Characterization of peripheral white blood cells transcriptome to unravel the regulatory signatures of bovine subclinical mastitis resistance.

Subclinical bovine mastitis is a pathogenic infection of the breast characterized by a marked decrease in milk production and quality. As it has no obvious clinical symptoms, diagnosis and treatment are challenging. Therefore, searching for biomarkers in cows' peripheral white blood cells is valuable for preventing and treating subclinical mastitis. Thus, in this study, the transcriptome of peripheral blood from healthy and subclinical mastitis cows was characterized to find the regulatory signatures of bovine subclinical mastitis using RNA-seq. A total of 287 differentially expressed genes (DEGs) and 70 differentially expressed lncRNAs (DELs) were detected, and 37 DELs were documented near known Quantitative Trait Loci (QTL) associated with the mastitis of cows. Bioinformatic analysis indicated that lncRNAs MSTRG25101.2, MSTRG.56327.1, and MSTRG.18968.1, which are adjacent to the SCS QTL and SCC QTL, may be candidate lncRNAs that influence the pathogenesis of mastitis in cows by up-regulating the expression of genes TLR4, NOD2, CXCL8, and OAS2. Moreover, the alternative splicing (AS) pattern of transcriptional sequence differences between healthy cows and subclinical mastitis cows suggested a molecular mechanism of mastitis resistance and susceptibility. A total of 2,212 differential alternative splicing (DAS) events, corresponding to 1,621 unique DAS genes, were identified in both groups and significantly enriched in immune and inflammatory pathways. Of these, 29 DAS genes were subject to regulation by 32 alternative splicing SNPs, showing diverse and specific splicing patterns and events. It is hypothesized that the PIK3C2B and PPRPF8 splice variants associated with AS SNPs (rs42705933 and rs133847062) may be risk factors for susceptibility to bovine subclinical mastitis. Altogether, these key blood markers associated with resistance to subclinical mastitis and SNPs associated with alternative splicing of genes provide the basis for genetic breeding for resistance to subclinical mastitis in cows.

Novel Streptococcus uberis sequence types causing bovine subclinical mastitis in Hainan, China.

AIM: To determine the molecular epidemiology, genotypes and phenotypes of the major species of Streptococcus associated with bovine subclinical mastitis in Hainan, China. METHODS AND RESULTS: In total, 150 subclinical mastitis milk samples were collected from two large dairy farms in Hainan. On the basis of biochemical tests and 16S rDNA sequencing, 39 samples were Streptococcus positive and the most frequently isolated species was Streptococcus uberis (n = 29, 74.4%). According to multilocus sequence typing (MLST), and assays of biofilm formation, antimicrobial susceptibility, resistance and virulence genes, the S. uberis isolates were clustered into nine new sequence types (STs; ST986-ST994) but were not merged into a clonal group (except for ST991 [CC143]). All isolates produced biofilm, but most weakly. The dominant virulence pattern was hasABC + sua + gapC + oppF + pauA + mtuA + cfu (27/29, 91.1%), based on the 11 virulence genes tested. The majority of isolates (88.46%) carried at least one resistance gene, and more than half (58.62%) were multidrug-resistant. The main resistance genes were linB (65.5%), ermB (37.9%) and tetS (34.5%), among the six antibiotic resistance genes and 11 antimicrobials tested. CONCLUSION: Environmental S. uberis is important in bovine subclinical mastitis in Hainan. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus uberis isolates in Hainan, China, show distinct MLST, virulence and antibiotic resistance characteristics.

Evaluation of sodium lauryl sulfate for the development of cow-side mastitis screening test.

BACKGROUND AND AIM: Subclinical mastitis (SCM) is an economically important disease for dairy cattle worldwide; therefore, regular screening is imperative to detect SCM at an early stage so as to control it. The study was conducted to compare the test characteristics of sodium lauryl sulfate (SLS) as a test reagent to detect SCM in dairy cows. MATERIALS AND METHODS: First, 106 milk samples of dairy cows were subjected to available indirect screening tests (white side test [WST], surf field mastitis test, Leucocytest, and Immucell) considering somatic cell count (SCC) as gold standard test. Then 273 milk samples were allowed to react with different concentrations of SLS with or without sodium hydroxide (NaOH) and indicators (bromothymol blue [BTB] and bromocresol purple [BCP]). RESULTS: SLS with or without NaOH yielded best reaction with the milk samples similar to Leucocytest. It was observed that the reaction of milk samples with SLS added with indicators (BTB and BCP) was easier to visualize than without indicators. SLS 3%+NaOH 1.5% with BTB and SLS 2% with BCP had high sensitivity, specificity, and substantial agreement with SCC. The area under the receiver operating characteristics curve of SLS 2% with BCP and SLS 3%+NaOH 1.5% with BTB was 0.917 and 0.875, respectively. CONCLUSION: It may be concluded that SLS 3%+NaOH 1.5% with BTB and SLS 2% with BCP may be the potential reagents for the development of an effective cow-side test to detect SCM, as the main ingredient SLS is considerably cheap and readily available in developing countries.

Milk Lactose as a Biomarker of Subclinical Mastitis in Dairy Cows.

