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1.
J Dairy Sci ; 103(8): 7407-7410, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32600771

RESUMO

The most clinically relevant staphylococci in veterinary medicine are those that are coagulase-positive, namely Staphylococcus aureus. During microbiological udder health monitoring (2009-2018), a new S. aureus strain (coagulase-positive and maltose-negative) was discovered as an emerging udder pathogen during routine examinations of South African dairy herds. This study challenged the conventional microbiological diagnosis of staphylococci by comparing its results to those of the MALDI-TOF mass spectrometry and 16S rRNA sequencing. Both of these tests confirmed that the maltose-negative staphylococcus (MNS), identified as Staphylococcus pseudintermedius by conventional microbiology, was S. aureus ST2992. Multi locus sequence typing was performed on 3 of the MNS isolates and indicated that these isolates were of single origin. These strains tested positive for both MALA and MALR genes (control: S. aureus ATCC 25923). Although the α-glucosidase gene was present, it was not expressed phenotypically. The latter may be attributed to the abnormal stop codon identified in the MALA gene sequence of S. aureus ST2992 (GenBank accession number, MN531305). The newly identified MNS has a field behavior different to that of maltose-positive S. aureus, and more similar to the low virulence of non-aureus staphylococci.


Assuntos
Mastite Bovina/microbiologia , Staphylococcus aureus/isolamento & purificação , Animais , Bovinos , Coagulase/análise , Feminino , Maltose/análise , Glândulas Mamárias Animais/microbiologia , Espectrometria de Massas , Tipagem de Sequências Multilocus , RNA Bacteriano , RNA Ribossômico 16S , Staphylococcus/classificação , Staphylococcus/isolamento & purificação , Staphylococcus aureus/química , Staphylococcus aureus/classificação
2.
J Dairy Sci ; 103(8): 7276-7287, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32505410

RESUMO

The objective of this randomized controlled trial was to assess the efficacy of an on-farm culture system using Petrifilm (3M, London, ON, Canada) for targeted treatment decisions at the quarter level at dry-off and its effects on dry period intramammary infections (IMI) and udder health and milk production in the subsequent lactation. A total of 568 cows (2,247 quarters) from 9 dairy herds with bulk tank somatic cell count <250,000 cells/mL in Québec, Canada, were systematically enrolled and randomly allocated to 4 groups: 2 quarter-based selective (QSDCT) groups, using results of quarter-milk culture on Petrifilm, and 2 blanket dry cow therapy (BDCT) groups. The 2 QSDCT groups consisted of (1) antimicrobial to infected quarters and internal teat sealant (ITS) to healthy quarters (QSDCT/ITS); and (2) antimicrobial and ITS to infected quarters and ITS to healthy quarters (QSDCT+ITS/ITS). The 2 BDCT groups were (1) antimicrobial alone to all quarters (BDCT); and (2) antimicrobial and ITS to all quarters (BDCT+ITS). Quarter milk samples were collected at dry-off and after calving for routine bacteriological culture at the laboratory to monitor IMI; data on milk production, somatic cell count, and clinical mastitis recorded up to 120 d in milk were retrieved from health and DHI records. The probability of avoiding antimicrobial treatment in QSDCT groups was estimated at 48.3% (95% confidence interval: 35.7, 60.9). There was no significant difference between the 4 treatment groups regarding acquisition of new IMI (15.9, 13.2, 15.8, and 15.1% probability for BDCT, BDCT+ITS, QSDCT/ITS, and QSDCT+ITS/ITS, respectively) or persistence of existing IMI (3.2, 2.1, 3.4, and 2.7% probability, respectively) over the dry period. In the subsequent lactation, there was no difference between groups regarding incidence of clinical mastitis (2.4, 3.7, 2.9, and 1.7% respectively for BDCT, BDCT+ITS, QSDCT/ITS, and QSDCT+ITS/ITS), mean milk somatic cell score (1.7, 2.0, 2.0, and 2.0 respectively), or mean daily milk production (43.8, 44.2, 43.2, and 42.6 kg/d, respectively) during the first 120 d in milk. In conclusion, QSDCT using the Petrifilm on-farm culture system to detect infected quarters at dry-off is an interesting option to decrease antibiotic use without any negative effects on udder health or milk production in the first 120 d of the subsequent lactation compared with BDCT.


Assuntos
Antibacterianos/uso terapêutico , Contagem de Colônia Microbiana/veterinária , Mastite Bovina/microbiologia , Leite/microbiologia , Animais , Bovinos , Contagem de Células/veterinária , Fazendas , Feminino , Incidência , Lactação , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/tratamento farmacológico , Mastite Bovina/epidemiologia , Leite/metabolismo , Quebeque/epidemiologia
3.
J Dairy Sci ; 103(8): 7249-7259, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32475664

RESUMO

The mode of action of bismuth subnitrate in teat sealant formulations as a preventative for intramammary infections during the dry period is unknown. Although previous studies proposed an action mechanism-creating a physical barrier in the teat canal to prevent bacterial invasion-it has not been proven experimentally. We hypothesized that bismuth subnitrate has an inhibitory effect on bacterial growth, in addition to its barrier effect. The objective of this study was to assess the effect of bismuth subnitrate on bacterial growth of major mastitis-causing agents. A strain of Streptococcus uberis (SR115), 2 strains of Staphylococcus aureus (SA3971/59 and SA1), and a strain of Escherichia coli (P17.14291) were tested in vitro for their ability to grow in the presence or absence of bismuth subnitrate. Disk diffusion testing, impedance measurement, and evaluation of bacterial growth in shaking conditions were the methods used to test this hypothesis. A reduction of growth in the presence of bismuth subnitrate occurred for all the strains tested. However, we observed strain and species variations in the extent of growth inhibition. These results suggest that an inhibitory effect on bacterial growth by bismuth subnitrate could partially explain the efficacy of bismuth-based formulations for preventing intramammary infections over the dry period. Further research is required to test the effect of teat sealant formulations on bacterial growth.


