Expression-based analysis of genes related to single nucleotide polymorphism hits associated with bovine leukemia virus proviral load in Argentinean dairy cattle.

In dairy cattle infected with bovine leukemia virus (BLV), the proviral load (PVL) level is directly related to the viral transmission from infected animals to their healthy herdmates. Two contrasting phenotypic groups can be identified when assessing PVL in peripheral blood of infected cows. A large number of reports point to bovine genetic variants (single nucleotide polymorphisms) as one of the key determinants underlying PVL level. However, biological mechanisms driving BLV PVL profiles and infection progression in cattle have not yet been elucidated. In this study, we evaluated whether a set of candidate genes affecting BLV PVL level according to whole genome association studies are differentially expressed in peripheral blood mononuclear cells derived from phenotypically contrasting groups of BLV-infected cows. During a 10-mo-long sampling scheme, 129 Holstein cows were phenotyped measuring anti-BLV antibody levels, PVL quantification, and white blood cell subpopulation counts. Finally, the expression of 8 genes (BOLA-DRB3, PRRC2A, ABT1, TNF, BAG6, BOLA-A, LY6G5B, and IER3) located within the bovine major histocompatibility complex region harboring whole genome association SNP hits was evaluated in 2 phenotypic groups: high PVL (n = 7) and low PVL (n = 8). The log2 initial fluorescence value (N0) transformed mean expression values for the ABT1 transcription factor were statistically different in high- and low-PVL groups, showing a higher expression of the ABT1 gene in low-PVL cows. The PRRC2A and IER3 genes had a significant positive (correlation coefficient = 0.61) and negative (correlation coefficient = -0.45) correlation with the lymphocyte counts, respectively. Additionally, the relationships between gene expression values and lymphocyte counts were modeled using linear regressions. Lymphocyte levels in infected cows were better explained (coefficient of determination = 0.56) when fitted a multiple linear regression model using both PRRC2A and IER3 expression values as independent variables. The present study showed evidence of differential gene expression between contrasting BLV infection phenotypes. These genes have not been previously related to BLV pathobiology. This valuable information represents a step forward in understanding the BLV biology and the immune response of naturally infected cows under a commercial milk production system. Efforts to elucidate biological mechanisms leading to BLV infection progression in cows are valuable for BLV control programs. Further studies integrating genotypic data, global transcriptome analysis, and BLV progression phenotypes are needed to better understand the BLV-host interaction.

Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA.

Quantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement.

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs.

BOLA-DRB3 gene polymorphisms influence bovine leukaemia virus infection levels in Holstein and Holstein × Jersey crossbreed dairy cattle.

Bovine leukemia virus (BLV) infections, causing persistent lymphocytosis and lethal lymphosarcoma in cattle, have reached high endemicity on dairy farms. We observed extensive inter-individual variation in the level of infection (LI) by assessing differences in proviral load in peripheral blood. This phenotypic variation appears to be determined by host genetics variants, especially those located in the BoLA-DRB3 MHCII molecule. We performed an association study using sequencing-based typed BOLA-DRB3 alleles from over 800 Holstein and Holstein × Jersey cows considering LI in vivo and accounting for filial relationships. The DBR3*0902 allele was associated with a low level of infection (LLI) (<1% of circulating infected B-cells), whereas the DRB3*1001 and DRB3*1201 alleles were related to a high level of infection (HLI). We found evidence that 13 polymorphic positions located in the pockets of the peptide-binding cleft of the BOLA-DRB3 alleles were associated with LI. DRB3*0902 had unique haplotypes for each of the pockets: Ser13 -Glu70 -Arg71 -Glu74 (pocket 4), Ser11 -Ser30 (pocket 6), Glu28 -Trp61 -Arg71 (pocket 7) and Asn37 -Asp57 (pocket 9), and all of them were significantly associated with LLI. Conversely, Lys13 -Arg70 -Ala71 -Ala74 and Ser13 -Arg70 -Ala71 -Ala74 , corresponding to the DRB3*1001 and *1201 alleles respectively, were associated with HLI. We showed that the specific amino acid pattern in the DRB3*0902 peptide-binding cleft may be related to the set point of a very low proviral load level in adult cows. Moreover, we identified two BOLA-DRB3 alleles associated with a HLI, which is compatible with a highly contagious profile.

