FOOT-AND-MOUTH DISEASE IN EXPERIMENTALLY INFECTED MULE DEER (ODOCOILEUS HEMIONUS).

The only known outbreak of foot-and-mouth disease (FMD) in wildlife in the US occurred in mule deer (Odocoileus hemionus) in California in 1924-25. There is little recorded information on the pathogenesis and epidemiology of the disease in deer in that outbreak. In this experimental study, we compared the susceptibility of mule deer to FMD virus (FMDV) serotype O to that of cattle (Bos taurus). We also determined the potential for intra- and interspecies transmission of FMDV serotype O in mule deer and cattle, and assessed conventional laboratory tests in their ability to detect FMDV in mule deer. Two mule deer and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. The inoculated steer and deer were kept in the same room with contact animals of both species. Exposed contact animals were moved to rooms with unexposed animals after becoming febrile. All mule deer (n=14) and cattle (n=6) developed clinical signs and lesions consistent with FMDV infection. Deer had a high prevalence of myocarditis and high mortality. Virus was transmitted between mule deer, from cattle to mule deer, and from mule deer to cattle. Virus and antibodies against nonstructural FMDV proteins in mule deer and cattle were detected by conventional laboratory tests. Virus shedding was detected by PCR and virus isolation up to 9 d postexposure in deer.

Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses.

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91-99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28-1.06 and 0.01-0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.

FOOT-AND-MOUTH DISEASE IN A SMALL SAMPLE OF EXPERIMENTALLY INFECTED PRONGHORN (ANTILOCAPRA AMERICANA).

There is limited information on the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American wildlife and none concerning pronghorn ( Antilocapra americana ). In an experimental study of 13 pronghorn and six steers ( Bos taurus ), we compared the susceptibility of pronghorn to FMD virus (FMDV) strain O, with that of cattle ( Bos taurus ). We also determined the potential for intra- and interspecies transmission of FMDV strain O in pronghorn and cattle, assessed the application of conventional laboratory tests in their suitability to detect FMDV infection in pronghorn, and evaluated the potential role of pronghorn as efficient long-term carriers of FMDV. After acclimation to containment at Plum Island Animal Disease Center, two pronghorn and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were housed with contact animals. When contact-exposed animals developed fever they were placed in rooms with previously unexposed animals. All inoculated and exposed cattle and pronghorn developed clinical disease typical of FMD. Pronghorn developed severe foot lesions and mild to moderate oral lesions, primarily on the tongue. Duration of clinical signs in both species was 2-3 wk with foot abnormalities evident to the end of the study (51 d postexposure). Other lesions included pancreatitis, myositis of the tongue, and secondary lesions including pleuritis, pneumonia, decubital ulcers, and tenosynovitis. Virus transmission occurred between pronghorn, from cattle to pronghorn, and from pronghorn to cattle. Conventional laboratory tests detected virus and antibodies against nonstructural and structural FMDV proteins in pronghorn and cattle. Virus was present in some animals for 1 wk but was not detectable by virus isolation or PCR at 3 wk postinfection or afterward.

Analysis of clinical samples for early detection of classical swine fever during infection with low, moderate, and highly virulent strains in relation to the onset of clinical signs.

Classical swine fever (CSF) is a transboundary viral disease affecting swine. The clinical course of disease and the best diagnostic samples for early detection were examined using low, moderate, and highly virulent strains of CSFV inoculated into 8-12 week old domestic pigs. Clinical signs were monitored and recorded. Nasal swabs, tonsil scrapings, blood and tonsils were tested using virus isolation, immunohistochemistry, and real-time reverse transcriptase PCR (rRT-PCR).Severe clinical signs appear 3 days post infection (dpi) with the highly virulent strain, correlating with positive tonsil scrapings, tonsil and blood by virus isolation and rRT-PCR (83-100%), whereas nasal swabs become comparable by 5dpi (89-100%). The moderate strain caused less severe clinical signs between 5 and 7dpi, with tonsil scrapings, tonsil and blood positive by 7dpi (83-100%), and nasal swabs were comparable at 10dpi (67-90%). The low virulent strain showed mild clinical signs at 7dpi, with blood, tonsil and tonsil scrapings positive by virus isolation and rRT-PCR. Except for one sample at 10dpi, nasal swabs remained negative throughout the course of infection. This study indicates that irrespective of virulence, whole blood and tonsil scrapings are the sample of choice for early detection of CSFV in live pigs.

