Antibody response against three widespread bovine viruses is not impaired in Holstein cattle carrying bovine leukocyte antigen DRB3.2 alleles associated with bovine leukemia virus resistance.

Due to the wide dissemination of bovine leukemia virus (BLV) infection among dairy cattle, control and eradication programs based on serological detection of infected cattle and subsequent culling face a major economic task. In Argentina, genetic selection of cattle carrying alleles of the bovine leukocyte antigen (BoLA) DRB3.2 gene associated with BLV-infection resistance, like *0902, emerges as the best additional tool toward controlling virus spread. A potential risk in expanding or segregating BoLA selected populations of cattle is that it might increase susceptibility to other common viruses. Special concern raises the strong association found between low proviral load and low antibody titer against major BLV structural proteins. This phenomenon might depend on host genetic factors influencing other viruses requiring, unlike BLV, strong and long-lasting humoral immune response to prevent infection. In this study, we demonstrate that there is no association among neutralizing antibody titers against foot and mouth disease virus, bovine viral diarrhea virus, or bovine herpesvirus type 1 and polymorphism of the BoLA DRB3.2 gene. Conversely, there is strong association between BoLA DRB3.2*0902 and low antibody titers against 2 BLV structural proteins--env gp51 and gag p24--to date, the best BLV resistance marker. There is also significant association between low antibody titers against gp51 and p24 and BoLA DRB3.2*1701 and low antibody titers against p24 and BoLA DRB3.2*1101 or 02. Our data suggest that increasing BoLA-selected BLV-resistant cattle or segregating BoLA-associated alleles to BLV susceptibility would not affect the resistance or the predisposition to bovine viral diarrhea virus, bovine herpesvirus type 1, or foot and mouth disease virus infection.

Association of BLV infection profiles with alleles of the BoLA-DRB3.2 gene.

Bovine leukaemia virus (BLV) causes lymphosarcoma and persistent lymphocytosis (PL). Some MHC class II gene polymorphisms have been associated with resistance and susceptibility to the development of lymphosarcoma and PL, as well as with a reduced number of circulating BLV-infected lymphocytes. Previously, 230 BLV-infected Holstein cattle were classified into two infection profiles characterized by low and high proviral loads (LPL and HPL respectively). Here, the influence of the polymorphism at the BoLA-DRB3.2* gene of these animals was examined. After genotyping, the association between the BoLA-DRB3.2* alleles and the BLV infection profile was determined as the odds ratio (OR). Two subtypes of allele *11 were identified (ISAG*0901 and *0902). Allele ISAG*0902 showed a stronger association with the LPL profile (OR = 8.24; P < 0.0001) than allele *11 itself (OR = 5.82; P < 0.0001). Allele ISAG*1701 (*12) also showed significant association with the LPL profile (OR = 3.46; P < 0.0055). Only one allele, ISAG*1501 or 03 (*16), showed significant association with HPL (OR = 0.36; P < 0.0005). The DRB3.2* alleles were assigned to three categories: resistant (R), susceptible (S) and neutral (N). Based on their DRB3 genotypes, cattle were classified as homozygous or heterozygous. The RR and RN genotypes were associated with the LPL profile, while the SS and NS genotypes were associated with the HPL profile. The RS genotype could not be associated with any particular profile. Our results show that allele ISAG*0902 appears to be the best BLV resistance marker in Holstein cattle.

Detection of bovine viral diarrhea virus by amplification on polycation-treated cells followed by enzyme immunoassay / Detección del virus de la diarrea viral bovina por amplificación sobre células tratadas con policationes seguida de enzimoinmunoensayo