Validation of an r3AB1-FMDV-NSP ELISA to distinguish between cattle infected and vaccinated with foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed livestock which has a drastic economic impact for affected countries. Although FMDV is distributed worldwide, many regional programs have been effective eradicating this agent. In Argentina, as in many other regions of South America, the combination of a systematic vaccination plan, together with an effective detection system capable of differentiating infection from vaccination, has been successful for eradicating this agent from the country. The properties of recombinant 3AB1 FMDV non-structural protein (r3AB1 FMDV-NSP), as a marker for the detection of antibodies to differentiate between cattle infected and vaccinated with FMDV, have been described previously. The goal of the present study was to validate the 3AB1 ELISA using a well characterized serum panel from Argentina (n=559) including eight national and one international reference sera. Overall, the 3AB1 ELISA demonstrated good feasibility, repeatability, reproducibility, analytical sensitivity and specificity, and accuracy. The results from the 3AB1 ELISA when compared with those obtained from the OIE index test (NCPanaftosa screening) showed a similar performance of both tests [diagnostic sensitivity=84% (C.I.=79-88%) and 80% (C.I.=75-85%), respectively; and diagnostic specificity=98.6% (C.I.=97-100%) and 95% (C.I.=91-98%), respectively]. The present work proposes the 3AB1 ELISA as an alternative to imported kits for FMD internal screening and transboundary sero-surveillance.

Immunochromatographic lateral flow test for detection of antibodies to Equine infectious anemia virus.

The purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (ICLF) test for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera. Viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. The performance of rp26-ICLF was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (AGID) test used as a standard of comparison according to international guidelines. The values obtained for comparative diagnostic sensitivity (98.3%), diagnostic specificity (87.4%) and concordance (92.4%) were similar to those reported for other ICLF tests for animal infectious diseases. Very good repeatability and reproducibility, as well as a total agreement with blind previous results from three proficiency test panels, were obtained, thus indicating that rp26-ICLF is a precise test. The end point of the twofold serial dilution of serum samples was the same as, and even better than, the AGID test, thus demonstrating the same analytical sensitivity as that of the reference method for EIA diagnosis. No cross-reactivity was observed when serum samples from horses with other infectious diseases were analyzed. rp26-ICLF proved to be a precise and rapid test suitable for field screening in veterinary practice, since minimal equipment and operator expertise are required. However, further research should be carried out to increase the level of sensitivity.

Detection of bovine leukemia virus specific antibodies using recombinant p24-ELISA.

We developed an indirect ELISA test (rp24-ELISA) for the detection of antibodies against the p24 capsid protein of bovine leukemia virus (BLV) in bovine serum samples. A recombinant p24 (rp24) was expressed in the Escherichia coli expression system, purified in a nickel charged resin and used as antigen in the ELISA test. A cut-off point was established considering the distribution of reactivity values obtained by rp24-ELISA on a set of 555 field serum samples that were stated as double positive (DP) or double negative (DN) by the combination of commercial agar gel immunodiffusion (AGID) assay and gp51-ELISA tests results. The rp24-ELISA showed good concordance with the agar gel immunodiffusion assay, when 710 serum samples were analyzed. In addition, rp24-ELISA demonstrated to be a precise assay with good repeatability and reproducibility and a better analytical sensitivity than AGID. From 67 discordant rp24-ELISA-AGID sera, 4 negative reactors were from the DP group, 28 positive samples were from the DN group and 35 positive samples were stated as negative by AGID but positive by the gp51-ELISA. Samples from this last subgroup were sent to an OIE reference laboratory where 28 samples were stated as negative, 5 samples were stated as positive and 2 as inconclusive. Complete concordance with blind previous results from an international proficiency test panel confirmed the capability of the assay to discriminate between infected and non-infected animals. In conclusion, the rp24-ELISA developed and standardized demonstrated to have good analytical characteristics to be considered for screening of BLV.

Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies.

We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.

Standardization and validation of an agar gel immunodiffusion test for the diagnosis of equine infectious anemia using a recombinant p26 antigen.

