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Staphylococcus is a very prevalent etiologic agent of bovine mastitis, and antibiotic resistance contributes to the successful colonization and dissemination of these bacteria in different environments and hosts on dairy farms. This study aimed to identify the antimicrobial resistance (AMR) genotypes and phenotypes of Staphylococcus spp. isolates from different sources on dairy farms and their relationship with the use of antibiotics. An antimicrobial susceptibility test was performed on 349 Staphylococcus strains (S. aureus, n = 152; non-aureus staphylococci (NAS), n = 197) isolated from quarter milk samples (QMSs) from cows with subclinical mastitis (176), the teats of cows (116), the milking parlor environment (32), and the nasal cavities of milk workers (25). Resistance and multidrug resistance percentages found for S. aureus and NAS were (S. aureus = 63.2%, NAS = 55.8%) and (S. aureus = 4.6%, NAS = 11.7%), respectively. S. aureus and NAS isolates showed resistance mainly to penicillin (10 IU) (54.1% and 32.4%) and ampicillin (10 mg) (50.3% and 27.0%) drugs. The prevalence of AMR Staphylococcus was higher in environmental samples (81.3%) compared to other sources (52.6-76.0%). In S. aureus isolates, the identification of the blaZ (83.9%), aacAaphD (48.6%), ermC (23.5%), tetK (12.9%), and mecA (12.1%) genes did not entirely agree with the AMR phenotype. We conclude that the use of ß-lactam antibiotics influences the expression of AMR in Staphylococcus circulating on dairy farms and that S. aureus isolates from the environment and humans may be reservoirs of AMR for other bacteria on dairy farms.

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Staphylococcus aureus is one of the most prevalent pathogens causing bovine mastitis in the world, in part because of its ease of adaptation to various hosts and the environment. This study aimed to determine the prevalence of S. aureus in Colombian dairy farms and its relationship with the causal network of subclinical mastitis. From thirteen dairy farms enrolled, 1288 quarter milk samples (QMS) and 330 teat samples were taken from cows with positive (70.1%) and negative California Mastitis Test (CMT). In addition, 126 samples from the milking parlor environment and 40 from workers (nasal) were collected. On each dairy farm, a survey was conducted, and the milking process was monitored on the day of sampling. S. aureus was identified in 176 samples, i.e., 138 QMS, 20 from teats, 8 from the milking parlor environment, and 10 from workers' nasal swabs. Isolates identified as S. aureus underwent proteomics (clustering of mass spectrum) and molecular (tuf, coa, spa Ig, clfA, and eno genes) analysis. Regarding proteomics results, isolates were distributed into three clusters, each with members from all sources and all farms. Concerning molecular analysis, the virulence-related genes clfA and eno were identified in 41.3% and 37.8% of S. aureus isolates, respectively. We provide evidence on the circulation of S. aureus strains with limited variability among animals, humans, and the environment. The parameters with the lowest compliance in the farms which may be implicated in the transmission of S. aureus are the lack of handwashing and abnormal milk handling.

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Compost-bedded pack barns (CB) are receiving increasing attention as a housing system that can potentially improve the welfare of dairy cows. This study characterized the frequency and profile of pathogens isolated from clinical (CM) and subclinical (SCM) mastitis in dairy cows housed in CB. It evaluated the association between mastitis occurrence and bedding characteristics in CB systems. Over six months, seven dairy herds were visited monthly for milk and bedding sample collections. Milk samples from mastitis cases were submitted to microbiological identification by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS). Bedding samples were submitted to physical-chemical (pH, organic matter, moisture, and carbon to nitrogen ratio) and microbiological counting (total bacterial counts, coliforms, streptococci, and staphylococci) analyses. Regression analysis was used to determine the association between mastitis occurrence and CB characteristics. Our results showed that Escherichia coli and environmental streptococci were the most frequently isolated pathogens from CM cases, while Staphylococcus chromogenes and contagious pathogens (Staphylococcus aureus and Streptococcus agalactiae) were the most commonly isolated from SCM cases. Bedding moisture content was positively associated with the incidence of CM. The bedding carbon to nitrogen ratio was negatively associated with the incidence of SCM, and the bedding total bacteria counts tended to be associated with the incidence of SCM. Bedding counts of coliforms positively associated with the prevalence of SCM. Our results can support decision-makers in the dairy industry seeking strategies for bedding management and mastitis control.