Assuntos
Bismuto/farmacologia , Escherichia coli/efeitos dos fármacos , Mastite Bovina/prevenção & controle , Leite/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus/efeitos dos fármacos , Animais , Bovinos , Escherichia coli/crescimento & desenvolvimento , Feminino , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus/crescimento & desenvolvimento
4.
J Dairy Sci ; 103(8): 7302-7314, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32475666

RESUMO

Mastitis is one of the major causes for antimicrobial use on dairy cattle farms. On farms with an automatic milking system (AMS), diagnostics differ from those with a conventional milking system (CMS), with potentially a different attitude toward mastitis treatment. This may result in differences in antimicrobial usage (AMU) between these 2 types of farms. The aims of this study were (1) to compare AMU between AMS and CMS farms, (2) to identify variables associated with AMU in both types of herds, and (3) to describe the distribution of mastitis-causing pathogens and their antimicrobial resistance patterns. Data on AMU was collected for 42 AMS and 254 CMS farms in the Netherlands and was expressed as animal-defined daily dose (ADDD). The ADDD variables were total usage (ADDDTOTAL), intramammary usage during lactation (ADDDIMM), usage for dry cow therapy (ADDDDCT), and usage by injection (ADDDINJ). Eighteen AMS farms and 24 CMS farms participated in a survey on factors potentially related to AMU. These farmers collected 5 quarter milk samples from quarters with clinical mastitis or high somatic cell count, which were subjected to bacteriological culture and antimicrobial susceptibility testing. In addition, routinely collected udder health data of these farms were used in the analysis. Nonlinear principal component analysis (NLPCA) was used to explore associations between AMU, udder health, and questionnaire variables. The ADDDTOTAL and ADDDDCT were comparable between AMS and CMS farms, whereas ADDDIMM tended to be lower and ADDDINJ higher on AMS farms than on CMS farms. The NLPCA yielded 3 principal components (PC) that explained 48% of the variation in all these variables. The AMS farms were not distinguished from CMS farms in the principal component space. The 3 PC represented different aspects of udder health, ADDDTOTAL, and treatment strategy. Differences in treatment strategy were unrelated to total antimicrobial usage or overall udder health. The distribution of mastitis-causing pathogens and their antimicrobial resistance were comparable between AMS and CMS farms. In conclusion, our study shows that AMU on AMS farms was similar to that of CMS farms, but AMS farmers tend to apply more injectable and fewer intramammary treatments during lactation than CMS farmers. Across both farm types, farmers' attitudes toward udder health in general and toward mastitis treatment are associated with AMU.


Assuntos
Anti-Infecciosos/uso terapêutico , Mastite Bovina/tratamento farmacológico , Leite/metabolismo , Animais , Atitude , Bovinos , Indústria de Laticínios , Fazendeiros , Fazendas , Feminino , Lactação , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Leite/microbiologia , Países Baixos
5.
J Dairy Sci ; 103(8): 7416-7424, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32475678

RESUMO

The objective of this study was to evaluate the antibacterial activities of extract derived from moringa leaves. In particular, the effect of moringa extract (Mor) on adhesion and invasion of Escherichia coli O55, Enterococcus faecalis, Staphylococcus simulans, and Serratia liquefaciens was evaluated in bovine mammary epithelial cells (MAC-T). Broth microdilution method, minimum inhibitory concentration and minimum bactericidal concentration assays, adhesion and invasion assays, and real-time PCR were performed. The minimum inhibitory concentration and minimum bactericidal concentration of Mor ranged from 12.5 to 50 mg/mL on 18 out of 27 tested isolates. Treatment of E. coli O55 with Mor (100 and 200 µg/mL) inhibited the adhesion and invasion on MAC-T cells via downregulation of adhesion factors (i.e., papC, f17c-A, and eaeA). Also, when MAC-T cells were pretreated with Mor (200 µg/mL, 12 h) and incubated with E. coli O55, Enterococcus faecalis, Staphylococcus simulans, or Serratia liquefaciens, both E. coli O55 and Enterococcus faecalis showed a significant decrease in adhesion and invasion. Staphylococcus simulans exhibited decreased adhesion and increased invasion. Serratia liquefaciens showed increased adhesion and decreased invasion. In addition, Mor increased mRNA expression of antioxidant enzymes (e.g., heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase 1) in MAC-T cells. In conclusion, 12.5 to 50 mg/mL of Mor exhibited antibacterial activity against 18 out of 27 tested isolates. Also, pretreatment of 200 µg/mL of Mor to MAC-T cells modulated adhesion and invasion of E. coli O55 and other mastitis-associated pathogens. Furthermore, Mor increased antioxidant capacities in MAC-T cells, but further in vivo studies are needed.