We developed and validated an agar gel immunodiffusion test (AGID) test for the diagnosis of equine infectious anemia (EIA) using as antigen the p26 protein of equine infectious anemia virus (EIAV) produced in the Escherichia coli expression system. The developed rp26-AGID test showed an excellent diagnostic relative sensitivity (100%) and specificity (100%) compared to a commercial AGID assay when 1855 field serum samples were analyzed. In addition, the rp26-AGID demonstrated to be a precise assay with excellent repeatability and reproducibility. In the analytical sensitivity trial, positive sera showed nearly the same endpoint dilutions for both compared tests. No positive-reactions were observed with 35 serum samples with antibodies related to other endemic agents and also with severely hemolysed samples, demonstrating that the rp26-AGID has an excellent analytical specificity. Complete concordance with blind previous results from five proficiency test panels confirmed the capability of the assay of accurate detection of EIAV antibodies. This is the first time that a recombinant AGID assay able to identify EIAV infections has been standardized and validated in Argentina according to international guidelines. Taking into account the results obtained, the p26-AGID could be adopted as an official test method for the diagnosis and control of EIA in this country.

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector.

We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4 microg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.

Seroprevalence of bovine leukemia virus in dairy cattle in Argentina: comparison of sensitivity and specificity of different detection methods.

Bovine leukemia virus (BLV) is a retrovirus that induces a chronic infection in cattle, which develop in three possible pathological forms: asymptomatic course, persistent lymphocytosis (PL) and lymphosarcoma. Once infected, cattle remain virus carriers for life and start to show a serological reaction within a few weeks after infection. Eradication and control of the disease is based on early diagnostic and segregation of the carriers. The agar gel immunodiffusion (AGID) test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA) has replaced the AGID for large scale testing. Although Argentina has over 60 million cattle population, no nationwide studies have been conducted yet to determine the prevalence of the infection. To estimate the rate of BLV infection in dairy cattle in Argentina, a survey for specific antibodies in >10,000 serum samples from animals over 18 months old, belonging to 363 different herds from the largest dairy production areas of the country, was carried out in our laboratory, along 1999. For this purpose, we developed an ELISA to detect serum antibodies against the BLV virus. The cut-off of the ELISA was established over 339 serum samples, using polymerase chain reaction and southern blot (PCR-SB) as confirmatory test. The sensitivity and specificity of the ELISA was of 97.2 and 97.5%, respectively, while the local official AGID test showed a sensitivity of 79.7% and specificity of 99.0%. To know the seroprevalence of BLV on dairy herds, and also the incidence of the infection within the herd, the serological survey was based on individual serum samples. The results show that the prevalence of infected individuals is of 32.85%, while the percentage of infected herds, harboring one or more infected animals, is of 84%. These results indicate a medium level of seropositive animals when taken individually, but a high prevalence of infected farms, which has been notoriously increased in the last 15 years as shown when compared with previous data from particular geographic areas, indicating that BLV constitutes a serious sanitary problem for dairy producers in Argentina. They also indicate the poor sensitivity of the official AGID test used in the country.

Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants.

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1.

The utilization of transgenic plants expressing recombinant antigens to be used in the formulation of experimental immunogens has been recently communicated. We report here the development of transgenic plants of alfalfa expressing the structural protein VP1 of foot and mouth disease virus (FMDV). The presence of the transgenes in the plants was confirmed by PCR and their specific transcription was demonstrated by RT-PCR. Mice parenterally immunized using leaf extracts or receiving in their diet freshly harvested leaves from the transgenic plants developed a virus-specific immune response. Animals immunized by either method elicited a specific antibody response to a synthetic peptide representing amino acid residues 135-160 of VP1, to the structural protein VP1, and to intact FMDV particles. Additionally, the immunized mice were protected against experimental challenge with the virus. We believe this is the first report demonstrating the induction of a protective systemic antibody response in animals fed transgenic plants expressing a viral antigen. These results support the feasibility of producing edible vaccines in transgenic forage plants, such as alfalfa, commonly used in the diet of domestic animals even for those antigens for which a systemic immune response is required.