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Extended-spectrum ß-lactamase (ESBL)-producing Gram negative bacteria are becoming increasingly important in veterinary and human medicine because they can hydrolyze the third generation ß-lactams, penicillins, and monobactams. The aim of this study was to identify ESBL-producing Enterobacteriaceae in raw cow milk samples from northeast Brazil. Twenty-six bacterial isolates belonging to the Enterobacteriaceae family were obtained from milk samples from 257 cows with subclinical mastitis. Using microbiological tests, 53.85% (14/26) were identified as Escherichia coli, 15.38% (4/26) as Proteus mirabilis, 26.92% (7/26) as Klebsiella spp., and 3.85% (1/26) as Citrobacter spp. Of all the isolates, 61.54% (16/26) were positive in the ESBL screening test, of which 12.5% (2/16) were positive in the double-disc synergy test using three types of cephalosporins and amoxicillin/clavulanic acid. The two isolates were identified as Klebsiella spp. Among all the isolates, 53.85% (14/26) were positive for one or both ESBL-encoding genes, blaSHV and blaTEM; among these, 71.43% (10/14) were identified as E. coli. This study demonstrates that ESBL-producing bacteria can be found in raw cow milk from northeast Brazil. Cows with subclinical mastitis should be recognized as reservoirs of these strains, which can propagate to humans.

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In this study, we evaluated the main risk factors for the occurrence of bovine mastitis, in the southeastern of Pará, in the Brazilian Amazon. We surveyed 91 dairy farmers to identify management practices and bovine breed characteristics. From each farm, 50 mL of milk sample was collected for microbiological analysis and somatic cell count (SCC). Depending on the management practices and breed, a logit model was used to determine the odds ratio of subclinical mastitis (SCM) occurrence. In irrigated pastures, an SCM-associated risk factor, the occurrence of SCM was 5.03 times higher than that in the non-irrigated pastures. Similarly, in Girolando breed herds, the occurrence of SCM increased by 5.8 times compared to the crossbred herds. Moreover, the occurrence of mastitis was 33 times higher in farms using common cloths for drying teats than in farms using paper towels. Therefore, adoption of better management practices can lead to SCC reduction, milk quality improvement and a guarantee to contain SCC within prescribed Brazilian limits for the Amazon region.

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Raw milk samples were collected from 200 dairy cows belonging to Girolando 1/2, Gyr, Guzera, and Holstein breeds, and the bacterial diversity was explored using 16S rRNA amplicon sequencing. SCC analysis showed that 69 animals were classified as affected with subclinical mastitis. The milk bacterial microbiome was dominated by Firmicutes, Proteobacteria, and Actinobacteria, with an increase of Firmicutes in animals with subclinical mastitis and Proteobacteria in healthy animals. At the family and genus level, the milk bacterial microbiome was dominated by Staphylococcus, Acinetobacter, Pseudomonas, members of the family Enterobacteriaceae, Lactococcus, Aerococcus, members of the family Rhizobiaceae, Anaerobacillus, Streptococcus, members of the family Intrasporangiaceae, members of the family Planococcaceae, Corynebacterium, Nocardioides, and Chryseobacterium. Significant differences in alpha and beta diversity analysis suggest an effect of udder health status and breed on the composition of raw bovine milk microbiota. LEfSe analysis showed 45 and 51 discriminative taxonomic biomarkers associated with udder health status and with one of the four breeds respectively, suggesting an effect of subclinical mastitis and breed on the microbiota of milk in cattle.