Assuntos
Antibacterianos/isolamento & purificação , Aderência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Glândulas Mamárias Animais/microbiologia , Moringa/química , Extratos Vegetais/farmacologia , Animais , Antibacterianos/farmacologia , Bovinos , Enterococcus faecalis , Células Epiteliais/microbiologia , Infecções por Escherichia coli/veterinária , Feminino , Mastite Bovina/microbiologia
6.
J Dairy Sci ; 103(8): 7322-7330, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32534929

RESUMO

The aim of this study was to evaluate the effect of sampling technique and milk fraction on bovine milk microbiota data and to compare the microbiota in milk to microbiota on the teat end and in the teat canal. Representative milk samples are highly important for assessment of bacteriological findings and microbiota in milk. Samples were obtained from 5 healthy lactating dairy cows at udder quarter level during 1 milking. Swab samples from the teat end and teat canal, and milk samples collected using different techniques and in different milk fractions were included. Milk was collected by hand stripping and through a teat canal cannula before and after machine milking, through a trans-teat wall needle aspirate after milking, and from udder quarter composite milk. The microbiota of the samples was analyzed with sequencing of the V1-V3 region of the 16S rRNA gene. In addition, somatic cell counts and bacterial cultivability were analyzed in the milk samples. Microbiota data were analyzed using multivariate methods, and differences between samples were tested using analysis of similarity (ANOSIM). Differences between samples were further explored via individual studies of the 10 most abundant genera. The microbiota on the teat end, in the teat canal, and in udder quarter composite milk, collected using a milking machine, differed in composition from the microbiota in milk collected directly from the udder quarter. No differences in milk microbiota composition were detected between hand-stripped milk samples, milk samples taken through a teat canal cannula, or milk samples taken as a trans-teat wall needle aspirate before or after milking. We conclude that for aseptic milk samples collected directly from the lactating udder quarter, sampling technique or milk fraction has minor effect on the microbiota composition.


Assuntos
Bactérias/isolamento & purificação , Bovinos/microbiologia , Microbiota , Leite/microbiologia , Manejo de Espécimes/veterinária , Animais , Bactérias/genética , Indústria de Laticínios , Feminino , Lactação , Glândulas Mamárias Animais/microbiologia , Pele/microbiologia
7.
Anim Sci J ; 91(1): e13406, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32578314

RESUMO

The present study assessed the effects of intramammary infusion of Bifidobacterium breve (B. breve) on mastitis-causing pathogens and on the somatic cell counts (SCC) in lactating cows with chronic subclinical mastitis. The bacteriological cure rates of 42 quarters from 42 cows infected with Staphylococcus aureus, Corynebacterium bovis, coagulase-negative staphylococci, and environmental streptococci were 18.2% (2/11), 14.3% (1/7), 58.8% (10/17), and 28.6% (2/7), respectively, on day 14 after B. breve infusion. In a second trial, B. breve was infused into 18 quarters from 18 cows with chronic subclinical mastitis from which pathogens had not been isolated; the rates of quarters showing SCC > 50 × 104 cells/ml prior to B. breve infusion that decreased to < 30 × 104 cells/ml after infusion were significantly (p < .01) increased to 61.1% (11/18) on day 14 compared to that prior to infusion (0/18). The intramammary infusion of B. breve appears to be a non-antibiotic approach for elimination of minor pathogens and decreasing SCC in quarters with chronic subclinical mastitis in dairy cows.


Assuntos
Bifidobacterium breve , Infecções por Corynebacterium , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Mastite Bovina/terapia , Infecções Estafilocócicas , Animais , Bovinos , Corynebacterium , Infecções por Corynebacterium/veterinária , Feminino , Infusões Intralesionais , Infecções Estafilocócicas/veterinária , Staphylococcus aureus , Resultado do Tratamento
8.
J Appl Microbiol ; 129(5): 1102-1119, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32416020

RESUMO

Staphylococcus aureus is an important agent of contagious bovine intramammary infections in dairy cattle. Its ability to persist inside the udder is based on the presence of important mechanisms such as its ability to form biofilms, polysaccharide capsules small colony variants, and their ability to invade professional and nonprofessional cells, which will protect S. aureus from the innate and adaptive immune response of the cow, and from antibiotics that are no longer considered to be sufficient against S. aureus bovine mastitis. In this review, we present the recent research outlining S. aureus persistence properties inside the mammary gland, including its regulation mechanisms, and we highlight alternative therapeutic strategies that were tested against S. aureus isolated from bovine mastitis such as the use of probiotic bacteria, bacteriocins and bacteriophages. Overall, the persistence of S. aureus inside the mammary gland remains a pressing veterinary problem. A thorough understanding of staphylococcal persistence mechanisms will elucidate novel ways that can help in the identification of novel treatments.


Assuntos
Mastite Bovina/microbiologia , Mastite Bovina/terapia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia , Animais , Bacteriocinas/uso terapêutico , Bovinos , Feminino , Glândulas Mamárias Animais/microbiologia , Terapia por Fagos/veterinária , Probióticos/uso terapêutico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/terapia , Staphylococcus aureus/isolamento & purificação
9.
PLoS One ; 15(5): e0231168, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365127

RESUMO

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows. The objective of this study was to establish a rat model of mastitis induced by S. aureus infection and to explore changes in the proteomes of mammary tissue in different udder states, providing a better understanding of the host immune response to S. aureus mastitis. On day 3 post-partum, 6 rats were randomly divided into two groups (n = 3), with either 100 µL of PBS (blank group) or a S. aureus suspension containing 2×107 CFU·mL-1 (challenge group) infused into the mammary gland duct. After 24 h of infection, the rats were sacrificed, and mammary gland tissue was collected. Tandem mass tag (TMT)-based technology was applied to compare the proteomes of healthy and mastitic mammary tissues. Compared with the control group, the challenge group had 555 proteins with significant differences in expression, of which 428 were significantly upregulated (FC>1.2 and p<0.05) and 127 were downregulated (FC>0.83 and p<0.05 or p<0.01). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that upregulated differentially significant expressed proteins (DSEPs) were associated with mainly immune responses, including integrin alpha M, inter-α-trypsin inhibitor heavy chain 4, and alpha-2-macroglobulin. This study is the first in which a rat model of S. aureus-induced mastitis was used to explore the proteins related to mastitis in dairy cows by TMT technology, providing a model for replication of dairy cow S. aureus-induced mastitis experiments.


