Effects of vaccination with Brucella melitensis, strains Rev 1 ΔeryCD and Rev 1, on the reproductive system of young male goats.

The present study evaluates the effects of vaccination with Brucella melitensis strains Rev 1 ΔeryCD and Rev 1 on the reproductive system of male goats. Three groups, each of them consisting of 15 six-month-old brucellosis-free male goats, were studied. The first group was vaccinated with the Rev 1 ΔeryCD strain, the second group received Rev 1 and the third group was inoculated with sterile physiological saline solution. The dose of both strains was of 1×109CFU/ml. Over the course of the five months of this study, three males from each group were euthanized every month. Their reproductive tracts, spleens, and lymph nodes were collected to analyze serology, bacteriology PCR, histology, and immunohistochemistry. Results show that vaccination with B. melitensis strains Rev 1 ΔeryCD and Rev 1 does not harm the reproductive system of male goats. Strain B. melitensis Rev 1 ΔeryCD displayed a lower capacity to colonize the reproductive tract than strain Rev 1, which was attributed to its limited catabolic action toward erythritol.

Presence of Chlamydia abortus in colostrum, milk, and vaginal discharge samples of sheep

Background: The main transmission route of Chlamydia abortus is by ingesting the microorganism that has been eliminated in vaginal secretions, placental membranes or abortions that contaminate the environment and, possibly, through milk and colostrum. Elimination through vaginal secretions is well documented. However, there are no reports about isolation and identification of C. abortus in the colostrum or milk of infected sheep, so it is important to determine whether or not C. abortus may be present in these secretions, which are the only food of lambs. Objective: To detect C. abortus in colostrum, milk, and vaginal secretions of sheep with a history of reproductive disorders. Methods: Colostrum, milk, and vaginal exudates were collected from 66 sheep. The samples were inoculated in mouse fibroblast cell cultures and the presence of C. abortus determined by direct immunofluorescence. Results: 19 out of 66 colostrum samples (28.7%), 14 out of 66 milk samples (21.2%) and 17 out of 66 vaginal swabs (25.7%) were positive for C. abortus. The 50 samples positive for isolation and detected by immunofluorescence, together with 42 negative samples were subjected to qPCR to amplify a fragment of the ompA gene from C. abortus. Thirty-eight of the 92 samples processed by this technique were positive for C. abortus. Conclusion: The results demonstrated the presence of C. abortus in a high proportion in colostrum, milk and vaginal secretions of infected sheep. To the best of our knowledge, this is the first field study confirming the presence of C. abortus in colostrum, which shows that excretion of Chlamydia by lactogenesis could occur in the first hours after birth.

Antecedentes: La principal vía de transmisión de C. abortus es la ingestión del microorganismo que ha sido eliminado en las secreciones vaginales, membranas placentarias, abortos y, posiblemente, a través de la leche y el calostro. La eliminación a través de secreciones vaginales está bien documentada. Sin embargo, no existen reportes del aislamiento e identificación de C. abortus en el calostro o la leche de ovejas infectadas, por lo que es importante determinar si la bacteria puede o no estar presente en estas secreciones, que son el único alimento de los corderos. Objetivo: Detectar la presencia de C. abortus in calostro, leche y secreciones vaginales de ovejas con antecedentes de problemas reproductivos. Método: Con el propósito de aislar e identificar C. abortus en estas secreciones, se recolectó calostro, leche y exudado vaginal de 66 ovejas. Las muestras fueron inoculadas en cultivos celulares de fibroblastos de ratón y se determinó la presencia de la bacteria por inmunofluorescencia directa. Resultados: Fueron positivas 19 de 66 muestras de calostro (28,7%), 14 de 66 muestras de leche (21,2%) y 17 de 66 hisopos vaginales (25,7%). Las 50 muestras positivas al aislamiento y detectadas por inmunofluorescencia, junto con 42 negativas se sometieron a qPCR para amplificar un fragmento del gen ompA de C. abortus; 38 de las 92 muestras procesadas por esta técnica fueron positivas para C. abortus. Conclusión: Los resultados del presente estudio demostraron la presencia de C. abortus en una alta proporción en el calostro, la leche y las secreciones vaginales de ovejas infectadas. Este es el primer estudio de campo que confirma la presencia de C. abortus en calostro, lo que demuestra que la excreción de clamidia por lactogénesis podría ocurrir en las primeras horas después del nacimiento.

