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.

Deletion in KARLN intron 5 and predictive relationship with bovine tuberculosis and brucellosis infection phenotype.

Kalirin (gene: KALRN) is a Rho-GEF kinase linked to neurodegenerative diseases in humans. Unexpectedly, various polymorphisms in KALRN gene were previously associated with resistance to bacterial infections in ruminants. In this study, we evaluated the effect of the rs384223075 (RS-075) deletion in KALRN intron 5 on the occurrence of Mycobacterium bovis and Brucella abortus infections in cattle. We performed two separate case-control association analyses: one for bovine tuberculosis (bTB) using 308 Holstein and Jersey cows from three herds with prevalence between 5 and 15% for this infection; and another for brucellosis using 140 Holstein and beef crossbred cows from two herds with high prevalence for brucellosis (> 30%). In the bTB analysis, the RS-075 deletion frequency was higher among cases than controls (p = 0.0001), and the absence of the RS-075 deletion allele was associated with negative PPD-skin test results (p = 0.0009) at genotype level. On the contrary, RS-075 was not associated with Brucella spp. serological status (p = 0.72) but, unexpectedly, the deletion allele was more frequent among controls than cases in the beef crossbred herd (0.31 vs. 0.14, p = 0.02). In concordance with this observation, in vitro assays showed that the RS-075 deletion could be linked to an enhanced cellular response to bacterial antigens and unspecific stimulation in mononuclear cells derived from beef crossbred cows, specifically the reactive nitrogen species production (p = 0.008) and proliferation capacity (p = 0.018). This study is consistent with other reports that support an important role of the KALRN gene and its polymorphisms in the host response to intracellular pathogens.

Methodology for the identification of relevant loci for milk traits in dairy cattle, using machine learning algorithms.

Machine learning methods were considered efficient in identifying single nucleotide polymorphisms (SNP) underlying a trait of interest. This study aimed to construct predictive models using machine learning algorithms, to identify loci that best explain the variance in milk traits of dairy cattle. Further objectives involved validating the results by comparison with reported relevant regions and retrieving the pathways overrepresented by the genes flanking relevant SNPs. Regression models using XGBoost (XGB), LightGBM (LGB), and Random Forest (RF) algorithms were trained using estimated breeding values for milk production (EBVM), milk fat content (EBVF) and milk protein content (EBVP) as phenotypes and genotypes on 40417 SNPs as predictor variables. To evaluate their efficiency, metrics for actual vs. predicted values were determined in validation folds (XGB and LGB) and out-of-bag data (RF). Less than 4500 relevant SNPs were retrieved for each trait. Among the genes flanking them, signaling and transmembrane transporter activities were overrepresented. The models trained:•Predicted breeding values for animals not included in the dataset.•Were efficient in identifying a subset of SNPs explaining phenotypic variation. The results obtained using XGB and LGB algorithms agreed with previous results. Therefore, the method proposed could be applied for future association studies on milk traits.

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.

Single nucleotide polymorphisms from candidate genes associated with nematode resistance and resilience in Corriedale and Pampinta sheep in Argentina.

Selective breeding of genetically resistant animals is considered a promising strategy to face the problem of nematode resistance to anthelmintics and mitigate concerns about the presence of chemical residues in animal food products and the environment. Gastrointestinal nematode resistance is a complex, multifactorial trait related to host immunity. However, the mechanisms underlying host resistance and response to infection remain to be fully elucidated. In this context, the objective of this study was to provide insight into the chromosomal regions determining nematode resistance and resilience in Corriedale and resistance in Pampinta sheep breeds. A total of 170 single nucleotide polymorphisms (SNP) from 76 candidate genes for immune response were studied in 624 Corriedale and 304 Pampinta animals. Lambs underwent artificial or natural challenges with infective larvae mainly from Haemonchus contortus. Fecal egg counts, estimated breeding values for fecal egg counts, and rate of packed cell volume change and FAMACHA© score change over the challenge were used, when available, as indicators of host parasite resistance or resilience. Phenotype-genotype association studies were conducted and significance values obtained were adjusted for multiple testing errors. Eight SNPs, located on OARs 3, 6, 12, and 20, reached significance in Corriedale sheep under artificial challenge. Those SNP represent allelic variants from the MHC-Ovine Lymphocyte Antigen-DRA, two C-type lectin domain families, the Interleukin 2 receptor ß, the Toll-like receptor 10, the Mannan binding lectin serine peptidase 2, and the NLR family, CARD domain containing 4 genes. On Pampinta lambs under natural challenge, we found three significant SNPs, located in the TIMP metallopeptidase inhibitor 3, the FBJ murine osteosarcoma viral oncogene homolog, and the Interleukin 20 receptor alpha genes, on OARs 3, 7, and 8, respectively. The results obtained herein confirm genomic regions previously reported as associated with nematode resistance in other sheep breeds, reinforcing their role in host response to parasites. These findings contribute to gain knowledge on parasite resistance and resilience in Corriedale sheep and report for the first time SNPs associated with resistance to gastrointestinal parasite infections in Pampinta breed.