A bovine viral diarrhea virus (BVDV) amplification method combined with an enzyme immunoassay was developed to detect BVDV antigens in seropositive cattle. Reconstitution assays conducted by adding decreasing amounts of BVDV (Singer strain) to Madin-Darby bovine kidney (MDBK) cells showed that the sensitivity threshold of the combined assay was 10-7 TCID50. BVDV amplification was carried out in polycation (DEAE-Dextran and polybrene)- treated MDBK cells. Treated cells were able to replicate both ether-treated virus and neutralizing antibody-coated virus. Ammonium chloride decreased virus replication in polycation-treated cells, suggesting viral penetration by endocytosis. BVDV detection was tested in leukocytes from 104 seropositive cattle from 2 unvaccinated commercial closed dairy herds with high seroprevalence. Lysates and co-cultures of peripheral blood leukocytes (PBL) were tested, directly or after up to 6 blind passages in normal or polycation-treated cells. BVDV was detected in 10/104 cattle after only one co-culture of PBL in treated cells. No virus was detected in whole blood or plasma samples. BVDV positive and negative cattle were retested three times, achieving consistent results. The finding of immune carriers supports the possibility that these animals may constitute an epidemiological risk.

Se desarrolló un método de detección de antígenos del virus de la diarrea viral bovina (BVDV) combinando amplificación viral con enzimoinmunoensayo. El método combinado presentó una sensibilidad de 10-7 TCID50 en ensayos con diluciones decrecientes de BVDV cepa Singer sobre la línea celular MDBK. La amplificación del título viral se efectuó sobre células MDBK tratadas con policationes Estas células replicaron tanto el BVDV tratado con éter como el unido a anticuerpos. La replicación viral en las células tratadas disminuyó ante la presencia de cloruro de amonio, lo que sugiere la penetración viral por endocitosis. El BVDV se determinó en leucocitos de 104 bovinos seropositivos de dos rodeos en producción, cerrados y con alta seroprevalencia. Los leucocitos de sangre periférica (LSP) fueron lisados y analizados directamente o luego de hasta 6 pasajes ciegos sobre células normales o tratadas con policationes. El BVDV se detectó en 10 de los 104 animales después de solamente un cultivo de LSP en células tratadas. No se pudo detectar presencia viral en las muestras de sangre o plasma. Los estudios se repitieron tres veces en animales BVDV positivos y negativos, con resultados consistentes. El hallazgo de bovinos seropositivos portadores del virus indica la posibilidad de que estos animales puedan significar un riesgo epidemiológico.

Detection of bovine viral diarrhea virus by amplification on polycation-treated cells followed by enzyme immunoassay.

A bovine viral diarrhea virus (BVDV) amplification method combined with an enzyme immunoassay was developed to detect BVDV antigens in seropositive cattle. Reconstitution assays conducted by adding decreasing amounts of BVDV (Singer strain) to Madin-Darby bovine kidney (MDBK) cells showed that the sensitivity threshold of the combined assay was 10(-7) TCID50. BVDV amplification was carried out in polycation (DEAE-Dextran and polybrene)-treated MDBK cells. Treated cells were able to replicate both ether-treated virus and neutralizing antibody-coated virus. Ammonium chloride decreased virus replication in polycation-treated cells, suggesting viral penetration by endocytosis. BVDV detection was tested in leukocytes from 104 seropositive cattle from 2 unvaccinated commercial closed dairy herds with high seroprevalence. Lysates and co-cultures of peripheral blood leukocytes (PBL) were tested, directly or after up to 6 blind passages in normal or polycation-treated cells. BVDV was detected in 10/104 cattle after only one co-culture of PBL in treated cells. No virus was detected in whole blood or plasma samples. BVDV positive and negative cattle were retested three times, achieving consistent results. The finding of immune carriers supports the possibility that these animals may constitute an epidemiological risk.

Detection of bovine viral diarrhea virus (BVDV) in seropositive cattle.

Detection of bovine virus diarrhoea virus (BVDV) in one vaccinated beef cattle and three non-vaccinated dairy herds was investigated on peripheral blood leukocytes (PBL) with or without previous treatment followed by a capture ELISA (cELISA). Using the combination of PHA and polycation treatment, PBL from 229 seropositive cattle were studied and could be classified in four different states of BVDV infection. Lysed PBL from four animals were directly positive in cELISA (Category I), PBL of 17 animals were positive after PHA stimulation (Category II), 15 animals were positive only after PHA stimulation plus polycation treatment (Category III), while virus could not be detected in 193 seropositive cattle. Wild-type BVDV strains were isolated by co-culture on polycation-treated MDBK cells from 11 of these seropositive animals. BVDV antibodies of these same animals were able to neutralize their own virus, indicating that virus persists in PBL in spite of strain-specific antibodies. No apparent change of leukocyte subpopulations could be detected in any category of virus-positive animals. Thus, BVDV may be present in the PBL of some cattle, even in the presence of a specific active immune response.

