Clinical Mastitis Cases Research Articles

Treating clinical mastitis in dairy herds: a role for on farm culture?

The focus on the rational use of antibiotics in dairy herds has meant that mastitis control has received renewed interest, particularly around prevention of new infections and alternative treatment strategies. The former has seen the development of a new industry initiative (QuarterPRO) to help advisors and farmers reduce the rate of new infections and index cases. The latter has seen widespread uptake of the selective use of intramammary antibiotic for infected cows at drying-off, a reminder to the veterinary profession as well as clients about the general lack of evidence for the use of parenteral antibiotic in combination with intramammary antibiotic for mild and moderate clinical mastitis events, and finally the potential to select intramammary antibiotic treatment for clinical mastitis using on farm culture (OFC). It is important that veterinary surgeons are aware of the research around the development and use of OFC and the suitability of individual herds for this approach. Whereas antibiotics may not be required for mild clinical mastitis events caused by some pathogens, particularly Escherichia coli, it is important that clinical mastitis events (particularly first cases in a lactation cycle) caused by Gram-positive pathogens such as Streptococcus spp. and Staphylococcus aureus are treated with intramammary antibiotic to optimise chance of cure and reduce risk of transmission. With this in mind, we need to consider individual herd cell count data, herd pathogen profile and ensure careful monitoring of outcomes for clinical mastitis cases in herds that are selecting treatment based on OFC, particularly for index (first) cases in a cow's lactation.

Genomic comparisons and phylogenetic analysis of mastitis-related staphylococci with a focus on adhesion, biofilm, and related regulatory genes

Mastitis is a common and costly disease on dairy farms, commonly caused by Staphylococcus spp. though the various species are associated with different clinical outcomes. In the current study, we performed genomic analyses to determine the prevalence of adhesion, biofilm, and related regulatory genes in 478 staphylococcal species isolated from clinical and subclinical mastitis cases deposited in public databases. The most prevalent adhesin genes (ebpS, atl, pls, sasH and sasF) were found in both clinical and subclinical isolates. However, the ebpS gene was absent in subclinical isolates of Staphylococcus arlettae, S. succinus, S. sciuri, S. equorun, S. galinarum, and S. saprophyticus. In contrast, the coa, eap, emp, efb, and vWbp genes were present more frequently in clinical (vs. subclincal) mastitis isolates and were highly correlated with the presence of the biofim operon (icaABCD) and its transcriptional regulator, icaR. Co-phylogenetic analyses suggested that many of these adhesins, biofilm, and associated regulatory genes could have been horizontally disseminated between clinical and subclinical isolates. Our results further suggest that several adhesins, biofilm, and related regulatory genes, which have been overlooked in previous studies, may be of use for virulence profiling of mastitis-related Staphylococcus strains or as potential targets for vaccine development.

Longitudinal study of Staphylococcus aureus genotypes isolated from bovine clinical mastitis

Bovine clinical mastitis is an important problem for the dairy industry, and Staphylococcus aureus is a common mastitis-causing pathogen in many countries. Detailed knowledge on genetic variation of Staph. aureus strains within the bovine population, including changes over time, can be useful for mastitis control programs, because severity of disease and effects on milk production are at least partly strain-associated. Therefore, the major aim of this study was to compare sequence types of Staph. aureus isolated from cases of bovine clinical mastitis from 2002 to 2003 with sequence types of a more recent set of isolates collected from 2013 to 2018, using core genome multi-locus sequence typing (cgMLST). We also wanted to compare antibiotic resistance genes of isolates from the 2 sets, to identify changes that may have occurred over time in the Staph. aureus population. A total of 157 isolates of Staph. aureus, almost equally distributed between the 2 time periods, were subjected to high-throughput sequencing and cgMLST. The results showed that the most prevalent sequence types found among the 2002 to 2003 isolates belonged to the clonal complexes CC97, CC133, and CC151, and that those complexes still dominated among the isolates from 2013 to 2018. However, a population shift from CC133 to CC97 and CC151 over time was observed. Likewise, no important differences in prevalence of antibiotic resistance genes were found between the 2 sets of isolates. As expected, genes belonging to the major facilitator superfamily of transporter proteins, and multidrug and toxic compound extrusion transporters, were very common. Moreover, several genes and mutations conferring resistance to fosfomycin were present, but not in CC97 isolates. The β-lactamase gene blaZ was found in only 3 out of 81 isolates from 2002 to 2003 and 1 out of 76 isolates in 2013 to 2018. In conclusion, the results indicate that mastitis-associated Staph. aureus strains circulating among dairy cows in Sweden exhibit a remarkable genotypic persistence over a time frame of close to 15 yr.

