Seropositivity for Bovine Viral Diarrhea and Enzootic Bovine Leukemia viruses in Blanco Orejinegro cattle in Colombia and infection associated-factors / Soropositividade para o vírus da diarréia viral bovina e leucemia bovina enzoótica em bovinos Blanco Orejinegro na Colômbia e fatores associados à infecção

ABSTRACT Serological controls for diseases of viral origin in animal production systems and the identification of factors associated with infections are decisive elements to establish prevention and control measures. The aim of this study was to establish the serological status for Bovine Viral Diarrhea (BVD) and Enzootic Bovine Leukemia (EBL) viruses in Blanco Orejinegro (BON) cattle from Colombia, and to identify the factors associated with seropositivity. A cross-sectional study was conducted with a total of 498 animals of all age groups and physiological states of the BON breed were selected, belonging to 14 herds located in 6 states of Colombia, in which a survey with 27 questions was conducted. By means of the chi-square test, possible factors associated with seropositivity against the 2 viruses were identified. A sample of 4 ml of blood was taken from each animal to extract plasma and make indirect Elisa tests to detect antibodies against both pathogens. General seropositivity of 27,1% was obtained for EBL, finding as factors associated with seropositivity the inadequate disposition of placental tissues after delivery of the cows and the non-performance of serological tests on new animals entering the herd. For BVD, seropositivity obtained was 50,6%, and the factors associated with seropositivity identified were having had a history of the disease in the herd, and using semen from bulls that are not known to be free for the infection. We suggest establishing control measures considering the factors associated with each viral infection to limit their expansion in the BON cattle production systems of Colombia.

RESUMO Os controles sorológicos para doenças de origem viral nos sistemas de produção animal e a identificação de fatores associados à infecção são elementos decisivos para estabelecer medidas de prevenção e controle. O objetivo deste estudo foi estabelecer o status sorológico dos vírus da Diarréia Viral Bovina (BVD) e da Leucemia Bovina Enzoótica (EBL) em bovinos Blanco Orejinegro (BON) da Colômbia e identificar os fatores associados à soropositividade. Foram selecionados 498 animais de todas as faixas etárias e estados fisiológicos da raça BON, de 14 rebanhos localizados em seis regiões da Colômbia, nos quais foi realizada uma pesquisa para identificar possíveis fatores associados à soropositividade contra os dois vírus. Foi retirada uma amostra de 4 ml de sangue de cada animal para extrair plasma e fazer testes ELISA indiretos para detectar anticorpos contra os dois patógenos. Obteve-se soropositividade geral de 27,1% para EBL, encontrando como fatores associados à soropositividade a disposição inadequada dos tecidos placentários após o parto das vacas e a não realização de testes sorológicos em novos animais que entraram no rebanho. Para a BVD, a soropositividade obtida foi de 50,6%, e os fatores associados à soropositividade identificados foram: histórico de doença no rebanho e uso de sêmen de touros que não são reconhecidos como livres da infecção. Sugerimos o estabelecimento de medidas de controle considerando os fatores associados a cada infecção viral para limitar sua expansão nos sistemas de produção de gado BON da Colômbia.

Análisis genético de cinco polimorfismos de nucleótido simple de caseínas lácteas obtenidos con chips genómicos en ganado Holstein de Antioquia, Colombia / Genetic analysis of five single nucleotide polymorphisms of dairy caseins obtained with genomic chips in Holstein cattle from Antioquia, Colombia