Bovine subclinical mastitis can cause great harm to dairy herds because of its negative impact on milk production and quality and cow health. Improved diagnostic tools are needed to maximise the control of subclinical mastitis distribution and ensure the high quality of milk as an industrial product. Between 2015 and 2020, seventy-two dairy herds were screened for bovine subclinical mastitis causative agents to identify the relationship between seasons, lactose levels and subclinical mastitis infection. The predominant species found in the milk samples were mixed microbiota, coagulase-negative Staphylococcus and Staphylococcus aureus. Yeasts were found exclusively in autumn, while Enterococcus spp. and Escherichia coli were only found in summer and autumn. A negative correlation was detected between milk lactose and number of somatic cells in milk (-0.471; p < 0.001). The lactose levels in milk were closely associated with the prevalence (%) of subclinical mastitis pathogens, such as Streptococcus agalactiae (y = -1.8011x + 10.867, R2 = 0.9298), Staph. aureus (y = -3.5216x + 25.957, R2 = 0.8604) and other Streptococci (y = -0.5956x + 7.6179, R2 = 0.6656). These findings suggest that milk lactose may be used as a biomarker of suspected udder inflammation in modern health prevention programmes for cows to reduce the prevalence of subclinical mastitis pathogens in dairy cattle herds.

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis / Expressão dos genes icaA e icaD na formação de biofilme em isolados de Staphylococcus aureus de mastite subclínica bovina

Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.(AU)

Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.(AU)

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis

ABSTRACT: Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.

RESUMO: Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.

Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts.

This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P < 0.0001) compared with the other assessed genes (cna, ebpS and fnbB). The higher frequency of occurrence (P < 0.005) of the bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process.

Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms

The aim of this study was to investigate the phenotypic and genotypic characteristics of Streptococcus uberis isolated from subclinical mastitis (SCM) cases, and to examine the possible association between both characteristics. A total of 32 S. uberis were isolated from 772 quarter milk samples (SCM > 250,000 cells/ml) collected from 195 cows selected randomly from 18 dairy farms located in Argentina. The S. uberis strains were characterized phenotypically by the presence of virulence factors as plasminogen activator factor (PAF), hyaluronidase (HYA), capsule (CAP) and CAMP factor, and were further characterized genotypically by pulsed-field gel electrophoresis (PFGE). S. uberis strains expressed plasminogen activator factor, hyaluronidase or capsule (65.5 %, 56.3 %, 59.4 %, respectively), but only 25 % of isolates were CAMP factor positive. Thirteen different virulence profiles were identified on the basis of the combination of virulence factors. Eighteen PFGE patterns with 90% of similarity were identified among 32 S. uberis. A great diversity of virulence profiles and PFGE patterns were present among dairy farms. S. uberis strains with the same PFGE pattern showed different virulence profiles. Bovine S. uberis strains causing SCM included in the present study showed heterogeneity in regard to their phenotypic and genotypic characteristics, and the PFGE patterns are not associated with the virulence profiles.

Caracterización fenotípica y genotípica de Streptococcus uberis aislados de mastitis bovina subclínica en tambos de Argentina. El objetivo de este estudio fue investigar las características fenotípicas y genotípicas de Streptococcus uberis aislados de casos de mastitis subclínica (MSC) y examinar la posible asociación entre ambas características. Un total de 32 cepas de S. uberis fueron aisladas de 772 muestras de leche de cuartos mamarios (MSC > 25 0000 células/ml) colectadas de 195 vacas seleccionadas al azar pertenecientes a 18 tambos lecheros localizados en Argentina. Las cepas de S. uberis fueron caracterizadas fenotípicamente sobre la base de la presencia de factores de virulencia tales como el factor activador del plasminógeno (FAP), la hialuronidasa (HIA), la cápsula (CAP) y el factor CAMP. Además, fueron caracterizadas genotípicamente por electroforesis de campos pulsados (PFGE). Las cepas de S. uberis expresaron el factor activador del plasminógeno, la hialuronidasa o la cápsula (65,5 %, 56,3 % y 59,4 %, respectivamente), pero solo el 25 % fueron CAMP positivas. Sobre la base de la combinación de los factores de virulencia se identificaron 13 perfiles de virulencia diferentes. Asimismo, se identificaron 18 patrones de PFGE con un 90 % de similitud entre las 32 cepas de S. uberis. Se presentó una gran diversidad de perfiles de virulencia y patrones de PFGE entre los tambos. Cepas con el mismo patrón de PFGE presentaron perfiles de virulencia diferentes. Las cepas de S. uberis causantes de MSC en bovinos incluidas en el presente estudio mostraron heterogeneidad con respecto a sus características fenotípicas y genotípicas, y los patrones de PFGE no estuvieron asociados con los perfiles de virulencia.

Invasive potential of biofilm-forming Staphylococci bovine subclinical mastitis isolates

Staphylococcus (S.) aureus is a common infectious agent of bovine chronic mastitis, a disease that is difficult to eradicate. The abilities of Staphylococci to be internalized and form a biofilm can contribute to host immunological defence evasion that subsequently impairs antimicrobial therapy. The invasive capability of six S. aureus field isolates with different biofilm-forming profiles was compared in vitro using a bovine mammary epithelial cell line. This was further confirmed in primary cell cultures using fluorescent rRNA probes against S. aureus. The results suggest that S. aureus invasion levels are not related to biofilm formation.