Assuntos
Glândulas Mamárias Animais/metabolismo , Mastite/metabolismo , Proteoma/análise , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Feminino , Glândulas Mamárias Animais/microbiologia , Glândulas Mamárias Animais/patologia , Mastite/microbiologia , Mastite/patologia , Gravidez , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Infecções Estafilocócicas/patologia , Espectrometria de Massas em Tandem
10.
J Dairy Sci ; 103(6): 5376-5386, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32229113

RESUMO

We analyzed a large number of immune response parameters from quarter milk samples with distinct bacteriological and quarter somatic cell count (qSCC) statuses. Furthermore, we sought to explore and identify displayed immune response patterns in milk samples from mammary glands with nonspecific mastitis. Thus, 92 quarter milk samples from 28 cows were stratified into 4 groups, as follows: (1) 49 culture-negative control quarters with a low qSCC (<1 × 105 cells/mL) from 19 dairy cows (so-called healthy quarters); (2) 15 culture-negative quarters with high qSCC (>2 × 105 cells/mL; so-called quarters with nonspecific mastitis) from 10 dairy cows; (3) 8 culture-positive quarters with low qSCC (noninflammatory quarters with low qSCC) from 5 dairy cows; and (4) 20 culture-positive quarters with high qSCC (so-called truly infected quarters) from 8 dairy cows. Using flow cytometry, we evaluated the percentage of milk neutrophils and their viability, intracellular reactive oxygen species production, phagocytosis, and the expression of CD62L, CD11b, and CD44 for each of the 4 quarter strata. Furthermore, the percentage of monocyte/macrophages, B cells, and T lymphocyte subsets were evaluated by flow cytometry. Milk samples from bacteriologically negative quarters (both with a low and elevated qSCC) had a lower qSCC than those with bacteriologically positive outcomes (both with a low and elevated qSCC). As expected, the healthy quarters showed the lowest percentage of neutrophils and also showed a higher percentage of milk monocytes/macrophages and lower percentage of T lymphocytes than truly infected quarters. The most prominent result of the present study is that quarters with nonspecific mastitis showed the highest percentage of milk CD4+ T lymphocytes. The healthy quarters had a lower percentage of apoptotic neutrophils than noninflammatory and truly infected quarters, although it did not differ from those from the quarters with nonspecific mastitis. Our study supports the role of differential cell counting in the diagnosis of mastitis, as the milk leukocyte populations markedly fluctuate under healthy and inflammatory conditions. Furthermore, an increase in milk CD4+ T cells was associated with nonspecific mastitis, suggesting an increase in this leukocyte subpopulation is correlated with low bacterial shedding. Our study allows us to go further in our understanding of mammary gland immunity, providing further insights on potential protective mammary gland immunity, which we hypothesize can open new avenues for the development of novel targets that can promote bovine udder health.


Assuntos
Mastite Bovina/imunologia , Leite , Animais , Linfócitos B , Bovinos , Contagem de Células/veterinária , Feminino , Citometria de Fluxo/veterinária , Contagem de Linfócitos , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/diagnóstico , Mastite Bovina/microbiologia , Leite/citologia , Leite/microbiologia , Neutrófilos , Fagocitose , Linfócitos T
11.
J Dairy Sci ; 103(6): 5431-5439, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32229116

RESUMO

Automatic flushing of milking clusters between milking events is a control measure aimed at reducing transmission of mastitis pathogens from infectious milk to a subsequently milked cow. We evaluated the effect of flushing with cold water and flushing with water containing peracetic acid (PAA) on the concentration of Staphylococcus aureus in teat cup liners. Thirty-two clusters in a swing-over milking parlor (Dairymaster, Causeway, Ireland) were subjected to a simulated milking with S. aureus-contaminated milk. Sixteen clusters were not flushed (controls), whereas 8 clusters were flushed with cold water (966 ± 32 mL) and 8 clusters were flushed with water containing PAA (200 mL/mL). A random teat cup in each cluster was sampled by rinsing with a phosphate buffer. Teat cup samples were cultured on the day following collection on Baird-Parker plates to determine the concentration of S. aureus. In teat cup samples from control clusters, the mean concentration of S. aureus was 2.8 × 105 cfu/mL. The concentration of S. aureus was zero in teat cup samples from clusters flushed with cold water. In teat cup samples from clusters flushed with water containing PAA, the concentration of S. aureus was in general reduced compared with control clusters, but S. aureus was not removed completely. However, the automatic cluster flushing did not function properly when clusters were flushed with water containing PAA; thus, results reflected the effect of inadequate function rather than the effect of adding disinfectant to the flushing water. Before the main study, we conducted a pilot study to evaluate whether teat cup sampling with swabs and sample analysis with quantitative PCR were appropriate methods for the main study. Specifically, we evaluated the effect of swab sample mass on detection of S. aureus by quantitative PCR in the laboratory, Further, we compared PCR and bacterial culture on detection of S. aureus in a suspension following disinfection of the suspension with PAA. We sampled 20 identical S. aureus suspensions for culture and PCR by swabs before and after disinfection with PAA. Swab sample mass was determined by differential weighing and contributed to 46% of the variation observed in detection of S. aureus by PCR. Following disinfection with PAA, S. aureus remained detectable by PCR, although culturability ceased. Based on these results, we sampled teat cups in the main study with a buffer rinse and quantified S. aureus in the samples by bacterial culture. We concluded that automatic cluster flushing with cold water was effective in removing S. aureus from teat cup liners and that addition of PAA was therefore not necessary.