RESUMEN Los polimorfismos genéticos asociados con las caseínas de la leche son de gran importancia, ya que pueden ser usados como marcadores genéticos para mejorar el rendimiento productivo en los hatos lecheros. El objetivo de este estudio fue evaluar la diversidad y estructura genética de 5 SNP de caseínas de la leche, obtenidos con chips genómicos en vacas y toros de raza Holstein en Antioquia (Colombia). Fueron muestreados 113 animales de raza Holstein en 3 regiones del departamento de Antioquia (norte, centro y oriente) y un cuarto grupo de sementales comerciales. Los animales fueron genotipificados con chips genómicos de alta densidad (Illumina BovineHD e Illumina SNP50 v2), a partir de los cuales se identificaron 5 SNP (ARS-BFGL-NGS-8140, BTA-77380-no-rs, BTA-32346-no-rs, BTB-00821654 y ARS-BFGL-NGS-15809). Para cada SNP se realizó un análisis genético mediante un análisis de varianza molecular (amova) usando el software GenAIEx 6.501. Los SNP con mayor heterocigosidad total (HT) fueron ARS-BFGL-NGS-8140 y BTA-32346-no-rs, con resultados cercanos al 45%; sin embargo, la Ht para ARS-BFGL-NGS-15809, BTA-77380-no-rs y BTB-00821654 estuvo por debajo del 15%. El SNP con mayor diversidad genética fue BTA-32346-no-rs (Ho-He = 0,06; p < 0,05). En esta investigación se evaluó una subpoblación de toros comerciales extranjeros, en la cual se obtuvieron frecuencias alélicas y genotípicas similares a las obtenidas para las subpoblaciones locales, sugiriendo que los alelos de los toros muy posiblemente están fijados en dichas subpoblaciones, por lo que la estructura y diversidad genética tienden a ser bajas en la muestra de estudio.

ABSTRACT Genetic polymorphisms associated with milk caseins have a great importance since they can be used as genetic markers to improve productive performance in dairy herds. The main goal of the present study was to evaluate the diversity and genetic structure of 5 SNPs of milk caseins, obtained with genomic chip in Holstein cows and bulls from Antioquia (Colombia). 113 Holstein animals were sampled in 3 regions of Antioquia (north, center, and east), and a fourth group of commercial sires. Animals were geno-typed with high-density SNP chips (Illumina BovineHD and Illumina SNP50 v2), from which 5 SNPs were identified (ARS-BFGL-NGS-8140, BTA-77380-no-rs, BTA-32346-no-rs, BTB-00821654 and ARS-BFGL-NGS-15809). For each SNP, a genetic analysis was performed by means of an analysis of molecular variance (AMOVA) using the GenAIEx 6.501 software. The SNPs with the highest total heterozygosity (Ht) were ARS-BFGL-NGS-8140 and BTA-32346-no-rs, with results close to 45%; however, the HT for ARS-BFGL-NGS-15809, BTA-77380-no-rs, and BTB-00821654 were below 15%. The SNP with the highest genetic diversity was BTA-32346-no-rs (Ho-He = 0,06; p < 0,05). In this research a subpopulation of foreign commercial bulls was evaluated, in which similar allelic and genotypic frequencies to those for local subpopulations were obtained, suggesting that the alleles of the bulls are very possibly fixed in these subpopulations, so that the structure and genetic diversity tend to be low in the study sample.

IFN-γ mRNA expression is lower in Holstein cows infected with bovine leukemia virus with high proviral load and persistent lymphocytosis.

Bovine leukemia virus (BLV) is a retrovirus that affects primarily milky cows. Animals serologically positive to BLV show a Th1 cytokine profile with a predominance of interferon gamma (IFN-γ). IFN-γ has antiviral activity through mechanisms such as resistance to infection, inhibition of viral replication and apoptosis. The objective of this work was to determine the transcription levels of IFN-γ and its relationship with proviral load and persistent lymphocytosis in a population of Holstein cows of the province of Antioquia, Colombia. IFN-γ transcription levels were evaluated by qPCR in 140 Holstein cows. A one-way analysis of variance and a Student's t test were used to evaluate the differences between the means. The amount of IFN-γ mRNA found in BLV-positive cows was lower than in BLV-negative cows. Moreover, in the group of infected cows a lower level of IFN-γ mRNA expression was found in BLV and persistent lymphocytosis cows (BLV+PL) compared with BLV and aleukemia cows (BLV+AL). The level of IFN-γ mRNA expression was lower in cows with high proviral load (HPL) compared to cows with low proviral load (LPL). BLV infection is related to abnormal expression of IFN-γ mRNA, although IFN-γ has antiviral activity, its expression is affected by high proviral load. Keywords: cytokine; immune system; leukemia; bovine leukemia virus.