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The aim of this study was to identify emergent pathogens associated with bovine mastitis in northeastern Brazil and to characterize them for phenotypic and genotypic resistance to antimicrobials. A total of 321 milk samples from cows with subclinical mastitis were collected, and the isolates obtained in culture were identified using matrix-associated laser desorption-ionization - time of flight mass spectrometry. Phenotypic and genotypic antimicrobial resistance tests were performed. We identified 72 bacteria considered emergent in the study region: Enterococcus faecalis (26.3%; 19/72), Streptococcus agalactiae (22.2%; 16/72), Enterococcus faecium (20.0%; 15/72), Escherichia coli (6.9%; 5/72), 6.9% (5/72) Lactococcus garvieae (6.9%; 5/72), Acinetobacter baumannii (5.5%; 4/72), Bacillus subtilis (1.3%; 1/72), Kocuria marina (1.3%; 1/72), Macrococcus caseolyticus (1.3%; 1/72), Microbacterium resistens (1.3%; 1/72), Micrococcus luteus (1.3%; 1/72), Streptococcus dysgalactiae (1.3%; 1/72), Streptococcus hyovaginalis (1.3%; 1/72) and Streptococcus pluranimalium (1.3%; 1/72). The antibiogram revealed the following resistance profiles: ampicillin (77.7%; 56/72), cefoxitin (69.4%; 50/72), erythromycin (61.1%; 44/72), oxacillin (63.8%; 46/72), penicillin (79.1%; 57/72), tetracycline (63.8%; 46/72), gentamicin (25.0%; 18/72), and vancomycin (20.8%; 15/72). Of the isolates, 83.4% (60/72) showed multiple resistance to antimicrobials. The tetM gene was identified in 43.0% (31/72) of the isolates, followed by tetL (31.9%; 23/72), and blaZ (26.3%; 19/72). 83.4% (60/72) of the isolates presented a multiple antimicrobial resistance index higher than 0,2. Emergent bacteria with zoonotic and multiresistant potential occur in cows with mastitis in northeastern Brazil. It is necessary to monitor the occurrence of these and other bacteria in livestock environments and develop control strategies to prevent their spread.

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Subclinical mastitis is a very important disease for dairy cattle farms. As a result of the deterioration of milk quality, it causes economic losses in enterprises. Milk samples of 180 cows were divided into five groups according to the somatic cell count. Some chemical properties and composition were measured in milk samples. The ratio of solid-non-fat, mineral matter, lactose, and protein decreased as the somatic cell count of the milk increased (P<0.05). No statistical difference was observed between the fat content of milk and the level of somatic cell count (P>0.05). Although specific gravity decreases (P<0.05), the electrical conductivity increases (P<0.001) in milk with high levels of somatic cell count. The milk quality of dairy cattle deteriorates due to the high somatic cell count. Therefore, regular monitoring of somatic cell count is recommended in dairy farms to monitor changes in fat, protein, solid-non-fat, lactose, and mineral matter ratios as well as some chemical properties that make up the milk composition.

A mastite subclínica é uma doença muito importante para as fazendas de gado leiteiro. Como resultado da deterioração da qualidade do leite, ela causa perdas econômicas nas empresas. Amostras de leite de 180 vacas foram divididas em cinco grupos de acordo com o nível de contagem de células somáticas (SCC). Algumas propriedades químicas e composição foram medidas em amostras de leite. A proporção de sólido sem gordura, matéria mineral, lactose e proteína diminuiu à medida que a SCC do leite aumentava (P<0,05). Nenhuma diferença estatística foi observada entre o conteúdo de gordura do leite e o nível de contagem de células somáticas (P>0,05). Embora a gravidade específica diminua (P<0,05), a condutividade elétrica aumenta (P<0,001) no leite com altos níveis de contagem de células somáticas. A qualidade do leite do gado leiteiro se deteriora devido à alta contagem de células somáticas. Portanto, o monitoramento regular da SCC é recomendado nas fazendas de leite para monitorar mudanças nas proporções de gordura, proteína, sólidos não gordurosos, lactose e matéria mineral, bem como algumas propriedades químicas que compõem a composição do leite.