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.

Association of an IRF3 putative functional uORF variant with resistance to Brucella infection: A candidate gene based analysis of InDel polymorphisms in goats.

Brucellosis is an important zoonotic disease caused by infection with Brucella spp. It generates major economic losses in livestock production worldwide. Goats are the principal hosts of B. melitensis, the main infection agent of caprine and human brucellosis. The selection of resistance-related genes is considered one of the best long-term means to improve control to bacterial infection in domestic ruminants. We performed a candidate gene association study to test if six short insertion/deletion polymorphisms (InDels) at bacterial-infection related genes influence the resistance to Brucella infection in female creole goats. InDels (IRF3-540: rs660531540, FKBP5-294: rs448529294, TIRAP-561: rs657494561, PTPRT-588: rs667380588, KALRN-989: rs667660989 and RAB5a-016: rs661537016) were resolved by PCR-capillary electrophoresis in samples from 64 cases and 64 controls for brucellosis. Allelic frequencies were significantly different between cases and controls at IRF3-540 and KALRN-989 (p = 0.001 and 0.005). Indeed, the minor alleles (a and k) at InDels IRF3-540 and KALRN-989 were more frequent among controls than cases, providing evidence that these alleles confer protection against Brucella infection. Moreover, IRF3-540 a-containing genotypes (Aa and aa) were associated with absence of Brucella-specific antibodies in goats (p = 0.003; OR = 3.52; 95% CI = 1.55-7.96), and more specifically, a-allele was associated with resistance to Brucella infection in a dose-dependent manner. Also, we observed that the IRF3-540 deletion (a-allele) extends a conserved upstream ORF by 75 nucleotides to the main ORF, and thus it may decrease gene expression by reducing translation efficiency from the main ORF. These results suggest a potential functional role of IRF3-540 deletion in genetic resistance to Brucella infection in goats.

Single nucleotide polymorphisms in candidate genes associated with milk yield in Argentinean Holstein and Holstein x Jersey cows.

BACKGROUND: Research on loci influencing milk production traits of dairy cattle is one of the main topics of investigation in livestock. Many genomic regions and polymorphisms associated with dairy production have been reported worldwide. In this context, the purpose of this study was to identify candidate loci associated with milk yield in Argentinean dairy cattle. A database of candidate genes and single nucleotide polymorphisms (SNPs) for milk production and composition was developed. Thirty-nine SNPs belonging to 22 candidate genes were genotyped on 1643 animals (Holstein and Holstein x Jersey). The genotypes obtained were subjected to association studies considering the whole population and discriminating the population by Holstein breed percentage. Phenotypic data consisted of milk production values recorded during the first lactation of 1156 Holstein and 462 Holstein x Jersey cows from 18 dairy farms located in the central dairy area of Argentina. From these records, 305-day cumulative milk production values were predicted. RESULTS: Eight SNPs (rs43375517, rs29004488, rs132812135, rs137651874, rs109191047, rs135164815, rs43706485, and rs41255693), located on six Bos taurus autosomes (BTA4, BTA6, BTA19, BTA20, BTA22, and BTA26), showed suggestive associations with 305-day cumulative milk production (under Benjamini-Hochberg procedure with a false discovery rate of 0.1). Two of those SNPs (rs43375517 and rs135164815) were significantly associated with milk production (Bonferroni adjusted p-values < 0.05) when considering the Holstein population. CONCLUSIONS: The results obtained are consistent with previously reported associations in other Holstein populations. Furthermore, the SNPs found to influence bovine milk production in this study may be used as possible candidate SNPs for marker-assisted selection programs in Argentinean dairy cattle.

Genome-wide scan for commons SNPs affecting bovine leukemia virus infection level in dairy cattle.

