María Elisa Etcheverrigaray

Isolation of Equine Herpesvirus–2 from the Lung of an Aborted Fetus

This study describes the isolation of equine herpesvirus–2 (EHV-2) from the lung of an aborted equine fetus in Argentina. The isolated virus was confirmed as EHV-2 by indirect immunofluorescence using a rabbit anti–EHV-2 polyclonal antiserum and by virus-neutralization test using an equine polyclonal antibody against EHV-2. Restriction endonuclease DNA fingerprinting with BamHI also confirmed the identity of the virus as EHV-2. Furthermore, viral nucleic acid was detected by polymerase chain reaction from the original lung sample and from the DNA obtained from cells infected with the virus isolate. This work constitutes the first reported isolation of EHV-2 from an aborted equine fetus. The presence of EHV-2 in the lung of the aborted fetus would indicate that this virus is capable of crossing the placental barrier. However, no cause–effect relationship was established between the EHV-2 isolate and the abortion.

Diversity of genomic electropherotypes of naturally occurring equine herpesvirus 1 isolates in Argentina

The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, BamHI and BglII, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype.

A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Enzootic bovine Leukosis: development of an indirect enzyme linked immunosorbent assay (I-Elisa) in seroepidemiological studies

Bovine Leukemia Virus (BLV) is the etiologic agent of Enzootic Bovine Leukosis, a retrovirus exogenous to the bovine species. Once infected, there is no detectable viraemia but instead there is a strong and persistent immunological response to BLV structural proteins, essentially the gp51 envelope glycoprotein and the mayor core protein p24. We describe the test procedure of an indirect ELISA (I-ELISA) using polyclonal reagents for the detection of antibodies to BLV. For comparison, the sera were simultaneously tested by agar gel immunodiffussion (AGID) test, which is currently used as diagnostic standard for BLV infection. The antigen applied does not require a high degree of purification and the data from the analysis of the negative sera showed that the establishment of a cut-off level corresponding to 3 times the standard deviation (SD) above the mean for the negative control set of sera provided acceptable specificity, reducing the risk of false positives results. A comparison of the results obtained by AGID test and I-ELISA showed that considering a total of 465 serum samples, all of the 234 samples (50%) that were positive by AGID were positive to the I-ELISA. Of 225 serum samples which yielded negative results in the AGID test, 69 (15%) were found to be positive by the I-ELISA and 156 (33%) were negative by both techniques. Few sera (2%) that were non-specific by AGID were defined as negative or positive by I-ELISA.

Evaluation of a blocking ELISA using a urease conjugate for the detection of antibodies to pseudorabies virus

A blocking enzyme-linked immunosorbent assay (ELISA) using a urease conjugate (U-B-ELISA) was evaluated for screening sera for antibodies to pseudorabies virus under field conditions. A total of 764 serum samples were analyzed by U-B-ELISA. Of these, 264 were evaluated by both virus neutralization and U-B-ELISA, and the results were compared. U-B-ELISA showed 98.5% and 98.9% sensitivity and specificity, respectively. This test combines the sensitivity and specificity of the blocking ELISA format while allowing visual assessment of results.