Assuntos
Bovinos , Indústria de Laticínios/métodos , Desinfetantes/farmacologia , Desinfecção/métodos , Glândulas Mamárias Animais/microbiologia , Ácido Peracético/farmacologia , Staphylococcus aureus , Animais , Feminino , Higiene , Irlanda , Leite/microbiologia , Mamilos/microbiologia , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Manejo de Espécimes , Água
12.
J Dairy Sci ; 103(7): 6569-6575, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32331896

RESUMO

The main objective of this study was to evaluate the barrier characteristics of 3 external teat sealants for dry cows in preventing bacterial penetration by 3 common major mastitis pathogens (Escherichia coli, Staphylococcus aureus, and Streptococcus uberis) via a novel in vitro simulation model using rubber calf-feeding nipples. All feeding nipples were filled with a sterile cotton plug soaked in sterile broth heart infusion medium and were treated as follows: rubber teats 1 and 5 were sealed with Ubera Dry (Inovet, Arendonk, Belgium); rubber teats 2 and 6 were sealed with T-Hexx Dry (Huvepharma Livestock, St. Louis, MO); rubber teats 3 and 7 were sealed with Uddergold Dry (Ecolab Food and Beverage Division, St. Paul, MN); and rubber teats 4 and 8 remained unsealed and served as positive and negative controls, respectively. After the dips had dried, rubber teats 1 to 4 were immersed in a suspension of E. coli (experiment 1), Staph. aureus (experiment 2), or Strep. uberis (experiment 3; ≥1.5 × 108 cfu/mL) for 24 h, whereas rubber teats 5 to 8 were not exposed to the bacterial suspensions. All external teat sealants adhered well to the rubber teats. All cotton plugs collected from the teats that were not exposed to E. coli, Staph. aureus, or Strep. uberis (rubber teats 5 to 8) remained culture-negative, except for 1 (due to contamination). Of the rubber teats that were exposed to the major mastitis pathogens, all cotton plugs collected from the teats dipped with Ubera Dry and T-Hexx Dry remained culture-negative for the mastitis pathogen they were exposed to. The cotton plugs of the rubber teats that were sealed with Uddergold Dry and exposed to E. coli and Strep. uberis showed positive cultures for the respective bacteria, as demonstrated using strain-typing. The cotton plugs collected from the rubber teats that were not sealed with an external teat sealant and that served as positive controls became culture-positive for the mastitis pathogens they were exposed to. We conclude that Ubera Dry showed comparable and superior barrier performance against penetration of E. coli, Staph. aureus, and Strep. uberis compared with T-Hexx Dry and Uddergold Dry, respectively, under in vitro conditions using a novel in vitro simulation model. Although one should be aware that the method has not yet been validated to predict risk of intramammary infections, the proposed technique can be a meaningful starting point to evaluate and compare the barrier characteristics of external teat sealants in preventing bacterial penetration. A large-scale clinical trial is needed before any definite conclusions can be drawn as to the adherence, duration of adherence, barrier performance, and efficacy in protection against intramammary infections of the 3 external teat sealants under field conditions.


Assuntos
Anti-Infecciosos Locais/administração & dosagem , Escherichia coli/fisiologia , Mastite Bovina/prevenção & controle , Staphylococcus aureus/fisiologia , Staphylococcus/fisiologia , Animais , Bélgica , Bovinos , Indústria de Laticínios , Feminino , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Borracha
13.
J Dairy Sci ; 103(5): 4732-4737, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32113752

RESUMO

Staphylococcus aureus is an important intramammary pathogen for dairy cows that also is remarkably important for public health. Multiple virulence factors can be involved simultaneously during the pathogenesis of a staphylococcal disease, including adhesion proteins, extracellular enzymes, and toxins. The main objective of this study was to assess virulence factors that are associated with cow intramammary infection (IMI) and of human health concern among Staph. aureus isolates obtained from bulk tank milk (BTM) and adherences on milking equipment surfaces. A total of 166 Staph. aureus isolates from 23 dairy farms were characterized according to their virulence profiles. For virulence factors of importance in IMI, the presence of the virulence markers thermonuclease (nuc) and coagulase (coa) and virulence genes such as fibronectin (fnbA) and intercellular adhesion (icaA, icaD) were assessed. For virulence factors of public health concern, presence of antimicrobial resistance (mecA and mecC) and enterotoxin (sea and seb) genes were analyzed. Among all Staph. aureus isolates, 5 virulence profiles were found; the profile nuc(+)coa(+)fnbA(+)icaA(+)icaD(+)mecA(-)mecC(-)sea(-)seb(-) was the most frequently observed (21 out of 23 dairy farms). No differences were found between the virulence profile frequencies of Staph. aureus from BTM and adherences on milking equipment surfaces. The virulence profiles most frequently observed included genes involved in the adherence and biofilm-forming ability of Staph. aureus, which could represent a potential advantage for the bacterium during the early stages of IMI colonization and for persistence on surfaces. Our results indicate a greater frequency of virulence factors of importance for IMI pathogenesis than virulence factors of public health concern, consistent with the dairy origin of isolates. The mecA, mecC, and seb genes were not observed among Staph. aureus isolates analyzed in this study. However, the sea gene was detected in 3 Staph. aureus isolated from BTM, thus posing a potential public health threat. Our results emphasize the importance of understanding the epidemiology and dynamics of Staph. aureus on dairy farms as a tool for the improvement of udder health and milk safety.