BACKGROUND: Bovine leukemia virus (BLV) infection is omnipresent in dairy herds causing direct economic losses due to trade restrictions and lymphosarcoma-related deaths. Milk production drops and increase in the culling rate are also relevant and usually neglected. The BLV provirus persists throughout a lifetime and an inter-individual variation is observed in the level of infection (LI) in vivo. High LI is strongly correlated with disease progression and BLV transmission among herd mates. In a context of high prevalence, classical control strategies are economically prohibitive. Alternatively, host genomics studies aiming to dissect loci associated with LI are potentially useful tools for genetic selection programs tending to abrogate the viral spreading. The LI was measured through the proviral load (PVL) set-point and white blood cells (WBC) counts. The goals of this work were to gain insight into the contribution of SNPs (bovine 50KSNP panel) on LI variability and to identify genomics regions underlying this trait. RESULTS: We quantified anti-p24 response and total leukocytes count in peripheral blood from 1800 cows and used these to select 800 individuals with extreme phenotypes in WBCs and PVL. Two case-control genomic association studies using linear mixed models (LMMs) considering population stratification were performed. The proportion of the variance captured by all QC-passed SNPs represented 0.63 (SE ± 0.14) of the phenotypic variance for PVL and 0.56 (SE ± 0.15) for WBCs. Overall, significant associations (Bonferroni's corrected -log10p > 5.94) were shared for both phenotypes by 24 SNPs within the Bovine MHC. Founder haplotypes were used to measure the linkage disequilibrium (LD) extent (r2 = 0.22 ± 0.27 at inter-SNP distance of 25-50 kb). The SNPs and LD blocks indicated genes potentially associated with LI in infected cows: i.e. relevant immune response related genes (DQA1, DRB3, BOLA-A, LTA, LTB, TNF, IER3, GRP111, CRISP1), several genes involved in cell cytoskeletal reorganization (CD2AP, PKHD1, FLOT1, TUBB5) and modelling of the extracellular matrix (TRAM2, TNXB). Host transcription factors (TFs) were also highlighted (TFAP2D; ABT1, GCM1, PRRC2A). CONCLUSIONS: Data obtained represent a step forward to understand the biology of BLV-bovine interaction, and provide genetic information potentially applicable to selective breeding programs.

A haplotype at intron 8 of PTPRT gene is associated with resistance to Brucella infection in Argentinian creole goats.

Brucellosis is the leading zoonosis on a worldwide scale and constitutes a major public health threat in many regions of the world. Several molecular markers associated with natural resistance to intracellular bacterial infection have been identified. Recently seven single-nucleotide polymorphisms (SNPs) located in the PTPRT gene were associated with resistance to Mycobacterium bovis infection in cattle. Here, we perform a case-control study to test if polymorphisms at PTPRT intron 8 might influence the resistance or susceptibility to Brucella infection in goats. DNA samples from 22 seropositive (cases) and 22 seronegative (controls) for brucellosis, unrelated female creole goats, were included in the present study. Four previously reported polymorphisms (SNP1: rs643551276, SNP2: rs651618967, SNP3: rs662137815 and SNP4: rs657542977) and a new SNP (SNP5: chr13: 691695526) were detected by PCR-DNA sequencing method. Genotypic and allelic frequencies differed significantly between cases and controls at SNPs 1, 2, 4 and 5 (p≤0.001). Indeed, the SNP1 TT, SNP2 TT, SNP4 CC and SNP5 TT genotypes were associated with absence of Brucella-specific antibodies (ORs=0.019 to 0.045). Moreover, haplotype association analysis revealed a significant association of the TTCCT haplotype with protection to Brucella infection (p≤1×10-4; OR=18), including the major allelic variants associated with resistance. These results represent the first evidence of genetic association between polymorphisms in the PTPRT gene and absence of brucellosis in goats.

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)

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)

Bovine leukemia virus becomes established in dairy herds before the first lactation.

In this work, we studied seven groups of pregnant heifers from a consortium of dairy farms heavily infected with bovine leukemia virus (BLV). ELISA testing showed that the seroprevalence ranges of BLV in heifers between 36.1 and 66.5 %. No significant differences in proviral load were found when comparing heifers with adult cattle. Before their first delivery, more than 9.8 % of heifers show a high proviral load. Because BLV infection can occur during the first two years of life, the rationale of any strategy should be to take action as early as possible after birth.

Genetic Diversity and Population Structure in South African, French and Argentinian Angora Goats from Genome-Wide SNP Data.