Foot-and-mouth disease in North American bison (Bison bison) and elk (Cervus elaphus nelsoni): susceptibility, intra- and interspecies transmission, clinical signs, and lesions.

There is limited information about the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American bison (Bison bison) or elk (Cervus elaphus nelsoni). In these two experimental infection studies, we compared the susceptibilities of bison and elk to FMD virus (FMDV), respectively, with that of cattle; determined whether intra- and interspecies transmission could occur in bison and cattle, and elk and cattle; determined suitability of conventional available laboratory tests to detect FMDV infection in bison and elk; and investigated whether bison or elk are efficient long-term carriers of FMDV. In both studies, after a period of acclimation to the containment at Plum Island Animal Disease Center, animals were infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were kept with contact animals; subsequently, inoculated and/or exposed contact animals were placed in rooms with unexposed animals. All bison developed oral mucosal and foot lesions similar to those of cattle. Bison developed fever, lameness, inappetence, and ptyalism. Physical examinations on bison revealed numerous small vesicles and erosions affecting tongue, gingiva, muzzle, hard and soft palates, coronary bands, and interdigital skin. Inoculated elk developed transient fever and mild focal tongue and foot lesions. Contact elk developed neither clinical signs nor gross pathologic lesions of FMD. At necropsy, lesions in bison included numerous extensive vesicles, erosions, and/or ulcers in the oral cavities, feet, and rumen pillars depending on the stage of disease. Less extensive oral, foot, and rumen lesions were present in the inoculated elk. All bison and inoculated elk developed antibodies to FMDV and were positive for FMDV by reverse transcription-polymerase chain reaction (RT-PCR). Transmission occurred between cattle and bison, and bison and bison. It did not occur between elk and cattle. Elk-to-elk transmission studies resulted in only one contact elk developing serologic evidence of a subclinical infection. Other exposed elk developed neither clinical, pathologic, virologic, nor serologic evidence of disease. FMDV was not isolated from animals past 28 days postinfection.

A pandemic strain of calicivirus threatens rabbit industries in the Americas.

Rabbit Hemorrhagic Disease (RHD) is a severe acute viral disease specifically affecting the European rabbit Oryctolagus cuniculus. As the European rabbit is the predominant species of domestic rabbit throughout the world, RHD contributes towards significant losses to rabbit farming industries and endangers wild populations of rabbits in Europe and other predatory animals in Europe that depend upon rabbits as a food source. Rabbit Hemorrhagic Disease virus (RHDV) - a Lagovirus belonging to the family Caliciviridae is the etiological agent of RHD. Typically, RHD presents with sudden death in 70% to 95% of infected animals. There have been four separate incursions of RHDV in the USA, the most recent of which occurred in the state of Indiana in June of 2005. Animal inoculation studies confirmed the pathogenicity of the Indiana 2005 isolate, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages. Complete viral genome sequences of all USA outbreak isolates were determined and comparative genomics revealed that each outbreak was the result of a separate introduction of virus rather than from a single virus lineage. All of the USA isolates clustered with RHDV genomes from China, and phylogenetic analysis of the major capsid protein (VP60) revealed that they were related to a pandemic antigenic variant strain known as RHDVa. Rapid spread of the RHDVa pandemic suggests a selective advantage for this new subtype. Given its rapid spread, pathogenic nature, and potential to further evolve, possibly broadening its host range to include other genera native to the Americas, RHDVa should be regarded as a threat.

Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR.

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription-PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding beta-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.