Randomized controlled trial of pegbovigrastim as an adjunct therapy for naturally occurring severe clinical mastitis cases in dairy cows

The main objective of this study was to quantify the effect of pegbovigrastim (PEG) as an adjunct therapy for naturally occurring severe mastitis cases on survival, intramammary bacteriological cure, and subsequent milk production. A double-blinded, randomized controlled trial was conducted on a single commercial dairy farm. During the study period, all cows having a case of severe clinical mastitis, defined as the presence of abnormal milk and inflammation in one quarter or more combined with the presence of systemic signs (pyrexia, dehydration, or recumbency), were enrolled in the study. In addition to a standardized therapy combining systemic and intramammary antimicrobials as well as systemic anti-inflammatory drugs, cows received a subcutaneous injection of either 2.7 mL of 0.9% sterile saline (control group; CON) or 2.7 mL of PEG (PEG group). A milk sample for bacteriological analysis was taken before treatment was administered and a second sample was taken 14 d after enrollment. Survival (30 d post-treatment) and bacteriological cure (14 d post-treatment) were analyzed by survival analysis and chi-squared tests, respectively, whereas daily milk production was treated as a repeated measure in mixed regression models. The 77 cows enrolled in the study were of parity 2 to 4 (mean = 3) and between 3 and 302 d in milk (mean = 154). The bacteria identified in milk at enrollment were Klebsiella spp. (n = 48; 62%), Escherichia coli (n = 16; 21%), Enterobacter spp. (n = 10; 13%), or no growth (n = 3; 4%). The probability of survival during the first 30 d after treatment was higher in the PEG group (84%) than in the control group (46%). Daily milk production over the 30-d period following treatment and bacteriological cure 14 d post-treatment did not differ between groups. Overall, cows treated with PEG as an adjunct therapy for naturally occurring cases of severe clinical mastitis had a better survival than cows treated with saline.

Antimicrobial Resistance and Virulence Factor of Streptococcus dysgalactiae Isolated from Clinical Bovine Mastitis Cases in Northwest China.

ObjectiveStreptococcus dysgalactiae is a major pathogen in bovine mastitis. The purpose of this study was to survey the prevalence, antimicrobial resistance, as well as the spread of resistance and virulence-associated gene of S. dysgalactiae.MethodsA total of 60 S. dysgalactiae strains were obtained from 830 milk samples from Holstein cows with clinical mastitis. Antimicrobial resistance was examined by the disk diffusion method. Antimicrobial resistance and virulence genes were investigated by PCR, agarose gel electrophoresis and 16S rRNA gene sequencing.ResultsAll isolates were resistant to tetracycline and showed a high level of resistance to aminoglycoside antibiotics, where 81.67% of the strains were multi-resistant to these ten sorts of antibiotics. In addition, the most prevalent resistance gene in S. dysgalactiae was aphA-1 (98.33%), followed by blaTEM (96.67%), ermB (83.3%), aadA1/aadA2 (78.33%) and tetL (73.33%). Totally, seven virulence genes with 25 combination patterns were detected in these isolates, and each isolates harbored at least one virulence gene. 21.67% of the isolates carried three or more virulence genes, while one strain with seven virulence-related genes and belonged to cfb+lmb+eno+napr+bca+scpB+cyl.ConclusionThese findings indicate that S. dysgalactiae isolated from clinical bovine mastitis cases in Northwest China show a variety of molecular ecology and are highly resistant to antibiotics commonly used in dairy farms. This research will help investigators better understand the pathophysiology S. dysgalactiae in bovine mastitis and choose the appropriate antibiotics to treat mastitis.