Antecedentes: A principal via de transmissão da Chlamydia abortus é a ingestão do microrganismo que foi eliminado nas secreções vaginais, membranas placentárias ou abortos que contaminam o meio ambiente e, possivelmente, através do leite e colostro. A eliminação pelas secreções vaginais está bem documentada. No entanto, não há relatos de isolamento e identificação de C. Abortus no colostro ou leite de ovelhas infectadas, por isso é importante verificar se a bactéria pode estar ou não presente nessas secreções, único alimento dos cordeiros. Objetivo: Detectar a presença de C. Abortus no colostro, leite e secreções vaginais de ovelhas com histórico de distúrbios reprodutivos Métodos: Para isolar e identificar C. Abortus nessas secreções, foram coletados colostro, leite e exsudato vaginal de 66 ovelhas. As amostras foram inoculadas em cultura de células de fibroblastos de camundongo e a presença da bactéria determinada por imunofluorescência direta. Resultados: 19 de 66 amostras de colostro (28,7%), 14 de 66 amostras de leite (21,2%) e 17 de 66 esfregaços vaginais (25,7%) sendo positivos. As 50 amostras positivas para isolamento e detectadas por imunofluorescência, juntamente com as 42 negativas, foram submetidas a qPCR para amplificar um fragmento do gene ompA de C. Abortus. Trinta e oito das 92 amostras processadas por esta técnica foram positivas para C. Abortus. Conclusão: Os resultados do presente estudo demonstraram a presença de C. Abortus em alta proporção no colostro, leite e secreções vaginais de ovelhas infectadas. Este trabalho é o primeiro estudo de campo na literatura científica confirmando a presença de C. Abortus no colostro, o que mostra que a excreção da clamídia por lactogênese pode ocorrer nas primeiras horas após o nascimento.

Genetic analysis of the pX region of bovine leukemia virus genotype 1 in Holstein Friesian cattle with different stages of infection.

The pX genetic region of bovine leukemia virus (BLV) includes four genes with overlapping reading frames that code for the Tax, Rex, R3, and G4 proteins. These proteins are involved in the regulation of transcriptional and post-transcriptional viral expression, as well as having oncogenic potential. Our goal was to investigate the pathogenicity of the pX region of BLV genotype 1 in terms of lymphocytosis, lymphomas, and proviral DNA load. We screened 724 serological samples from mixed-age Holstein Friesian cattle from six states in Mexico. Peripheral blood leukocytes (PBLs) were isolated from whole blood with anticoagulant, and genomic DNA was extracted from the PBLs using a commercial kit. Then, a set of primers that hybridize in conserved regions of the BLV pX region were used, which allowed for PCR standardization to detect proviral DNA in infected cells. Positive amplicons were sequenced using the Sanger method, resulting in 1156-nucleotide-long final sequences that included the four pX region genes. The experimental group consisted of 30 animals. Twelve of these had lymphocytosis, six had lymphoma, and 12 were apparently healthy cattle without any signs of lymphocytosis or lymphoma. The presence of lymphoma was detected in six bovine tumor tissues using histopathology, and the presence of BLV was detected by in situ hybridization. Phylogenetic analysis demonstrated that the 30 sequences were associated with genotype 1, and the genetic distance between the sequences ranged from 0.2% to 2.09%. We identified two sequences in the G4 gene: one with a three-nucleotide deletion resulting in the loss of a leucine (AGU_7488L, in a cow with lymphocytosis), and one with a nine-nucleotide deletion resulting in the loss of leucine, proline, and leucine (AGU_18A, in a cow without lymphocytosis). Analysis of the PX region indicated that positive selection had occurred in the G4, rex, and R3 genes, and we found no difference in proviral DNA load between the studied groups. We were unable to establish an association between variations in the pX region and the development of lymphocytosis, lymphoma, asymptomatic status, or proviral DNA load in BLV-infected cattle.

Serotyping versus genotyping in infected sheep and goats with small ruminant lentiviruses.

Despite SRLV infection being endemic in Mexico, there is little information regarding which genotypes are present. We compared serotyping and PCR-sequencing results from sheep and goats infected with SRLV. We separated plasma and peripheral blood leukocytes (PBL) from 1940 blood samples from sheep and goats from 12 states across Mexico. To detect SRLV infection, we tested plasma samples using two commercial ELISA kits (VMRD and Eradikit SRLV Screening). Then, we serotyped the infecting virus (A/ B) using Eradikit SRLV Genotyping. PBL DNA was used to detect the proviral genome via PCR. Positive amplicons were sequenced to identify viral genotypes using a phylogenetic analysis. Also, we analysed for residues differences in the sequences of a capsid epitope between genotypes. The serological results indicated a higher detection of seropositive animals using the VMRD ELISA compared to Eradikit, with 21 % and 15.3 % more in sheep and goats respectively. Only 25.7 % of the ELISA serotyping results matched those from PCR-sequencing. PCR-sequencing was able to identify genotype A, B and coinfections in animals classified as indeterminate by the ELISA test. This lack of sensitivity may be related to the lack of epitopes from the matrix and transmembrane peptides used by ELISA screening. Sequences analysis revealed that SRLVs found in sheep cluster with genetic subtypes A2 and B1, while those in goats cluster with subtypes A1 and B1. Serotyping did not prove to be an adequate method for predicting the viral genotype (A and / or B) in infections caused by SRLV.