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The objective of this study was to evaluate the efficacy of the antibiotic therapy associated with vaccination on the microbiological cure rate of subclinical mastitis caused by Staphylococcus aureus in lactating dairy cows. A total of five herds, from which 72 cows (120 mammary quarters - MQ) were diagnosed with S. aureus subclinical mastitis, were included in this study. Cows were randomly allocated to one of three treatment groups: a) Control (no treatment); b) ATB (antibiotic therapy); and c) ATB+VAC (antibiotic therapy plus vaccination against S. aureus). Intramammary treatment consisted of twice-daily infusion of ampicillin 75mg + cloxacillin 200mg, for 5 days. Parenteral treatment was done by injection of a single dose (7.5mg/kg) of enrofloxacin, on the first day of the treatment protocol. Vaccinated cows received three doses of a commercial vaccine 14 days before treatment (d-14), on the first day of treatment protocol (d1), and 14 days after the treatment protocol (d+14). Non-treated cows had a lower cure rate (0.06) than cows treated with ATB (0.84) and ATB+VAC (0.85). No difference in cure rate was observed between cows treated with ATB and ATB+VAC. On the other hand, vaccinated cows had lower somatic cell count (SCC) after 28 days of the treatment protocols (4.76 log10) than non-treated cows (5.37 log10). In conclusion, treatment with intramammary ampicillin and cloxacillin, associated with intramuscular enrofloxacin presented a high cure rate for SCM caused by S. aureus during lactation. The use of vaccination against S. aureus in association with antibiotic therapy did not increase the cure rate of MQ during lactation, but it was effective in reducing the SCC when compared to non-treated MQ. Although to ensure that the decrease of the SCC in ATB+VAC group was associated with the vaccination, the study should have included an additional group of only vaccinated cows, without antimicrobial therapy, with was not done in the present study, and therefore is one of the limitations of the experimental protocol used.

O objetivo deste estudo foi avaliar a eficácia da antibioticoterapia associada à vacinação sobre a taxa de cura microbiológica de mastite subclínica causada por Staphylococcus aureus em vacas leiteiras em lactação. Foram selecionados 5 rebanhos, dos quais 72 vacas (120 quartos mamários, QM) foram diagnosticadas com mastite subclínica por S. aureus e foram alocadas aleatoriamente em um de três grupos de tratamento: a) Controle (sem tratamento); b) ATB (antibioticoterapia); e c) ATB+VAC (antibioticoterapia mais vacinação contra S. aureus). O tratamento intramamário consistiu em infusão de ampicilina 75 mg + cloxacilina 200 mg duas vezes ao dia, durante 5 dias. O tratamento parenteral foi feito por injeção de uma dose única (7,5 mg/kg) de enrofloxacina, no primeiro dia do protocolo de tratamento. As vacas vacinadas receberam três doses de uma vacina comercial, 14 dias antes do tratamento (d-14), no primeiro dia do protocolo de tratamento (d1) e 14 dias após o protocolo de tratamento (d+14). A taxa de cura das vacas não tratadas foi menor (0,06) do que das vacas tratadas com ATB (0,84) e ATB+VAC (0,85). Não foi observada diferença de taxa de cura entre vacas tratadas com ATB e ATB+VAC. Por outro lado, as vacas vacinadas apresentaram menor a contagem de células somáticas (CCS) após 28 dias de tratamento (4,76 log10) do que em vacas não tratadas (5,37 log10). Em conclusão, o tratamento com ampicilina e cloxacilina intramamária, associados à enrofloxacina intramuscular, apresentou alta taxa de cura para MSC causada por S. aureus durante a lactação. A utilização da vacinação contra S. aureus associada à antibioticoterapia não aumentou a taxa de cura dos QM durante a lactação, mas foi eficaz na redução do CCS quando comparada à QM não tratados. Entretanto, para ter certeza que a diminuição da CCS no grupo ATB+VAC estivesse associada à vacinação, o estudo deveria ter incluído um grupo adicional de apenas vacas vacinadas, sem terapia antimicrobiana, o que não foi feito no presente estudo e, portanto, é um das limitações do protocolo experimental utilizado.

