Modification of a previously patented method to unequivocally score A2-like and A1-like bovine ß-casein variants.

A growing interest in the production and commercialization of A2 cow's milk has been observed in many countries in the last few years due to the beneficial properties for human health attributed to A2 ß-casein variant. Methods of varying complexity and different equipment requirements have been proposed for the determination of the ß-casein genotype of individual cows. We proposed herein a modification of a previously patented method based on an amplification-created restriction site PCR followed by restriction fragment length polymorphism analysis. This method allows to identify and differentiate A2-like from A1-like ß-casein variants, after differential endonuclease cleavage flanking the nucleotide that determines the amino acid at position 67 of ß-casein. The advantages of this method are that it: • enables to unequivocally score A2-like as well as A1-like ß-casein variants, • can be performed at low cost in simply equipped molecular biology laboratories, and • can be scaled up to analyze hundreds of samples per day. For these reasons, and based on the results obtained from the analysis carried out in this work, it showed to be a reliable method for the screening of herds to selective breeding of homozygous cows and bulls for A2 or A2-like alleles.

Diversity of the BoLA-DRB3 gene in cattle breeds from tropical and subtropical regions of Argentina.

Bovine leukocyte antigens (BoLA) have been widely studied because of their primary function in the recognition of pathogens by the immune system. To date, however, the characterization of the BoLA-DRB3 gene in Latin American Zebu and mixed zebuine breeds is scarce. By a sequence-based typing method, here we sequenced exon 2 of BoLA class II DRB3 gene in 264 animals from the five most commonly used breeds in northern Argentina (Creole, Brahman, Braford, Brangus, and Nellore).The Bos taurus, Bos indicus, and mixed breeds analyzed here contained 61 previously reported alleles. Genetic diversity was high at both allelic and nucleotide sequence levels, particularly in the mixed breeds Braford and Brangus. In contrast to previous reports on DRB3 diversity, no evidence of balancing selection was found in our data. Differentiation among breeds was highly significant, as shown by FST (FST = 0.052, P < 0.001) and cluster analyses. In accordance with historical origin of the breeds, UPGMA trees and metric multidimensional scaling (MDS) analyses showed that Creole is distantly related to the other zebuine breeds. Among them, Brahman, Braford, and Brangus exhibited the closest affiliations. Despite the overall differentiation of the breeds, analysis of the peptide binding regions at the aminoacid level revealed that the key aminoacids involved in peptide recognition are greatly conserved suggesting little influence of domestication and breeding in functional MHC variability. In sum, this is the first report of BoLA-DRB3 diversity in pure and mixed Bos indicus cattle breeds from Argentina. Knowledge of BoLA-DRB3 variability in breeds adapted to tropical and subtropical environments contributes not only to the characterization of MHC diversity but also to the design of peptide-based vaccines.

Generalized glycogenosis in Brahman-derived breeds: diagnosis and prevalence in Argentina.

Generalized glycogenosis is a lethal autosomal recessive disease caused by a deficient activity of the acidic 1,4-α-glucosidase enzyme and characterized by an accumulation of glycogen within lysosomes. Three mutations in the GAA gene causing bovine generalized glycogenosis have been identified in two cattle breeds, Brahman and Shorthorn. The objective of this study was to evaluate the prevalence of carriers of the E7 mutation in the GAA gene in Argentinean Brahman-derived herds. A total of 930 Braford, 94 Brangus, and 8 Brahman samples were analyzed. The genotyping was done by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). We found that 12.02% (95% CI 12.00-12.04) of the total number of samples received were heterozygous (i.e., carriers) for the E7 mutation, while 12.58% (95% CI 12.56-12.60) of the Braford, 6.38% (95% CI 6.26-6.51) of the Brangus, and 12.50% (95% CI 9.82-15.18) of the Brahman samples were carriers of this loss-of-function allele. Neither breed nor sex were significantly associated to the presence of the mutation. The prevalence informed in this study is similar to the average prevalence reported for Australian Brahmans. The finding of heterozygous animals suggests that breeders and insemination centers should continue screening their herds to minimize the dissemination of this deleterious allele.

A novel association of BoLA DRB3 alleles in BLV infected cattle with different proviral loads / Uma nova associação de alelos de BoLA DRB3 em bovinos infectados com BLV com diferentes cargas provirais

Bovine leukemia virus (BLV) is associated with the most common neoplastic disease of cattle. BLV has a silent dissemination in the herd due to infected cell exchange, thus the concentration of BLV-infected cells in blood should play a major role in the success of viral transmission. Genes from Bovine leukocyte antigen (BoLA), the MHC system of cattle, are associated with genetic resistance and susceptibility to a wide range of diseases, and also with production traits. Some BoLA DRB3.2 allele polymorphisms in Holstein cattle have been associated with resistance or susceptibility to BLV-disease development, or with proviral load (PVL). This investigation studied 107 BLV-infected Argentinean Holstein dairy cows, all of them belonging to one herd. PVL was analysed by qPCR and animals were classified as high proviral load (HPVL, N = 88) and low proviral load (LPVL, N = 19), and BoLA DRB3.2 alleles were genotyped. Alleles BoLA DRB3.2*1501 and *1201 were significantly associated with HPVL (p = 0.0230 and p = 0.0111 respectively), while allele BoLA DRB3.2*0201 was significantly associated with LPVL (p = 0.0030). The present study aims at contributing to the knowledge of the association between BoLA polymorphism and development of a BLV infection profile. Genes that best explain the PVL in this population resulted BoLA DRB3.2*0201 (as a protection factor) and *1501 (as a risk factor). Allelic differences may play an important role in the development of effective immune responses. A better understanding of how BoLA polymorphism contributes to these responses and the establishment of a BLV status is desirable to schedule and evaluate control measures.(AU)

O vírus da leucemia bovina (BLV) está associado à doença neoplásica mais comum do gado bovino. O BLV tem uma disseminação silenciosa no rebanho devido à troca de células infectadas, assim, a concentração de células BLV infectadas no sangue deve desempenhar um papel importante no sucesso da transmissão viral. Os genes do antígeno leucocitário bovino (BoLA), sistema MHC do gado bovino, estão associados à resistência genética e à susceptibilidade a uma ampla gama de doenças, bem como às características da produção. Alguns polimorfismos de alelos de BoLA DRB3.2 em bovinos Holstein têm sido associados à resistência ou susceptibilidade ao desenvolvimento da doença BLV, ou com carga proviral (PVL). Esta investigação avaliou 107 vacas leiteiras da raça Holstein argentina infectadas com BLV e pertencentes a um único rebanho. A PVL foi analisada por qPCR, os animais foram classificados em alta carga proviral (HPVL, N = 88) e baixa carga proviral (LPVL, N = 19), e os alelos BoLA DRB3.2 foram genotipados. Os alelos BoLA DRB3.2*1501 e *1201 estavam significativamente relacionados à HPVL (p = 0,0230 e p = 0,0111, respectivamente), enquanto o alelo BoLA DRB3.2*0201, à LPVL (p = 0,0030). O objetivo deste estudo é contribuir para o conhecimento da associação entre o polimorfismo de BoLA e o desenvolvimento de infecção por BLV. Os genes que melhor explicam a PVL na população analisada resultaram em BoLA DRB3.2*0201 (como fator de proteção) e *1501 (como fator de risco). As diferenças alélicas podem desempenhar um papel importante no desenvolvimento de respostas imunitárias eficazes. Uma melhor compreensão de como o polimorfismo BoLA contribui para estas respostas e o estabelecimento de um estado BLV é desejável para agendar e avaliar as medidas de controle.(AU)