First evidence of bovine immunodeficiency virus infection in Mexican cattle.

This study set out to identify the presence of bovine immunodeficiency virus (BIV) in animals geographically located in Mexico. BIV was first discovered in the United States in a dairy cow with persistent lymphocytosis, lymphoid hyperplasia and lymphocytic encephalitis. Many studies indicate that BIV infection is globally distributed, but its presence in Mexico remains unknown. We collected 1,168 heparinized blood samples from cattle in ten states across the Mexican Republic, then separated plasma using centrifugation and tested for antibodies against BIV. We used an indirect ELISA based on the use of a synthetic peptide derived from transmembrane glycoprotein (gp45/TM). In order to identify the viral genome, we designed a synthetic gene as a PCR control, as well as a pair of oligonucleotides for amplifying a 519 bp product of the env gene which encodes the surface protein. Positive amplicons were purified and subjected to nucleotide sequencing. A total of 189 (28.94%) tested plasma samples suggest the presence of specific anti-BIV antibodies in all states studied except for Chiapas. Additionally, PCR results identified six positive cows in the states of Puebla and Coahuila. BIV in these cows was confirmed via nucleotide sequencing and in silico analysis of these samples. This is the first report of the presence of BIV in Mexican cattle.

Signature patterns in region V4 of small ruminant lentivirus surface protein in sheep and goats.

The env gene in Small Ruminant Lentiviruses (SRLV) encodes the surface glycoprotein (SU) that divides into conserved (C1-C4) and variable regions (V1-V5). SRLV region V4 has been found to be homologous to the V3 region of human lentivirus (HIV). HIV V3 is responsible for tropism and the development of nervous clinical patterns when there is a tendency to conserve amino acids in specific "signature pattern" positions. The goal of this study was to identify signature patterns in the V4 region of the SU, which is encoded by the SRLV env gene. Secondarily, to understand how these signature patterns are associated with different clinical status in naturally infected sheep and goats. Starting with 244 samples from seropositive animals from nine Mexican states, we amplified the V4 region using nested PCR and obtained 49 SRLV sequences from peripheral blood leukocytes. Based on phylogenetic analysis results, we identified three groups: asymptomatic genotypes A (Ssx GA) and B (Ssx GB), as well as animals with arthritic presentation, genotype B (A GB). Similarity levels between group sequences ranged from 67.9%-86.7%, with a genetic diversity ranging from 12.7%-29.5% and a dN / dS ratio that indicated negative selection. Analyses using Vespa and Entropy programs identified four residues at positions 54, 78, 79 and 82 in SU region V4 as possible signature patterns, although with variable statistical significance. However, position 54 residues "N" (p = 0.017), "T" (p = 0.001) and "G" (p = 0.024) in groups A GB, Ssx GA and Ssx GB respectively, best characterized the signature patterns. The results obtained identified a signature pattern related to different genotypes and clinical status by SRLV in sheep and goats.

Lack of association between amino acid sequences of the bovine leukemia virus envelope and varying stages of infection in dairy cattle.

We collected 724 blood samples from dairy cattle from six Mexican states, and tested them for the presence of antibodies against BLV using a commercial ELISA test. Our study groups consisted of 32 samples: 12 asymptomatic cows, 12 cows with lymphocytosis and 8 samples of tumor tissue of the abomasum and heart of cattle with lymphoma. We designed three pairs of primers to amplify the complete BLV env gene, and obtained a fragment of 1548 nucleotides in length with the sequenced products. According to the phylogenetic tree we constructed to identify the viral genotype, 96.87 % of the sequences grouped into genotype 1, while a single sample from a cow with lymphocytosis (3.13 %) was associated with genotype 3 sequences. The similarity between the Mexican BLV sequences ranged from 0.985-1.00. In addition, the proportion of non-synonymous and synonymous mutations indicated negative selection. We did not identify any conserved residues in the viral protein sequences that could be related to BLV infection stage in cattle. Proviral quantification was performed using quantitative polymerase chain reaction, and we used Mood´s median test as statistical analysis. We found no significant association between proviral load and phase of infection. The sequences showed high similarity without any association between BLV surface glycoprotein and the different infection stages, nor differences in the proviral load. BLV genotype 1 was identified as prevalent in the studied samples, and for the first time in Mexico, we identified BLV genotype 3 in cattle.