El componente racial influencia la resistencia a la infección con el virus de la leucosis bovina / The racial component influences resistance to infection with the bovine leukemia virus

RESUMEN El virus de la leucosis bovina (VLB) es un retrovirus que afecta principalmente el ganado lechero, reduciendo la producción de leche entre el 2,5 y 5%. La raza criolla colombiana Blanco Orejinegro (BON) es una raza rustica, bien adaptada, que ha mostrado resistencia in vitro a las infecciones ocasionadas por los virus de la fiebre aftosa y la estomatitis vesicular, así como las originadas por la bacteria Brucella abortus. El objetivo del presente estudio fue determinar si la raza BON y su cruce con Holstein son resistentes a la infección por el VLB. Se tomaron 124 muestras de sangre (59 Holstein, 40 BON y 25 BON x HOL) del mismo hato, se extrajo el DNA y se realizó una PCR-anidada correspondiente a una región del gen env de VLB. Se obtuvo un fragmento de 444 pb en los animales positivos. La prevalencia molecular del hato fue 33% para VLB. Se encontró diferencia significativa para infección por VLB entre los tres grupos raciales (p < 0,05). El porcentaje de infección fue del 55,9% para la raza Holstien, 5% para las vacas BON y 24% para el cruce BON x HOL; este último presentó una reducción en el porcentaje de infección del 32% respecto a la raza Holstein, lo cual puede ser atribuido a la presencia de genes de resistencia en la raza BON. Se comprobó que el nivel de infección es menor en vacas lecheras del cruce BON x HOL que en la raza lechera Holstein.

ABSTRACT The bovine leukemia virus (BLV) is a retrovirus that primarily affects dairy cattle, reducing milk production between 2.5 and 5%. The Colombian Blanco Orejinegro (BON) is a well-adapted, rustic, creole breed resistant to in vitro infections of Foot-and-mouth disease virus and vesicular stomatitis virus, as well as to Brucella abortus. This study aimed to determine if the crossing of BON and Holstein breeds is resistant to infection by BLV. Blood samples of 124 individuals (59 Holstein, 40 BON, and 25 BON x HOL) of the same herd were taken. The DNA was extracted, and a nested PCR was performed related to a region of the env gene of BLV. A fragment of 444 bp was obtained for positives animals. The molecular in-herd prevalence was 33% for BLV. A significant difference for BLV infection was found among the groups (p<0.05). The infection rate for the Holstein group was 55.9%, for BON cattle 5%, and for BON x HOL cattle 24%. The latter showed a reduction in the infection rate of 32% to the Holstein breed, which can be attributed to the presence of resistance genes in the BON breed. It was found that the level of infection is lower in BON x HOL cattle in contrast with Holstein dairy cows.

Resveratrol-cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol-methyl ß-cyclodextrin (RV-CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV-CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV-CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT-qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV-CD and embryo development and blastocysts gene expression by RT-qPCR were studied. A group without RV-CD (control- ) and a group with cyclodextrin (control+ ) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p < .05). Blastocysts produced by IVC with resveratrol showed that RV-CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p < .05). RV-CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts.

Genotyping of SNPs associated with meat tenderness: comparison of two PCR-based methods.

Single nucleotide polymorphisms (SNPs) carried in calpain (CAPN1), calpastatin (CAST), and leptin (LEP) genes are associated with meat tenderness. Due to the economic importance of this meat quality attribute, the development of fast, reliable, and affordable methods to identify bovine carriers of favorable alleles is of great importance for genetic improvement. Currently, PCR-RFLP is accepted as the standard gold method for genotyping SNPs associated with meat tenderness. But these SNPs can be detected by other techniques as high-resolution melting (HRM) analysis - a post-PCR method - that offers several advantages and has great application potential in the meat industry. In this study, we standardized, validated, and compared the performance of PCR-HRM to that of PCR-RFLP in genotyping bovine SNPs associated with meat tenderness: CAPN4751, CAPN316, CAST2959, CAST282, LEPE2FB, and LEPE2JW. We analyzed genotypes of a total of 380 bovines, 110 Bos taurus and 270 Bos indicus. Results obtained with PCR-HRM were consistent with those found by PCR-RLFP. Furthermore, HRM was found to be highly sensitive, and our results confirmed the repeatability (intra-assay precision) and reproducibility (inter-assay precision) of this assay. An internal control for endonuclease activity was created using site-directed mutagenesis to generate an additional enzymatic restriction point useful to discriminate SNP alleles. Our results show that PCR-HRM is an efficient method that produces reliable and rapid results. However, should be had in account that the method of DNA extraction, the quality and quantity of DNA, analyst-related variations, and primer design may generate challenges for allele discrimination.

