Development of a novel diagnostic test for detection of bovine viral diarrhea persistently infected animals using hair

The purpose of this study was to determine whether manually plucked hairs might serve as an alternative sample for a quantitative real time polymerase chain reaction (qRT-PCR) testing. Twenty three, 1~3 week old, non-bovine viral diarrhea virus (BVDV) vaccinated calves, found to be positive for BVDV by immunohistochemical staining, were selected and hairs were manually plucked from the ear. qRT-PCR was performed on samples consisting of more than 30 hairs (30~100) and whole blood. All 23 animals were positive for the virus by qRT-PCR performed on the whole blood and when samples of more than 30 hairs were assayed. Additionally, qRT-PCR was performed on groups of 10 and 20 hairs harvested from 7 out of 23 immunohistochemical staining-positive calves. When groups of 20 and 10 hairs were tested, 6 and 4 animals, respectively, were positive for the virus

ON THE IMMUNOLOGICAL RESPONSE TO SKIN TEST ANTIGENS FOR PSEUDORABIES IN SWINE

Procedures were developed for the production of two effective pseudorabies virus (PrV) skin test antigens, one consisting of chemically inactivated virions, the other of viral nucleocapsids. In vivo methods used to determine the immunological basis of a cutaneous response to the virion antigen in PrV-infected swine suggested that the reaction was due to the cell-mediated immune system. The evidence was obtained through experiments to demonstrate: (1) passive transfer of immunity, which did not materialize; (2) antihistamine blocking of the PrV skin test and of the tuberculin purified protein derivative reaction, which did not eventuate in either case; and (3) through histopathological studies on biopsies of skin at test sites, which revealed a cellular response typical of delayed type hypersensitivity. The nucleocapsid antigen reaction was similar to the cutaneous response induced by the virion antigen. False positive results did not occur with either test antigen. Three vaccinal doses of the virion antigen and the nucleocapsid antigen were given intramuscularly on three separate occasions to PrV-susceptible swine. The virion antigen elicited a detectable humoral immune response after the second injection as determined by the standard serum neutralization (SN) test and an enzyme-linked immunosorbent assay (ELISA) for pseudorabies. The nucleocapsid antigen did not cause a humoral response detectable by the SN test; however, anti-PrV antibodies were measurable by the ELISA in a few of the principals. The nucleocapsid antigen principals, as a whole, did not differ significantly from the control animals. Both antigens appeared similar in their ability to induce a cutaneous cell-mediated immune response in PrV-infected swine. Either antigen provided a fast, effective, and reproducible method for the evaluation of the immune status of swine herds. Use of the nucleocapsid antigen should avoid the problem of seroconversion as determined by the SN test that is encountered with the virion antigen

Discovery of a bovine enterovirus in alpaca.

A cytopathic virus was isolated using Madin-Darby bovine kidney (MDBK) cells from lung tissue of alpaca that died of a severe respiratory infection. To identify the virus, the infected cell culture supernatant was enriched for virus particles and a generic, PCR-based method was used to amplify potential viral sequences. Genomic sequence data of the alpaca isolate was obtained and compared with sequences of known viruses. The new alpaca virus sequence was most similar to recently designated Enterovirus species F, previously bovine enterovirus (BEVs), viruses that are globally prevalent in cattle, although they appear not to cause significant disease. Because bovine enteroviruses have not been previously reported in U.S. alpaca, we suspect that this type of infection is fairly rare, and in this case appeared not to spread beyond the original outbreak. The capsid sequence of the detected virus had greatest homology to Enterovirus F type 1 (indicating that the virus should be considered a member of serotype 1), but the virus had greater homology in 2A protease sequence to type 3, suggesting that it may have been a recombinant. Identifying pathogens that infect a new host species for the first time can be challenging. As the disease in a new host species may be quite different from that in the original or natural host, the pathogen may not be suspected based on the clinical presentation, delaying diagnosis. Although this virus replicated in MDBK cells, existing standard culture and molecular methods could not identify it. In this case, a highly sensitive generic PCR-based pathogen-detection method was used to identify this pathogen

Prepubertal Exposure to the Live Attenuated Avian Infectious Bronchitis Virus Induces Epididymal Stones in the Rooster after Puberty

Roosters immunized prepubertally with the avian infectious bronchitis virus (AIBV) have a high incidence of epididymal calcium stones, reduced daily sperm production and lower serum testosterone as adults. The aim of the present study was to determine when and how stones are formed following vaccination with AIBV. Specific pathogen free roosters were either immunized with the live attenuated AIBV (vaccinated group, n=38) or with the vehicle (nonvaccinated group, n=33) at 2, 6 and 10wk of age. Testes and epididymides were studied histologically at 12, 16, 20 and 26wk of age. Abnormalities were not observed in testes of either vaccinated or nonvaccinated group at any age observed. The epididymal region of the vaccinated group was normal until 16wk. However, aggregations of sperm, cell debris and macrophages and stones were present in the proximal efferent ductules in the vaccinated group at 20wk and 26wk of age. These efferent ductules showed expanded lumen, reduced mucosal folds and low columnar epithelium. Next to these affected ductules were normal efferent ductules. On the other hand, epididymal stones also occurred in the nonvaccinated group (2/8, 25%) at 26wk but the frequency was lower than that in the vaccinated group (8/14, 57%). These results indicate that epididymal stone formation begins with aggregations of sperm, cell debris and macrophages immediately after puberty, which is followed by calcium deposition. It is suggested that the prepubertal vaccination with AIBV accelerates epididymal stone formation

Genetic Characterization of H1N2 Influenza A Viruses Isolated from Pigs throughout the United States

An H1N2 influenza A virus was isolated from a pig in the United States for the first time in 1999 (A. I. Karasin, G. A. Anderson, and C. W. Olsen, J. Clin. Microbiol. 38:2453-2456, 2000). H1N2 viruses have been isolated subsequently from pigs in many states. Phylogenetic analyses of eight such viruses isolated from pigs in Indiana, Illinois, Minnesota, Ohio, Iowa, and North Carolina during 2000 to 2001 showed that these viruses are all of the same reassortant genotype as that of the initial H1N2 isolate from 1999

Neighbor-joining phylogenetic tree of the deduced amino acid sequences from the capsid gene.

<p>Enteroviruses representing the bovine enterovirus (BEV) in species EV-E and EV-F, porcine enterovirus (PEV)/Enterovirus G, and human enterovirus (HEV)/Enterovirus A–D groups are included and species and serotypes are indicated. The capsid sequences for the capped langur (JX538037) and unclassified AY24745 sequences (denoted with asterisks) are partial, so the correct placement of these sequences in these trees may change as more sequence data become available. The amino acid sequences were aligned with the Clustal W program, and bootstrap confidence values were determined by 1000 replications. The scale bar represents the number of amino acid substitutions per site.</p