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BACKGROUND: The possibility that maternal health status and breastfeeding practices contribute to growth faltering before 6 mo is underexplored. OBJECTIVES: This longitudinal study investigated whether indicators of subclinical mastitis (SCM) and breast inflammation, maternal fecal-oral contamination, and/or breastfeeding practices were associated with infant anthropometry or growth velocity before 6 mo. METHODS: Indigenous Mam-Mayan mother-infant dyads (n = 140) were recruited. Breast milk was collected at early (<6 wk) and established (4-6 mo) lactation when maternal and infant anthropometry were measured. Milk Na:K ratio as an indicator of SCM and concentrations of 4 proinflammatory cytokines were measured. Maternal stool samples were examined for the presence of intestinal parasites including nonpathogenic protozoa (Endolimax nana, Iodamoeba bütschlii, Entamoeba coli, Blastocystis hominis). Questionnaires characterized breastfeeding and hygiene practices. Multiple linear regression identified factors associated with infant growth attainment [weight-for-age z-score (WAZ), length-for-age z-score (LAZ), and head circumference-for-age z-score (HCAZ)] and growth velocity (expressed as change per day from early to established lactation). Multiple logistic regression identified factors associated with increased odds of underweight, stunting, and low head circumference at both lactation stages. RESULTS: A higher Na:K ratio, individual nonpathogenic protozoa, and specific breastfeeding and hygiene practices were associated with impaired growth before 6 wk and at 4-6 mo in exclusively breastfed infants. Growth velocity for weight was inversely associated with Entamoeba coli but cranial growth was associated positively with Iodamoeba bütschlii whereas feeding colostrum in early lactation was protective and decreased the odds of an HCAZ < -2 SD. Finally, the presence of SCM in early lactation increased the likelihood of both WAZ < -2 SD and LAZ < -2 SD by 6 wk. CONCLUSIONS: Prevention of SCM can improve early infant weight, but measures that promote the feeding of colostrum and reduce exposure to fecal-oral contamination might be required to minimize infant growth faltering in breastfed infants.

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The aim of this study was to investigate the potential risk factors for clinical and subclinical mastitis in smallholder dairy farms in Brazil. A prospective, repeated cross-sectional study was carried out between May 2018 and June 2019 on 10 smallholder dairy farms. Potential risk factors for subclinical and clinical mastitis at the herd and cow level were recorded through interviewing the owner and by observation. A combination of clinical udder examination and the Tamis (screened mug with a dark base) test (Tadabras Indústria e Comércio de Produtos Agrovetereinário LTDA, Bragrança Paulista, SP, Brazil) were applied to observe clinical mastitis, and the California Mastitis Test (Tadabras Indústria e Comércio de Produtos Agrovetereinário LTDA, Bragrança Paulista, SP, Brazil) was used to determine subclinical mastitis. A total of 4567 quarters were tested, 107 (2.3%) had clinical mastitis, while 1519 (33.2%) had subclinical mastitis. At the cow level, clinical mastitis risk was highest in mid-lactation (50-150 days in milk) with OR 2.62 with 95% confidence interval (CI) of 1.03-6.67, while subclinical mastitis was highest in late lactation (> 150 days in milk) with OR 2.74 (95% CI 2.05-3.63) and lower in primiparous (OR 0.54, 95% CI 0.41-0.71) than multiparous cows. At the herd level, using dry-cow treatment (OR 4.23, 95% CI 1.42-12.62) was associated with an increased risk of clinical mastitis. Milking clinical (OR 0.37, 95% CI 0.24-0.56) and subclinical cases last (OR 0.21, 95% CI 0.09-0.47) and cleaning the milking parlor regularly (OR 0.27, 95% CI 0.15-0.46) had decreased odds for subclinical mastitis, while herds with optimized feed had greater odds (OR 9.11, 95% CI 2.59-31.9). Prevalence of clinical mastitis was at its lowest at the first visit in June/July and highest at the last visit in April/June (OR 3.81, 95% CI 1.93-7.52). Subclinical mastitis also presented increased odds in the last visit (OR 2.62, 95% CI 2.0-3.36). This study has identified some risk factors for mastitis on smallholder farms but further research on more farms across more areas of Brazil is required to develop a targeted mastitis control program for smallholder farms.