Assuntos
Biofilmes/crescimento & desenvolvimento , Mastite Bovina/microbiologia , Leite/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Animais , Bovinos , Chile , Coagulase/genética , Indústria de Laticínios/instrumentação , Enterotoxinas/genética , Fazendas , Feminino , Glândulas Mamárias Animais/microbiologia , Nuclease do Micrococo/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/fisiologia , Virulência
14.
J Dairy Sci ; 103(5): 4088-4099, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32197847

RESUMO

Spore-forming bacteria, such as Paenibacillus spp. and Bacillus spp., can survive HTST pasteurization in spore form and affect the quality of dairy products (e.g., spoilage in fluid milk). With the demand for higher quality finished products that have longer shelf lives and that can be distributed further and to new markets, dairy processors are becoming interested in obtaining low-spore raw milk supplies. One method to reduce spores in the dairy system will require disrupting the transmission of spores from environmental locations, where they are often found at high concentrations (e.g., manure, bedding), into bulk tank raw milk. Previous research has suggested that cow hygiene factors (e.g., udder hygiene, level of spores in milk from individual cows) are important for the transmission of spores into bulk tank raw milk, suggesting that one potential strategy to reduce spores in bulk tank milk should target cow hygiene in the parlor. To that end, we conducted a study on 5 New York dairy farms over a 15-mo period to evaluate the effect of a combination of intervention strategies, applied together, on the levels of aerobic spores in bulk tank raw milk. The combination of interventions included (1) training milking staff to focus on teat-end cleaning during milking preparation, and (2) implementing changes in laundered towel preparation (i.e., use of detergent, chlorine bleach, and drying). Study design involved collecting bulk tank raw milk samples for a week before and a week after initiating the combination of interventions (i.e., training on the importance of teat-end cleaning and towel treatment). Observations on teat-end condition, udder hygiene scores, and number of kickoffs during milking were also collected for 24 h before and after implementation of the interventions. A total of 355 bulk tank raw milk samples were collected with mean mesophilic and thermophilic spore counts of 2.1 and 2.4 cfu/mL, respectively, before interventions were applied, and 1.6 and 1.5 cfu/mL, respectively, after the interventions were applied. These reductions represent decreases of 37 and 40% in bulk tank raw milk mesophilic spores and thermophilic spores, respectively. Importantly, spore reductions were observed during each of the 3 visits once the interventions were applied, and the largest reduction in spores was recorded for the first sampling after training the milking staff. Further, when a higher proportion of very rough teat ends was observed, bulk tank milk thermophilic spore counts were significantly higher. The intervention strategies tested here represent easy-to-execute cleaning strategies (e.g., focusing on teat-end hygiene and towel washing procedures) that can reduce bulk tank raw milk spore levels. Future studies should validate the effect of on-farm interventions for reduced spore raw milk on corresponding processed product quality and will need to verify the effects of these small changes on product shelf life.


Assuntos
Bovinos , Indústria de Laticínios/métodos , Higiene , Leite/microbiologia , Animais , Contagem de Colônia Microbiana , Feminino , Glândulas Mamárias Animais/microbiologia , New York , Paenibacillus , Pasteurização , Esporos Bacterianos
15.
J Dairy Sci ; 103(5): 4717-4731, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32171518

RESUMO

Mastitis is an important constraint to milk production in pastoralist camel (Camelus dromedarius) herds in Kenya. The objective of this study was to investigate the prevalence, risk factors, and bacterial panorama of subclinical mastitis (SCM) in pastoralist camel herds in Isiolo County, Kenya. Furthermore, antimicrobial susceptibility in udder pathogens was studied. A cross-sectional sample of 206 camels from 20 milking herds was screened using the California Mastitis Test (CMT), and quarter milk was subjected to bacterial culturing. Isolates were confirmed using MALDI-TOF mass spectrometry analysis, and antimicrobial susceptibility was determined using the broth microdilution method. Interviews focusing on herd management were conducted with camel owners. Subclinical mastitis, defined as a CMT score ≥ 3 (scale 1 to 5) and absence of clinical symptoms in the udder, were present in all visited herds. On the individual level, 46% of the camels had at least 1 quarter affected with SCM, and on the quarter level the prevalence was 26%. Intramammary infections (IMI) were common; out of 798 quarter milk samples, 33% yielded conclusive bacterial growth. The sensitivity and specificity of CMT for correctly identifying quarters with IMI were 82% and 92%, respectively. The most prevalent pathogen was Streptococcus agalactiae (72% of IMI-positive quarters), followed by non-aureus staphylococci (19%) and Staphylococcus aureus (13%). Antimicrobial susceptibility testing revealed that only a low proportion (4.9%) of Strep. agalactiae isolates was sensitive to tetracycline. For Staph. aureus, 59.1% of isolates exhibited sensitivity to penicillin. Skin lesions on the teats or udder were a risk factor for SCM. Increased age, parity, and stage of lactation were associated with increased risk of both SCM and IMI. Older camels with a blind teat or a previous history of mastitis were more likely to be infected with Strep. agalactiae. Hygiene routines for milking were largely absent in the observed herds, and knowledge of adequate milk handling was limited. The poor udder health is likely to depend on multiple factors, most prominently the within-herd maintenance of contagious udder pathogens, in combination with difficult sanitary conditions and lack of awareness among camel keepers. This study showed that in pastoralist camel herds around Isiolo town, SCM and IMI specifically caused by Strep. agalactiae are common udder health problems and are associated with increasing age, parity, and stage of lactation, and skin lesions on the teats and udder. Resistance to tetracycline in Strep. agalactiae was common. Control strategies specifically targeting SCM and adapted to pastorally managed camel herds need to be developed to reduce disease, combat antimicrobial resistance, and improve the livelihoods of pastoralists.