The Angora goat populations in Argentina (AR), France (FR) and South Africa (SA) have been kept geographically and genetically distinct. Due to country-specific selection and breeding strategies, there is a need to characterize the populations on a genetic level. In this study we analysed genetic variability of Angora goats from three distinct geographical regions using the standardized 50k Goat SNP Chip. A total of 104 goats (AR: 30; FR: 26; SA: 48) were genotyped. Heterozygosity values as well as inbreeding coefficients across all autosomes per population were calculated. Diversity, as measured by expected heterozygosity (HE) ranged from 0.371 in the SA population to 0.397 in the AR population. The SA goats were the only population with a positive average inbreeding coefficient value of 0.009. After merging the three datasets, standard QC and LD-pruning, 15 105 SNPs remained for further analyses. Principal component and clustering analyses were used to visualize individual relationships within and between populations. All SA Angora goats were separated from the others and formed a well-defined, unique cluster, while outliers were identified in the FR and AR breeds. Apparent admixture between the AR and FR populations was observed, while both these populations showed signs of having some common ancestry with the SA goats. LD averaged over adjacent loci within the three populations per chromosome were calculated. The highest LD values estimated across populations were observed in the shorter intervals across populations. The Ne for the Angora breed was estimated to be 149 animals ten generations ago indicating a declining trend. Results confirmed that geographic isolation and different selection strategies caused genetic distinctiveness between the populations.

Association of TNF-α gene promoter region polymorphisms in bovine leukemia virus (BLV)-infected cattle with different proviral loads.

Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine involved in the immune response against viral and other infections. Its expression levels are affected by a polymorphism in the promoter region of the gene. Bovine leukemia virus is a retrovirus that infects cattle and develops two different infection profiles in the host. One profile is characterized by a high number of proviral copies integrated into the host genome and a strong immune response against the virus, while the most relevant property of the other profile is that the number of copies integrated into the host genome is almost undetectable and the immune response is very weak. We selected a population of cattle sufficiently large for statistical analysis and classified them according to whether they had a high or low proviral load (HPL or LPL). Polymorphisms in the promoter region were identified by PCR-RFLP. The results indicated that, in the HPL group, the three possible genotypes were normally distributed and that, in the LPL group, there was a significant association between the proviral load and a low frequency of the G/G genotype at position -824.

Single nucleotide polymorphisms in candidate genes and their relation with somatic cell scores in Argentinean dairy cattle.

The prevention and control of bovine mastitis by enhancing natural defenses in animals is important to improve the quality of dairy products. Mastitis resistance is a complex trait which depends on genetic components, as well as environmental and physiological factors. The limitations of classical control measures have led to the search for alternative approaches to minimize the use of antibiotics by selecting naturally resistant animals. Polymorphisms in genes associated with the innate immune system are strong candidates to be evaluated as genetic markers. In this work, we evaluated a set of single nucleotide polymorphisms (SNPs) in candidate genes for health and production traits, and determined their association with the somatic cell score (SCS) as an indicator of mastitis in Argentinean dairy cattle. We evaluated 941 cows: Holstein (n = 677) and Holstein × Jersey (n = 264) crossbred, daughters from 22 bulls from 14 dairy farms located in the central dairy area of Argentina. Two of the 21 successfully genotyped markers were found to be significantly associated (p < 0.05) with the SCS: GHR_140 and OPN_8514C-T. The heterozygote genotype for GHR_140 showed a favorable effect in reducing the SCS. On the other hand, heterozygote genotypes for OPN8514C-T caused an increase in the SCS; moreover, combined genotypes for OPN SNPs showed an even larger effect. These findings can contribute to the design of effective marker-assisted selection programs.

Polymorphisms at the 3' untranslated region of SLC11A1 gene are associated with protection to Brucella infection in goats.