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A total of 695 Holstein Friesian lactating dairy cows were examined for the prevalence of subclinical mastitis (SCM) and its association with selected risk factors in a private dairy farm at Fayoum district, Egypt. 444 lactating cows with 1145 quarters were found positive for SCM with prevalence of 63.88% and 41.18% at cow and quarter level respectively based on California Mastitis Test (CMT), Electrical Conductivity (EC) that had a mean value of 6.27 ± 0.066 mS/cm and Somatic Cell Count (SCC) with a mean value of 8.8×105± 9.2×103 cells/ml. The risk factors investigated in this study revealed that the highest percentage of SCM was observed in winter, in cows at the late lactation stage with a percentage of 32.21 and 59.91 respectively. The results concluded that the young age cows were exposed to SCM more than old ones. While the occurrence of SCM was higher in cows with 2nd, 3rd and 1st than those with 4, 5, 6th parity number. The analytical results also revealed that there is a highly significant difference (p < 0.05) between numbers of parity. However, there is no significant difference (p > 0.05) between different stages of age, lactation and season as risk factors.(AU)

Um total de 695 vacas leiteiras Holstein Friesian lactantes foram examinadas para a prevalência de mastite subclí-nica (SCM) e sua associação com fatores de risco selecionados em uma fazenda privada de leite no distrito de Fayoum, Egito. 444 vacas em lactação com 1145 quartos foram consideradas positivas para SCM com prevalência de 63.88% e 41.18% no nível de vacas e quartos, respectivamente com base no California Mastitis Test (CMT), Condutividade Elétrica (EC) que teve um valor médio de 6.27 ± 0.066 mS / cm e Contagem de células somáticas (SCC) com um valor médio de 8.8 × 105± 9.2 × 103 células / ml. Os fatores de risco investigados neste estudo revelaram que o maior percentual de SCM foi observado no inverno, em vacas no final da lactação com um percentual de 32.21 e 59.91 respectivamente. Os resultados concluíram que as vacas jovens foram mais expostas ao SCM do que as vacas velhas. Enquanto a ocorrência de SCM foi maior nas vacas com 2, 3 e 1ª do que nas com 4, 5 e 6 números de paridade. Os resultados analíticos também revelaram que existe uma diferença altamente significativa (p <0.05) entre os números de paridade. No entanto, não há diferença significativa (p> 0.05) entre os diferentes estágios de idade, lactação e estação do ano como fatores de risco.(AU)

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A total of 695 Holstein Friesian lactating dairy cows were examined for the prevalence of subclinical mastitis (SCM) and its association with selected risk factors in a private dairy farm at Fayoum district, Egypt. 444 lactating cows with 1145 quarters were found positive for SCM with prevalence of 63.88% and 41.18% at cow and quarter level respectively based on California Mastitis Test (CMT), Electrical Conductivity (EC) that had a mean value of 6.27 ± 0.066 mS/cm and Somatic Cell Count (SCC) with a mean value of 8.8×105± 9.2×103 cells/ml. The risk factors investigated in this study revealed that the highest percentage of SCM was observed in winter, in cows at the late lactation stage with a percentage of 32.21 and 59.91 respectively. The results concluded that the young age cows were exposed to SCM more than old ones. While the occurrence of SCM was higher in cows with 2nd, 3rd and 1st than those with 4, 5, 6th parity number. The analytical results also revealed that there is a highly significant difference (p 0.05) between different stages of age, lactation and season as risk factors.

Um total de 695 vacas leiteiras Holstein Friesian lactantes foram examinadas para a prevalência de mastite subclí-nica (SCM) e sua associação com fatores de risco selecionados em uma fazenda privada de leite no distrito de Fayoum, Egito. 444 vacas em lactação com 1145 quartos foram consideradas positivas para SCM com prevalência de 63.88% e 41.18% no nível de vacas e quartos, respectivamente com base no California Mastitis Test (CMT), Condutividade Elétrica (EC) que teve um valor médio de 6.27 ± 0.066 mS / cm e Contagem de células somáticas (SCC) com um valor médio de 8.8 × 105± 9.2 × 103 células / ml. Os fatores de risco investigados neste estudo revelaram que o maior percentual de SCM foi observado no inverno, em vacas no final da lactação com um percentual de 32.21 e 59.91 respectivamente. Os resultados concluíram que as vacas jovens foram mais expostas ao SCM do que as vacas velhas. Enquanto a ocorrência de SCM foi maior nas vacas com 2, 3 e 1ª do que nas com 4, 5 e 6 números de paridade. Os resultados analíticos também revelaram que existe uma diferença altamente significativa (p 0.05) entre os diferentes estágios de idade, lactação e estação do ano como fatores de risco.

