute, however, and improved disease prophylaxis awaits a better understanding of protective immune responses.