Assuntos
Antibacterianos/farmacologia , Camelus/microbiologia , Farmacorresistência Bacteriana , Mastite/veterinária , Leite/microbiologia , Infecções Estafilocócicas/veterinária , Streptococcus/classificação , Animais , Estudos Transversais , Feminino , Geografia , Higiene , Quênia/epidemiologia , Lactação , Glândulas Mamárias Animais/microbiologia , Mastite/epidemiologia , Mastite/microbiologia , Leite/metabolismo , Prevalência , Fatores de Risco , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Streptococcus agalactiae/classificação , Tetraciclina/farmacologia
16.
J Dairy Sci ; 103(5): 4579-4587, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32147259

RESUMO

The first objective of this study was to determine whether differences would occur among teat end preparation techniques with regard to potential contamination of milk samples collected for bacterial culture. The second objective was to determine whether differences would be detected in genus or species of bacteria isolated from samples collected using the various methods as well as from contaminated or uncontaminated samples. Mammary quarter foremilk samples were collected from lactating dairy cattle at the University of Missouri Foremost Research Dairy Farm (Columbia). Four different teat end preparation methods were used to compare contamination rates in milk samples. Sampling techniques used before milk collection included (1) no preparation, (2) pre-milking disinfection and single-use towel drying of teats only, (3) scrubbing of the teat end with alcohol only, and (4) pre-milking disinfection, single-use towel drying, and scrubbing of the teat end with alcohol. Milk was plated on Columbia blood agar. Cultures were read at 48 h, with the number of morphologically different bacterial colony types quantified and isolated. Isolates were identified using MALDI-TOF mass spectrometry. Median numbers of colony types were compared among groups using Kruskal-Wallis ANOVA with post-hoc pairwise comparisons, and proportional data were compared using the chi-squared test. A total of 168 cows, including 665 quarters, were sampled, and 1,614 isolates resulted. Analysis with MALDI-TOF identified 29 unique genera and 81 unique species among the samples. More contaminated samples occurred in groups 1 and 2 compared with groups 3 and 4. Group 3 had more contaminated samples than group 4. The majority of Pseudomonas spp. isolates were identified within group 2. When applying previously described niches to Staphylococcus spp., environmental species were more likely to be identified among contaminated samples, whereas host-adapted species were more likely to be identified among uncontaminated samples. These data confirm that scrubbing the teat end with alcohol after pre-milking disinfection with an iodine-based teat disinfectant and drying of the teat minimizes contamination of the milk sample.


Assuntos
Bovinos/microbiologia , Desinfecção/métodos , Glândulas Mamárias Animais/microbiologia , Leite/microbiologia , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Desinfetantes , Feminino , Iodo , Lactação , Staphylococcus/isolamento & purificação
17.
Vet Res ; 51(1): 32, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122405

RESUMO

The presence of non-aureus staphylococci (NAS) in bovine rectal feces has recently been described. Similar to other mastitis causing pathogens, shedding of NAS in the environment could result in intramammary infection. The objective of this study was to investigate whether NAS strains present in feces can cause intramammary infection, likely via teat apex colonization. During a cross-sectional study in 5 dairy herds, samples were collected from the habitats quarter milk, teat apices, and rectal feces from 25%, 10%, and 25% of the lactating cows, respectively, with a cow serving as the source of one type of sample only. Samples from clinical mastitis cases were continuously collected during the 1-year study period as well. The 6 most prevalent NAS species, Staphylococcus (S.) chromogenes, S. cohnii, S. devriesei, S. equorum, S. haemolyticus, and S. hominis, were further subtyped by random amplification of polymorphic deoxyribonucleic acid polymerase chain reaction (RAPD-PCR), when the same NAS species was present in the same herd in the three habitats. For S. chromogenes, S. cohnii, S. devriesei, and S. haemolyticus, the same RAPD type was found in rectal feces, teat apices, and quarter milk, indicating that fecal NAS can infect the mammary gland. For S. hominis and S. equorum, we were unable to confirm the presence of the same RAPD types in the three habitats.


Assuntos
Doenças Mamárias/veterinária , Doenças dos Bovinos/microbiologia , Glândulas Mamárias Animais/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/isolamento & purificação , Animais , Bélgica , Doenças Mamárias/microbiologia , Bovinos , Estudos Transversais , Fezes/microbiologia , Feminino , Infecções Estafilocócicas/microbiologia , Staphylococcus/classificação
18.
Vet Microbiol ; 242: 108594, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32122598

RESUMO

Non-S. aureus staphylococci (NAS) are the most frequently isolated pathogens from bovine milk and can cause intramammary infections (IMI). They can also be found in teat canals, on bovine skin and in cows' environment, which may lead to unnoticed contamination of milk samples. The aim of this study was to investigate the role of NAS species as mastitis-causing pathogens or contaminants, and to identify possible differences between NAS species. A longitudinal study was conducted with consecutive milk sampling in five German dairy herds. Species identification was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Infections were distinguished from contaminations using two different definitions based on the repeated detection of an NAS species. Of 15 NAS species found, eight and ten, respectively, were associated with an IMI. Staphylococcus simulans and S. chromogenes were associated with IMI in more than 90 % of the findings. S. warneri, S. xylosus, S. microti, S. haemolyticus, and S. succinus seem to be frequent causes of IMI as well as contaminants. If a species-differentiation is available after cultivating NAS, the findings should be interpreted in consideration of the observations made in this study, whether it is more likely a question of a contaminant or a cause of intramammary infection. The bacteria shedding intensity of the NAS species with a more substantially adverse effect on udder health seems to be higher than that of the less important NAS pathogens.