Goats are susceptible to brucellosis and the detection of Brucella-infected animals is carried out by serological tests. In other ruminant species, polymorphisms in microsatellites (Ms) of 3' untranslated region (3'UTR) of the solute carrier family 11 member A1 (SLC11A1) gene were associated with resistance to Brucella abortus infection. Goats present two polymorphic Ms at the 3'UTR end of SLC11A1 gene, called regions A and B. Here, we evaluated if polymorphisms in regions A and/or B are associated with Brucella infection in goats. Serum (for the detection of Brucella-specific antibodies) and hair samples (for DNA isolation and structure analysis of the SLC11A1 gene) were randomly collected from 229 adult native goats from the northwest of Argentina. Serological status was evaluated by buffer plate antigen test (BPAT) complemented by the fluorescent polarization assay (FPA), and the genotype of the 3'UTR of the SLC11A1 gene was determined by capillary electrophoresis and confirmed by sequence analysis. Polymorphisms in regions A and B of the 3'UTR SLC11A1 gene were found statistically significant associated with protection to Brucella infection. Specifically, the association study indicates statistical significance of the allele A15 and B7/B7 genotype with absence of Brucella-specific antibodies (p=0.0003 and 0.0088, respectively). These data open a promising opportunity for limiting goat brucellosis through selective breeding of animals based on genetic markers associated with natural resistance to B. melitensis infection.

Evaluation of total white blood cell count as a marker for proviral load of bovine leukemia virus in dairy cattle from herds with a high seroprevalence of antibodies against bovine leukemia virus.

OBJECTIVE: To determine the reference interval for WBC counts in Holstein dairy cows from herds with high seroprevalence for anti-bovine leukemia virus (BLV) antibodies, analyze the correlation of total WBC counts and blood proviral load (bPVL) in BLV-infected animals, and determine whether total WBC count can be used a hematologic marker for in vivo infection. ANIMALS: 307 lactating cows from 16 dairy herds with high BLV seroprevalence. PROCEDURES: Blood samples were collected for assessment of plasma anti-BLV p24 antibody concentration (all cows), manual determination of WBC count (161 BLV-seronegative cows from 15 herds), and evaluation of bPVL (146 cows from another herd). RESULTS: The WBC count reference interval (ie, mean ± 2 SD) for BLV-seronegative dairy cows was 2,153 to 11,493 cells/µL. Of the 146 cows used to analyze the correlation between WBC count and bPVL, 107 (73%) had WBC counts within the reference interval; of those cows, only 21 (19.6%) had high bPVL. Most cows with high WBC counts (35/39) had high bPVL. Mean WBC count for cows with high bPVL was significantly higher than values for cows with low or undetectable bPVL. White blood cell counts and bPVL were significantly (ρ = 0.71) correlated. CONCLUSIONS AND CLINICAL RELEVANCE: These data have provided an updated reference interval for WBC counts in Holstein cows from herds with high BLV seroprevalence. In dairy cattle under natural conditions, WBC count was correlated with bPVL; thus, WBC count determination could be a potential tool for monitoring BLV infection levels in attempts to control transmission.

Bovine leukemia virus p24 antibodies reflect blood proviral load.

BACKGROUND: Bovine leukemia virus (BLV) is worldwide distributed and highly endemic in Argentina. Among the strategies to prevent BLV dissemination, a control plan based on the selective segregation of animals according to their proviral load (PVL) is promising for our dairy productive system. The objective of this work was to study the relationship between the blood PVL and the antibody level, in order to identify whether the individual humoral response, i.e. the anti-p24 or anti-whole-BLV particle, could be used as a marker of the blood level of infection and thus help to recruit animals that may pose a lower risk of dissemination under natural conditions. RESULTS: The prevalence of p24 antibodies on the 15 farms studied was over 66%. The prevalence of p24 and whole-BLV antibodies and PVL quantification were analyzed in all the samples (n = 196) taken from herds T1 and 51. ROC analysis showed a higher AUC for p24 antibodies than whole-BLV antibodies (Z(reactivity): 3.55, P < 0.001; Z(titer): 2.88, P < 0.01), and as consequence a better performance to predict the proviral load status in herd 51. No significant differences were found between the performance of p24 and whole-BLV antibodies in herd T1. A significant positive correlation was observed between PVL values and p24 antibody reactivity in both farms (r (T1) = 0.7, P < 0.001, r (51) = 0.71, P < 0.0001). The analysis was extended to the whole number of weak p24 antibody reactors (n = 311) of the other 13 farms. The mean of high PVL reactors within weak p24 reactors was 17.38% (SD = 8.92). In 5/15 farms, the number of weak p24 reactors with high PVL was lower than 10%. CONCLUSIONS: We found that the humoral response reflected the level of in vivo infection, and may therefore have useful epidemiological applications. Whereas the quantitative evaluation of blood proviral load using real-time PCR is expensive and technically demanding, the measurement of antibodies in blood by ELISA is relatively straightforward and could therefore constitute a cost-effective tool in a BLV control intervention strategy, especially in highly infected herds such as Argentinean dairy ones.