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Biofilm formation is a central feature to guarantee staphylococcal persistence in hosts and is associated with several diseases that are difficult to treat. In this research paper, biofilm formation and antimicrobial susceptibility were investigated in staphylococcal strains belonging to several species. These strains were isolated from the milk of cows with subclinical mastitis and most of them were coagulase-negative, with the prevalence of Staphylococcus chromogenes. High genetic diversity was observed among the strains by pulsed field gel electrophoresis. Antimicrobial resistance was assessed by disk diffusion and more than 50% of the strains were resistant to ampicillin and penicillin G, with multi-resistance profiles (13.6%) also being observed. Most strains (65.9%) formed biofilms when cultivated in BHI supplemented with 1% glucose. Most strains (72.7%) carried the intercellular adhesion gene (icaA), while less than half (36.3%) carried the biofilm-associated protein gene (bap). Concentrations of up to 10xMIC of erythromycin and tetracycline were not sufficient to suppress cell viability in preformed biofilms. Our results revealed that a genetically diverse group of biofilm-forming Staphylococcus species can be involved in subclinical mastitis. Since high antimicrobial concentrations cannot eradicate biofilm cells in vitro, their use in dairy animals may be ineffective in controlling infections, while supporting selection of resistant microorganisms. These data reinforce the need for alternative therapies aiming at disrupting biofilms for effective disease control.

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Systemic parameters and its relationship with Somatic Cell Count (SCC) in dairy cows were evaluated. Cows that presented subclinical mastitis (SMC; n = 16) and healthy cows (HC; n = 6) were selected and identified by the California Mastitis Test and SCC in milk. SCC results were logarithmically transformed into somatic cell score (SCS). HC presented SCS 〈 4.32 while SMC presented SCS 〉 4.32. Milk and blood samples were collected in three days: D1 (first day of sampling), D2 (48 h after D1) and D3 (7 days after D1), to determine White Blood Cells (WBC), albumin, total protein, total bilirubin and malondialdehyde (MDA). Results were expressed as mean ± standard deviation. It was considered significant at P < 0.05. The data of SCS on D1, D2 and D3 in SMC were: 6.8 ± 1.7, 6.4 ± 1.8, and 6.3 ± 2.0, respectively. In SMC the MDA (nmolL-1) were: D1 - 9.3 ± 2.6, D2 - 8.6 ± 2.4, and D3 - 11.5 ± 3.5. The MDA on D3 in SMC (11.5 ± 3.5) were increase when compared to HC (6.0 ± 1.3) (P < 0.001). No significant difference was found in WBC, TP, ALB, and TB between groups. It was observed a positive correlation between MDA-SCS (ρ = 0.4) and between WBC-SCS (ρ = 0.3) in the SMC group. It was concluded that the systemic repercussion damage in the mammary gland promoted by subclinical mastitis in dairy cows can be assessed using the MDA and WBC biomarkers.

RESUMO

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.

RESUMO

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)

Assuntos

RESUMO

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)

Assuntos

RESUMO

Abstract This study aimed to relate the serum levels of energy indicators (glucose and beta-hydroxybutyrate) and calcium to the occurrence of uterine and mammary diseases in the transition period of Holstein cows. One hundred and fifteen Holstein cows from a commercial herd were monitored starting one week before calving and during the first 2 weeks after calving. The presence of subclinical mastitis was monitored for 3 weeks postpartum using the California Mastitis Test (CMT) and somatic cell counting. Positive samples of subclinical mastitis were analyzed for bacterial identification. Clinical mastitis was monitored for 6 weeks postpartum, using data that were recorded by the farmer based on some established definitions. The presence of uterine disorders was identified for 3 weeks postpartum by clinical examination. Blood collections were made from all cows for determining calcium, glucose, and β-hydroxy-butyrate (BHB) the week before calving and twice during the postpartum (days 5 and 15 postpartum). A group of 70 healthy cows was used as control. Clinical mastitis was identified in 20% of the cows, subclinical mastitis in 10.4% and clinical metritis in 8.7% of the cows. The bacterial agents with the highest occurrence in subclinical mastitis cases were coagulase-negative Staphylococcus and Streptococcus. No relationships were observed between any of the biochemical metabolite studied and the uterine and mammary diseases in the cows during the studied period.