Assuntos
Contaminação de Alimentos/análise , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Leite/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/isolamento & purificação , Animais , Derrame de Bactérias , Bovinos , Contagem de Colônia Microbiana , Indústria de Laticínios , Feminino , Alemanha , Inflamação , Estudos Longitudinais , Mastite Bovina/diagnóstico , Infecções Estafilocócicas/diagnóstico , Staphylococcus/classificação , Staphylococcus aureus
19.
Vet Microbiol ; 242: 108608, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32122612

RESUMO

We aimed to identify the dynamics of the within-herd prevalence of Mycoplasma (M.) bovis intramammary infection (IMI) in four dairy herds, estimate prevalence of M. bovis in colostrum and clinical mastitis cases and compare M. bovis strains from calves' respiratory and cow clinical mastitis samples. Within a six-month study period, cow composite milk samples (CMS) were collected three times during routine milk recording, first milking colostrum samples from all calving cows and udder quarter milk samples from clinical mastitis cases. Calf respiratory samples were collected from calves with respiratory disease. Pooled milk samples were analysed for M. bovis with the Mastitis 4B polymerase chain reaction (PCR) test kit (DNA Diagnostic A/S). Prevalence estimates were calculated with Bayesian framework in R statistical programme. cg-MLST was used for M. bovis genotyping. In Herd I and II first testing M. bovis IMI within-herd prevalence (95 % credibility interval (CI)) was 4.7 % (2.9; 6.8) and 3.4 % (2.3; 4.6), changing to 1.0 % (0.1; 1.7) and 0.8 % (0.1; 1.4) in Herd I and 0.4 % (0.0; 0.7) in Herd II at the next samplings. In Herd III and IV first testing M. bovis IMI within-herd prevalence was 12.3 % (9.7; 15.2) and 7.8 % (6.2; 9.5), changing to 4.6 % (3.0; 6.4) and 3.2 % (1.9; 4.8) in Herd III and to 2.8 % (1.9; 3.8) and 4.9 % (3.6; 6.4) in Herd IV at the next samplings. The estimated prevalence of M. bovis in colostrum ranged between 1.7 % (0.2; 2.8) and 4.7 % (2.7; 7.1) and in clinical mastitis cases between 3.7 % (1.7; 6.4) and 11.0 % (7.5; 15.2) in the study herds. M. bovis strains isolated from cows and calves clustered within herds indicating possible transmission of M. bovis between dairy cows and calves. Prevalence of M. bovis in colostrum and clinical mastitis cases as well as the within-herd prevalence of M. bovis IMI was low in endemically infected dairy herds.


Assuntos
Glândulas Mamárias Animais/microbiologia , Mastite Bovina/epidemiologia , Mastite Bovina/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma bovis/genética , Animais , Técnicas de Tipagem Bacteriana , Teorema de Bayes , Bovinos , Colostro/microbiologia , Estudos Transversais , Indústria de Laticínios , Estônia/epidemiologia , Feminino , Genótipo , Tipagem de Sequências Multilocus , Infecções por Mycoplasma/epidemiologia , Mycoplasma bovis/classificação , Prevalência
20.
J Dairy Sci ; 103(4): 3493-3504, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32037181

RESUMO

Klebsiella pneumoniae, a common cause of clinical mastitis (CM) in dairy cows, can cause severe clinical symptoms. However, its pathogenicity in the bovine mammary gland is not well understood. Our objectives were to establish an in vitro infection model of K. pneumoniae on bovine mammary epithelial cells (bMEC) to assess (1) cytopathogenicity (adhesive and invasive ability, damage and apoptosis, pro-inflammatory effects) of K. pneumoniae on bMEC and (2) the role of hypermucoviscous (HMV) phenotype on cytopathogenicity. Two K. pneumoniae isolates from CM cows, 1 HMV and 1 non-HMV, were used to infect bMEC. Adhesion and invasion ability, release of lactate dehydrogenase (LDH), ultrastructural morphology, apoptosis, transcriptional expression of pro-inflammatory genes and production of pro-inflammatory cytokines were characterized at various intervals. Both K. pneumoniae isolates rapidly adhered to and invaded bMEC within 1 h post infection (pi), causing ultrastructural damage (swelling of mitochondria and vesicle formation on cell surface) after 3 h pi and apoptotic death after 9 h pi. In addition, K. pneumoniae promoted transcriptional expression of pro-inflammatory genes IL-6, IL-8, IL-1ß, and tumor necrosis factor (TNF)-α and production of IL-8, IL-1ß, and TNF-α cytokines. Compared with non-HMV K. pneumoniae, the HMV isolate had lower adhesive and invasive abilities but caused more serious cellular damage. In conclusion, K. pneumoniae was cytopathogenic on bMEC and induced a pro-inflammatory response; however, the HMV phenotype did not have a key role in pathogenicity. Therefore, more attention should be paid to milk loss, and targeted prevention and treatment strategies should be implemented in Klebsiella mastitis episodes.


Assuntos
Células Epiteliais/microbiologia , Klebsiella pneumoniae/patogenicidade , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Animais , Apoptose , Aderência Bacteriana , Bovinos , Linhagem Celular , Citocinas/metabolismo , Células Epiteliais/metabolismo , Feminino , Interleucinas/metabolismo , Klebsiella pneumoniae/isolamento & purificação , Glândulas Mamárias Animais/patologia , Leite/microbiologia , Mitocôndrias/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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