Prevalence and mechanisms of antibiotic resistance in Escherichia coli isolated from mastitic dairy cattle in Canada

BackgroundBovine mastitis is the most common infectious disease in dairy cattle with major economic implications for the dairy industry worldwide. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from dairy farms is vital not only for animal husbandry but also for public health.MethodsIn this study, the prevalence of AMR in 113 Escherichia coli isolates from cases of bovine clinical mastitis in Canada was investigated. Kirby-Bauer disk diffusion test with 18 antibiotics and microdilution method with 3 heavy metals (copper, zinc, and silver) was performed to determine the antibiotic and heavy-metal susceptibility. Resistant strains were assessed for efflux and ß-lactamase activities besides assessing biofilm formation and hemolysis. Whole-genome sequences for each of the isolates were examined to detect the presence of genes corresponding to the observed AMR and virulence factors.ResultsPhenotypic analysis revealed that 32 isolates were resistant to one or more antibiotics and 107 showed resistance against at least one heavy metal. Quinolones and silver were the most efficient against the tested isolates. Among the AMR isolates, AcrAB-TolC efflux activity and ß-lactamase enzyme activities were detected in 13 and 14 isolates, respectively. All isolates produced biofilm but with different capacities, and 33 isolates showed α-hemolysin activity. A positive correlation (Pearson r = + 0.89) between efflux pump activity and quantity of biofilm was observed. Genes associated with aggregation, adhesion, cyclic di-GMP, quorum sensing were detected in the AMR isolates corroborating phenotype observations.ConclusionsThis investigation showed the prevalence of AMR in E. coli isolates from bovine clinical mastitis. The results also suggest the inadequacy of antimicrobials with a single mode of action to curtail AMR bacteria with multiple mechanisms of resistance and virulence factors. Therefore, it calls for combinatorial therapy for the effective management of AMR infections in dairy farms and combats its potential transmission to the food supply chain through the milk and dairy products.

Microbial Aetiology, Antibiotic Susceptibility and Pathogen-Specific Risk Factors for Udder Pathogens from Clinical Mastitis in Dairy Cows.

Simple SummaryMastitis is among the diseases in dairy cows that most often require antibiotic treatment. In order to maintain optimal treatment, it is important to have updated knowledge about the causative agents and their antibiotic resistance patterns. This investigation aimed to reveal the most important bacterial pathogens and their resistance patterns in Sweden, and we also identified some risk factors for infection with certain pathogens. The bacteria that were the most common causes of mastitis were, in descending order, Staphylococcus aureus, Streptococcus dysgalactiae, Escherichia coli, and Streptococcus uberis. Only a few Gram-positive bacteria were resistant to penicillin, and in general, the occurrence of antibiotic resistance was low. Therefore, the potential for antibiotic treatment of bovine mastitis in Sweden is good.Mastitis is one of the most important infectious diseases and one of the diseases that causes the greatest use of antibiotics in dairy cows. Therefore, updated information on the bacteria that cause mastitis and their antibiotic susceptibility properties is important. Here, for the first time in over 10 years, we updated the bacterial findings in clinical mastitis in Swedish dairy cows together with their antibiotic resistance patterns and risk factors for each bacterial species. During the period 2013–2018, samples from clinical mastitis were collected, together with information on the cows and herds of origin. The samples were cultured, and a total of 664 recovered bacterial isolates were subjected to susceptibility testing. Staphylococcus aureus (S. aureus) was the most common pathogen and accounted for 27.8% of diagnoses, followed by Streptococcus dysgalactiae (S. dysgalactiae) (15.8%), Escherichia coli (E. coli) (15.1%), Streptococcus uberis (S. uberis) (11.4%), Trueperella pyogenes (T. pyogenes) (7.7%), non-aureus staphylococci (NAS) (2.8%), Klebsiella spp. (2.7%), Enterococcus spp. (1.3%), and Streptococcus agalactiae (S. agalactiae) (1.2%). Various other bacteria accounted for 2.6%. Staphylococci were, in general, susceptible to most antibiotics, but 2.6% of S. aureus and 30.4% of NAS were resistant to penicillin. No methicillin-resistant staphylococci were found. All S. agalactiae were susceptible to penicillin. Bimodal and trimodal MIC distributions for penicillin in S. dysgalactiae and S. uberis, respectively, indicate acquired reduced susceptibility in some isolates. The mostly unimodal MIC distributions of T. pyogenes indicate that acquired resistance does usually not occur in this species. Among E. coli, 14.7% were resistant to at least one antibiotic, most often ampicillin (8.7%), streptomycin (7.8%), or sulphamethoxazole (6.9%). Klebsiella spp. had low resistance to tetracycline (9.1%) but is considered intrinsically resistant to ampicillin. Pathogen-specific risk factors were investigated using multivariable models. Staphylococcus aureus, S. dysgalactiae, and T. pyogenes were more common, while E. coli was less common in quarters with more than one pathogen. S. aureus and T. pyogenes were mostly seen in early lactation, while E. coli was more common in peak to mid lactation and S. dysgalactiae in early to peak lactation. Trueperella pyogenes and Klebsiella spp. were associated with a previous case of clinical mastitis in the current lactation. Staphylococcus aureus was associated with tie stalls and T. pyogenes with loose housing. All pathogens except E. coli and S. dysgalactiae had a seasonal distribution. In conclusion, the aetiological agents for clinical bovine mastitis have remained relatively stable over the last 10–15 years, S. aureus, S. dysgalactiae, E. coli and S. uberis being the most important. Resistance to penicillin among Gram-positive agents was low, and in general, antibiotic resistance to other compounds was low among both Gram-positive and Gram-negative agents.