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle.

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.

[Bovine leukemia virus (BLV): prevalence in the Cuenca Lechera Mar y Sierras from 1994 to 1995]. / Virus de la leucosis bovina (BLV): prevalencia en la Cuenca Lechera Mar y Sierras entre 1994 y 1995.

The Cuenca Lechera Mar y Sierras (CLMS) includes about 300 dairy farms located in the counties of Tandil, Balcarce, Juarez, Ayacucho, General Pueyrredón, Gonzalez Chavez and Necochea, in the province of Buenos Aires. The purpose of this investigation was to determine the prevalence of infection caused by Bovine Leukemia Virus (BLV) in the CLMS. We investigated the presence of anti-BLV antibodies in 4,203 milk samples taken from 73 dairy farms belonging to the CLMS. An indirect ELISA, which is described and evaluated in this paper, was used to test the antibodies in milk. We classified the dairy farms according to their rate of infection. The percentage of dairy farms free of infection resulted in 31.50. On the other hand, 49.40% of the dairy farms showed a figure between 1% and 15% of infected cattle; 17.80% between 16% and 30%, and the remaining 1.30% turned out more than 30% of infected cattle. If compared with data obtained in the 1979-1981 period, which showed that 95.65% of the dairy farms was BLV-free, it is clear that a dramatic progress of the BLV infection has occurred for the last 15 years. Nevertheless, the CLMS is in a privileged position so as to incorporate an inexpensive control plan to eradicate the BLV infection, as almost 1/3 of its dairy farms is still BLV-free and 49.40% still has a low rate of BLV infection. Only about 20% of the dairy farms would require costly strategies of control.

Transmission of human T cell leukemia virus type I to sheep: antibody profile and detection of viral DNA sequences.

Lambs were inoculated intraperitoneally with either 1.8 x 10(7) live peripheral blood cells from an HTLV-I-infected person (five lambs) or with 8 x 10(7) live cells from the HTLV-I-producing cell lines MT-2 (four lambs) or C10 MJ (five lambs). Four control lambs were inoculated with minimal essential medium supplemented with fetal calf serum. The animals were monitored during a period of 24 months. Beginning at 5 to 12 months after inoculation, four of the five lambs inoculated with the fresh HTLV-I-infected peripheral blood cells began to develop detectable levels of antibodies to a recombinant HTLV-I gp21env antigen, as determined by an enzyme-linked immunoassay (ELISA). The anti-gp21 antibodies persisted for the remaining observation period. These antibodies were not detected in the sera from the other sheep. Absorption and blocking experiments demonstrated the specificity of the gp21 reactivity. This reactivity was also confirmed by Western blot (WB). With the exception of the serum of an MT-2-inoculated sheep that formed a weak band with p19 by WB, none of the sera of the four gp21-positive sheep or of the other experimental sheep reacted with other structural or regulatory HTLV-I proteins, as determined by ELISA, WB, and radioimmunoassay. PCR analyses demonstrated the presence of the HTLV-I provirus in peripheral blood leukocytes of the four sheep showing antibodies to gp21env. The remaining sheep were negative. PCR analyses failed to detect BLV sequences in any of the experimental sheep. None of the sheep showed clinical abnormalities during the observation period. The potential value of the sheep model for studying atypical virus-host interactions in infected people is discussed.

Identification of a type-specific protein that differentiates serologically between human T cell lymphotropic virus types I and II.