Detection of hepatitis E virus genome in pig livers in Antioquia, Colombia.

Hepatitis E is a form of endemic acute hepatitis found in humans in many countries worldwide and is caused by the hepatitis E Virus (HEV). Detection of HEV in pigs indicates that they may be carriers, possibly through zoonosis. The prevalence of HEV in pigs in Colombia is unknown. Studies in the US found that 11% of pig livers sold in grocery stores are contaminated with HEV. It is also known that HEV can be inactivated when cooked, as it is labile to high temperatures. The aim of this study was to determine HEV contamination in pig livers sold in Medellín, Antioquia. A total of 150 livers from 5 slaughterhouses and 100 livers in grocery stores from different social strata of the city of Medellin analyzed to detect a segment of the HEV open reading frame-1 using reverse transcription-polymerase chain reaction. The results showed that 41.3% of pig livers from slaughterhouses and 25% of livers from grocery stores tested positive for HEV. Thus, the HEV genome is present in pig livers sold in Antioquia, revealing the presence of this virus in pigs from Colombia and the need subject entrails to proper cooking processes before consumption. Further research is required to determine the role of this virus in public health and pork production in Colombia.

Effect of two single nucleotide polymorphisms on milk yield and composition.

The bovine prolactin (PRL) and bovine growth hormone (bGH) genes exhibit several polymorphisms. Some of them can be detected by molecular techniques using restriction endonucleases, such as RsaI for the PRL gene and MspI for the bGH gene. We examined the relationship between the PRL-RsaI and bGH-MspI polymorphisms and some economically important characteristics of Holstein cows. Research was conducted on 315 Holstein cows from 5 municipalities in the Department of Antioquia, Colombia. Individuals were genotyped using PCR/RFLP. The statistical analysis was carried out using generalized linear models and a regression analysis. Polymorphism of the bGH gene was found to have a significant association with the percentage of protein in milk and milk yield. Genotype (-/-) was favorable for dairy yield, while genotype (+/+) was favorable for protein percentage. The PRL gene showed no significant association with any of the evaluated characteristics. The bGH gene appears to be a candidate for the implementation of marker-assisted selection programs. To determine the effect of the prolactin gene, research should be conducted with a larger sample size and a group of animals with more balanced genotypes.

Apoptosis as pathogenic mechanism of infection with vesicular stomatitis virus. Evidence in primary bovine fibroblast cultures.

To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resistance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of Blanco Orejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible to VSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible to VSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among the fibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for a possible relationship between resistance/susceptibility and production of factors with antiviral activity; however fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis where induced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Using morphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levels of apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosis and the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanism of VSV.

Apoptosis as pathogenic mechanism of infection with vesicular stomatitis virus: evidence in primary bovine fibroblast cultures

To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resistance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of Blanco Orejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible to VSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible to VSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among the fibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for a possible relationship between resistance/ susceptibility and production of factors wi th antiviral activity; however fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis where induced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Using morphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levels of apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosis and the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanism of VSV.

Cellular receptors for foot and mouth disease virus.

Foot-and-mouth disease virus (FMDV), the prototype member of the Aphthovirus genus, is a single-stranded, positive-sense RNA genome virus, which affects many domestic livestock cloven-hoofed animals, causing substantial lost of milk in dairy cattle, reduction in the growth rate of meat animals, among others. It has been shown that the virus can enter to the cells using different pathways; the main one binding integrins via the clathrin-mediated endocytosis pathway, trafficking throughout the acidified endocytic vesicles, where its capsid rapidly dissociates, resulting in the release of the RNA genome, and the second one using heparan sulfate in which FMDV enters to the cells using the caveola-mediated endocytosis pathway and that caveolae can associate and traffic with endosomes. Different integrins had been involved as FMDV receptors (alphavbeta1, alphavbeta3, alpha5beta1, alphavbeta6, alphavbeta8); this review will try to resume the basic information about FMDV receptors from the last years to the present and will resume the most important in vitro and in vivo studies to elucidate the role of this receptor on the infection.