Polymorphism of bovine lipocalin-2 gene and its impact on milk production traits and mastitis in Holstein Friesian cattle

Background: Mastitis is one of the most serious diseases of dairy cattle, causing substantial financial losses. While predisposition to reduced somatic cell count in milk has been considered for in cattle breeding programs as the key indicator of udder health status, scientists are seeking genetic markers of innate immune response, which could be helpful in selecting cows with improved immunity to mastitis. Lipocalin-2 (LCN2) is a protein involved in the response of the immune system by eliminating iron ions which are necessary for the growth of pathogenic bacteria, so LCN2 may be considered as a natural bacteriostatic agent and could become a marker of infection. Results: A total of five SNPs were identified in LCN2 gene (one in the promoter, three in exon 1, and one in intron 1). A single haplotype block was identified. The locus g.98793763GNC was found to have a significant impact on protein levels in milk, and alleles of this locus were identified to have a significant positive dominance effect on this trait. None of the four analysed loci had a statistically significant impact on the milk yield, fat levels in milk or the somatic cell score. LCN-2 gene had no significant impact on the incidence of mastitis in the cows. Conclusions: Although the identified SNPs were not found to have any impact on the somatic cell count or the incidence of mastitis in cows, it seems that further research is necessary, covering a larger population of cattle, to confirm the association between lipocalin-2 and milk production traits and mastitis.

Caracterização fenotípica pela técnica de MALDI-TOF de Staphylococcus coagulase negativa isolados de mastite bovina em rebanhos leiteiros do Sul de Minas Gerais / Phenotypic characterization by the MALDI-TOF technique of Staphylococcus coagulase negative isolated from bovine mastitis in dairy herds of the South of Minas Gerais

O objetivo deste trabalho foi identificar espécies de Staphylococcus coagulase negativa em leite de vacas leiteiras com mastite por testes fenotípicos e pela técnica de MALDI-TOF MS. Foram utilizadas 85 isolados de estafilococos provenientes de leite de vacas com mastite. Todos os isolados foram caracterizados fenotipicamente por avaliação da morfologia das colônias, coloração de Gram, testes de oxidase, catalase e coagulase. Posteriormente, foram analisados os espectros de proteínas gerados no MALDI-TOF MS, seguida pela separação e detecção de íons pelo tempo de voo (TOF).Foram identificadas sete espécies, sendo a de maior ocorrência S. chromogenes 65 (76%), seguida por, S. hyicus 5 (6%), S. epidermidis 4 (5%).A técnica de MALDI-TOF demonstrou-se efetiva na caracterização de espécies de Staphylococcus causadores de mastite bovina.

Perfiles genéticos de Streptococcus uberis aislados de mastitis bovina / Genetic patterns of Streptococcus uberis isolated from bovine mastitis

El objetivo del presente estudio fue evaluar las relaciones genotípicas entre 40 Streptococcus uberis aislados de mastitis bovina mediante la técnica de electroforesis de campos pulsantes (pulsed-field gel electrophoresis [PFGE]). Además, se investigó la asociación entre los patrones de PFGE y los perfiles de virulencia. Los aislamientos mostraron 17 patrones de PFGE. Se encontraron diferentes cepas dentro de los tambos y en los distintos tambos, y un bajo número de aislamientos dentro del mismo tambo compartieron un perfil idéntico de PFGE. No se encontró ninguna asociación entre los patrones de PFGE y los perfiles de virulencia. Sin embargo, la detección de cepas particulares en algunos tambos podría indicar que algunas de ellas son más virulentas que otras. Sería importante avanzar en las investigaciones para identificar nuevos genes relacionados con la virulencia que podrían contribuir a la capacidad infecciosa de estas cepas

The aim of this study was to evaluate the genotypic relationships among 40 Streptococcus uberis isolated from bovine mastitis by using pulsed-field gel electrophoresis (PFGE). Additionally, the association between PFGE patterns and virulence profiles was investigated. The isolates exhibited 17 PFGE patterns. Different strains were found within and among herds; however, a low number of isolates within the same herd shared an identical PFGE type. No association between PFGE patterns and virulence profiles was found. However, the detection of specific strains in some herds could indicate that some strains are more virulent than others. Further research needs to be undertaken to elucidate new virulence-associated genes that might contribute to the capability of these strains to produce infection

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and alpha-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.

Characterization of single nucleotide polymorphisms [SNPs] in the 5'-flanking region of bovine lactoferrin gene in the local and Holstein cattle of Iran

Lactoferrin [Lf], an iron binding glycoprotein, has a variety of physiological roles and its foremost is antimicrobial properties. Therefore, the lactoferrin gene can be considered as a potential candidate gene for resistance to mastitis. This study was carried out to determine the haplotype frequency of the 5'-flanking region of bovine lactoferrin gene in local and Holstein cattle breeds of Iran using PCR-SSCPand DNA sequencing. Genomic DNA was isolated from 100 blood samples of two cattle breeds [50 Local, 50 Holstein]. Two new primer pairs were designed from Lf sequence to amplify a part of 5'-flanking region of the gene. The amplified fragment was screened by single strand conformation polymorphism [SSCP] and DNA sequencing. The multiple alignments were carried out for the nucleotide sequences of different SSCPpatterns. In silico analysis of identified single nucleotide polymorphisms [SNPs] within the 5'-flanking region of bovine Lf gene was screened, to identify any association on transcription factor binding affinity. Analysis of the whole samples revealed three SSCP patterns [A, B and C] for amplified fragment that C haplotype was the only variant identified in local breed samples. The bioinformatics analysis revealed that the Tto G trans version at position -602 created an AML-1 transcription binding site in combined genotype A. In -586 position, Tto C transition abolished binding site of AML-1 transcription factor. Therefore, to apply Lf gene for marker-assisted selection, additional studies are required to evaluate the functional role of these identified polymorphic sites on gene expression and somatic cell counts in cattle

Genotypic and phenotypic detection of capsular polysaccharides in Staphylococcus aureus isolated from bovine intramammary infections in Argentina

Staphylococcus aureus (n=157) isolated from intramammary infections in Argentine dairy areas were evaluated for presence of cap5 and cap8 loci. Isolates carrying cap5 and cap8 were serotyped using specific antisera. Sixty four percent of the isolates were genotyped as cap5 or cap8 and 50% of them expressed CP5 or 8.