Resumen Este estudio tuvo por objetivo relacionar valores séricos de indicadores energéticos (glucosa y beta-hidroxibutirato) y calcio con la ocurrencia de trastornos uterinos y mamarios durante el periodo de transición en vacas holstein. Ciento quince vacas holstein de un rebaño comercial fueron monitoreadas en la semana previa al parto y durante las primeras dos semanas después del parto. La presencia de mastitis subclínica fue monitoreada por tres semanas posparto mediante la prueba de mastitis California (CMT) y conteo de células somáticas. Las muestras positivas a mastitis subclínica fueron analizadas para identificación bacteriana. La mastitis clínica fue monitoreada durante seis semanas posparto, usando datos registrados en el rebaño. La presencia de trastornos uterinos fue identificada durante tres semanas posparto mediante examen clínico. Se realizaron muestras de sangre de todas las vacas para determinar calcio, glucosa y β-hidroxibutirato la semana anterior al parto y dos veces en el posparto (días 5 y 15). Un grupo de 70 vacas sanas fue usado como control. Un 20% de las vacas presentó mastitis clínica, el 10,4% mastitis subclínica y el 8,7% metritis clínica. Los agentes bacterianos con mayor ocurrencia en los casos de mastitis subclínica fueron Staphylococcus coagulasa-negativos y Streptococcus. No se observó relación de ninguno de los metabolitos bioquímicos con los trastornos uterinos o mamarios en las vacas durante el periodo estudiado.

RESUMO

Resumen El presente trabajo de investigación se llevó a cabo en un tambo del distrito de Nueva Italia, situado en el departamento Central. El objetivo de la investigación fue determinar la prevalencia de mastitis subclínica. Para el efecto, se contó con 147 animales de la especie bovina, hembras de razas holando, cruces jersey-holando y holando-gyr, de diferentes edades y periodos de lactación, con y sin síntomas de mastitis. Se realizó la prueba California Mastitis Test (CMT) y, de las 147 muestras analizadas, 110 dieron positivo a mastitis subclínica, lo que representa una prevalencia de 74,83 %. De los 551 cuartos estudiados, 315 fueron positivos, equivalentes al 57 %. Los cuartos posteriores resultaron ser el 50,6% de los cuartos totales. Según grados de CMT los resultados fueron: negativo, 43 % (236); trazas, 17 % (91); grado 1, 25 % (138); grado 2: 13,7 % (76), y grado 3: 1,8 % (10). La merma fue de 129,6 litros de leche por ordeñe. Teniendo en cuenta que el precio por litro de leche es Gs 2300 (USD 0,35), el total fue de Gs 298 080 (USD 45,6), que representa el 8,6 % de pérdida.

Abstract This research work was conducted in a dairy farm in the Nueva Italia district, located in the Central Province. The research aimed to determine the prevalence of subclinical mastitis therein. To do so, 147 female bovine subjects were studied, including Holstein specimens, Jersey-Holstein crossbreeds, and Holstein-Gyr crossbreeds, with different ages and lactation periods. There were both symptomatic and asymptomatic animals. A California Mastitis Test (CMT) was applied and out of the 147 sampled animals, 110 resulted positive to the disease, accounting for a prevalence of 74.83%. Out of the 551 studied rooms, 315 were positive, accounting for 57%. The rooms in the backside accounted for 50.6% of the total positive rooms. Based on the CMT grades, the results were as follows: negative, 43% (236); traces, 17% (91); grade 1.25% (138); grade 2: 13.7% (76), and grade 3: 1.8% (10). There was a decrease of 129.6 liters milk per milking session. Considering that per-liter price is Gs 2300 (USD 0.35), the total loss of the milk was Gs 298,080 (USD 45.6), equaling 8.6% of profit loss.