The 24-kDa band formed when sera of humans infected with human T cell lymphotropic virus type I (HTLV-1) were reacted with HTLV-I lysates in conventional Western blot (WB) assays was found to be composed of two immunologically unrelated proteins of 24- and 23-kDa. p24, but not p23, carries epitopes shared by the major core proteins of the other known transactivating C-type retroviruses. p23 is unrelated immunologically to the env and tax HTLV-I products but partly cross-reacts with HTLV-I p19. All HTLV-I and simian T cell leukemia virus type I sera tested reacted with p23. Reactivity with p23 was seen with some HTLV-I sera that did not react or reacted weakly with HTLV-I p24. No reactivity with p23 was seen among the 51 human HTLV-II sera tested nor among a large panel of control sera. Because of its type-specificity and strong immunogenicity, p23 provides a reliable serologic marker for the diagnosis of HTLV-I infection and for distinguishing between HTLV-I and -II.

Characterization of the blood lymphocyte population in cattle infected with the bovine leukemia virus.

Blood leukocytes of cattle characterized in terms of bovine leukemia virus (BLV) infection and persistent lymphocytosis (PL) were examined for the presence of lymphocyte subpopulation markers and viral antigens. The percentages of cells with surface and intracytoplasmic immunoglobulin M (IgM) and erythrocyte-antibody-rosetting cells agreed closely in all infected cattle. This correlation and the results of double labeling experiments indicate that virtually all the surface IgM-positive B-lymphocytes in the blood of these animals carry Fc receptors. In PL cattle, the percentages of surface IgM-positive cells were more than twice those of normal cells and accounted for all the increase in peripheral blood lymphocytes. B-cells accounted for most of the increase in peripheral blood lymphocytes seen in cattle with PL. In contrast, most BLV-infected, nonlymphocytotic cattle had normal percentages of B-cells. Thus, the expansion of the B-cell population in blood, while being a conspicuous characteristic of PL, is not necessarily a consequence of BLV infection per se. Comparisons of the percentages of IgM-positive and erythrocyte-antibody complement-rosetting cells, together with the results of double labeling experiments, indicate that about one-half the B-cells in the blood of cattle with PL lacked C-3 receptors. The proportion of these cells (most likely immature B-lymphocytes) was smaller in the blood of BLV-infected nonlymphocytotic cattle. Direct comparison showed that, in BLV-infected cattle with or without PL, and in BLV-free cattle, virtually all erythrocyte-rosetting blood cells had peanut agglutinin receptors. With only one exception, the numbers of erythrocyte-positive cells in the blood of BLV-infected cattle with or without PL were within normal values. The "null" blood cell population, estimated as the difference between the IgM-positive and erythrocyte-positive populations, was essentially unaffected in BLV-infected cattle without PL, but it was absent in PL cattle. The large majority of the B-lymphocytes present in the blood of cattle with PL were infected with BLV. The proportion of infected B-lymphocytes in the blood of BLV-positive nonlymphocytotic cattle was much lower. Even in cattle with low or moderate levels of BLV-infected blood lymphocytes, the percentages of these cells were remarkably constant during the 12-month period of the study. The data indicate that most of the BLV-infected B-lymphocytes of cattle with PL lack C-3 receptors.

An amplified immunoperoxidase assay to detect bovine leukemia virus expression: development and comparison with other assays.

An amplified immunoperoxidase (AIP) assay using an avidin:biotin complex was developed to detect bovine leukemia virus (BLV) antigen expression in lymphocytes which had been cultured 24 h and fixed with acetone. Nonspecific reactions were eliminated by absorbing the test serum with 100% horse or cow serum. DNA synthesis inhibition did not decrease the number of AIP-positive cells, and there were no apparent preferential losses of major lymphocyte subpopulations during culture. Both viable and nonviable BLV-expressing cells were detected. Thus, the number of AIP-positive cells seems to be a good estimate of the minimum number of infected lymphocytes present in the uncultured blood cells. In direct comparisons, twice as many BLV-expressing cells were detected with the AIP assay as with an indirect immunofluorescence test. The AIP assay is as sensitive as the syncytia infectivity assay and only slightly less sensitive than an immunoperoxidase infectivity assay for detecting BLV-infected lymphocytes in the blood of infected cattle that were in early stages of infection and/or had low titers of antiviral antibodies. The AIP assay is the most sensitive, rapid, and reproducible procedure available for the identification of individual cells infected with BLV. This assay may be of great value in studies on the biology of BLV infection.