Mode of action and in vitro susceptibility of mastitis pathogens to macedocin ST91KM and preparation of a teat seal containing the bacteriocin

Mastitis is considered to be the most economically costly disease affecting the dairy industry. Regular dosage of animals with antibiotics, including use of prophylactic concentrations, may select for resistant strains. The purpose of this study was to determine the mode of action of a new bacteriocin (macedocin ST91KM), to evaluate the antimicrobial resistance of mastitis pathogens to antibiotics commonly used in treatment remedies, and to introduce the possible use of an alternative antimicrobial agent. The bacteriocin macedocin ST91KM, produced by Streptococcus gallolyticus subsp. macedonicus ST91KM, is bactericidal to Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus associated with mastitis infections, including strains resistant to methicillin and oxacillin. Sensitive cells were deformed and secreted nucleotides, K+ and â-galactosidase when exposed to macedocin ST91KM. Adsorption of the peptide to target cells decreased in the presence of solvents, suggesting that receptors on the cell surfaces have lipid moieties. No adsorption was recorded in the presence of MgCl2, KI and Na2CO3, suggesting that ionic strength plays an important role. A teat seal preparation containing macedocin ST91KM effectively released the peptide and inhibited the growth of S. agalactiae. Macedocin ST91KM could form the basis for alternative dry cow therapy to prevent mastitis infections in dairy cows as it is effective against pathogens that display resistance to conventional antibiotic therapy.

Rep-pcr of Staphylococcus aureus strains isolated from bovine mastitis in Argentina

The aim of this study was to assess by rep-PCR the genetic relationshipof 52 S. aureus strains isolated from mammary infections collected infour herds located in the central dairy region of Argentina. Results were compared with the in vitro activity of antimicrobial drugs frequently used for treating bovine mastitis. Twelve different antimicrobials patterns were observed. Forty eight percent of the strains were susceptible to all antimicrobials tested. rep-PCR typing could successfully differentiate S. aureus strains of bovine origin. At afirst level of similarity (50%), it could be defined 5 clusters namely Ito V. Most of the strains (75%) were grouped in cluster I. The results may suggest that genotypes were similar in the different herds. Agreement between antibiotic patterns and rep-profiles was not observed for most isolates. The present report describes the genotypes responsible for the mastitis cases in the central dairy region of Argentina. A better knowledge of infective strains distribution indairy herds might help in formulating strategies to control of infection. Furthermore, antimicrobial susceptibility of S. aureus should be used as guide to select effective drugs to therapy in intramammary infections.

O objetivo do presente estudo foi determinar a relação genética entrerep-PCR de 52 linhagens Staphylococcus aureus isoladas de infecçõesmamárias em quatro fazendas leiteiras da região leiteira central daArgentina. Os resultados foram comparados com a atividade in vitrodos antimicrobianos freqüentemente utilizados no tratamento da mastite bovina. Foram observados 12 diferentes perfis deantimicrobianos. Do total de linhagens, 45% foram suscetíveis atodos os antibióticos ensaiados. A caracterizacão por rep-PCR podediferenciar com sucesso as linhagens de S. aureus de origem bovina.Num primeiro nível de similaridade (50%) foram definidos cincogrupos denominados de I a V. A maioria das estirpes (75%)agruparam-se no grupo I. Os resultados sugerem que os genotiposforam similares. Os genotipos não foram asociados com os perfis deantimicrobianos na maioria dos isolados. O presente estudo descreveos genotipos responsáveis pelos casos de mastites na região central daArgentina. O melhor conhecimento da distribução das linhagensinfectantes em fazendas leiteras poderia auxiliar na formulacão deestratégias para o controle de infecção. Além disso a suscetibilidadeaos antimicrobianos de linhagens de S. aureus deve ser usada paramontear a selecão de drogas efetivas para a terapia intramamária.

Characterization of lymphocyte subpopulations and major histocompatibility complex haplotypes of mastitis-resistant and susceptible cows

Bovine mastitis is an infectious disease with a major economic influence on the dairy industry worldwide. Many factors such as environment, pathogen, and host affect susceptibility or resistance of an individual cow to bovine mastitis. Recently, there has been considerable interest in defining genetic and immunological markers that could be used to select for improved disease resistance. In this study we have analyzed the lymphocyte subpopulations of mastitis-resistant and susceptible cows using monoclonal antibodies specific for bovine leukocyte differentiation antigens and flow cytometry. We have also used a microarray typing technique to define the bovine leukocyte antigen (BoLA) class I and class II haplotypes associated with resistance or susceptibility to bovine mastitis. A striking finding of the present study is that susceptibility to mastitis was associated with major histocompatibility complex (MHC) haplotypes that have only a single set of DQ genes. The study also revealed that susceptible cows had CD4:CD8 ratios of less than one in both their mammary gland secretions and peripheral blood. These results raise the possibility that the number of DQ genes that a cow has and/or a cow's CD4:CD8 ratio could be used as indicators of susceptibility to bovine mastitis.