Effects of prepartum vaccination timing relative to pen change with an acidogenic diet on serum and colostrum immunoglobulins in Holstein dairy cows

The objective of the present study was to assess the effects of prepartum vaccination timing relative to pen change with an acidogenic diet at 28 or 21 d before expected parturition (dpp) on colostral and serum IgG concentrations at calving in pregnant Holstein dairy cows. Pregnant multiparous Holstein cows (n = 308) from one large dairy herd were randomly allocated into 1 of 3 treatment groups at 35 ± 3 dpp: (1) vaccination at 28 dpp and pen change at 21 dpp (V28PC21; n = 108), (2) vaccination and pen change at 28 dpp (V28PC28; n = 99), and (3) vaccination and pen change at 21 dpp (V21PC21; n = 101). An acidogenic diet was fed when cows changed pens at 28 or 21 dpp. Blood and colostral samples were collected within 1 h following parturition. The total number of clinical mastitis (CM) cases within the first 150 d in milk (DIM) were recorded. The V28PC21 cows had greater colostral IgG concentrations at calving (160.4 ± 7.0 g/L) compared with V21PC21 cows (134.4 ± 7.0 g/L), and V28PC28 cows were intermediate (148.3 ± 7.2 g/L). At calving, V28PC21 cows had lower serum IgG concentrations (29.1 ± 1.2 g/L) compared with V21PC21 cows (32.2 ± 1.2 g/L) or V28PC28 cows (32.6 ± 1.3 g/L). Overall, 41% of V21PC21 cows received the booster vaccinations with at least 21 d before actual calving compared with V28PC21 or V28PC28 cows (88 and 86% respectively). The shorter the interval from prepartum booster vaccination to calving, the lower the colostral IgG at calving, regardless of treatment groups. Vaccinating at 28 dpp and pen change with an acidogenic diet at 21 dpp tended to reduce the rate of CM within the first 150 DIM compared with V21PC21. These findings provide evidence that vaccinating cows at 28 dpp, followed by pen change with an acidogenic diet at 21 dpp, improved the concentrations of colostral IgG at calving and tended to reduce the rate of CM. The interaction of prepartum vaccination timing relative to feeding an acidogenic diet should be considered when implementing an effective vaccination program to enhance overall herd health.

Comparative genomic analysis of Escherichia coli isolates from cases of bovine clinical mastitis identifies nine specific pathotype marker genes.

Escherichia coli is a major causative agent of environmental bovine mastitis and this disease causes significant economic losses for the dairy industry. There is still debate in the literature as to whether mammary pathogenic E. coli (MPEC) is indeed a unique E. coli pathotype, or whether this infection is merely an opportunistic infection caused by any E. coli isolate being displaced from the bovine gastrointestinal tract to the environment and, then, into the udder. In this study, we conducted a thorough genomic analysis of 113 novel MPEC isolates from clinical mastitis cases and 100 bovine commensal E. coli isolates. A phylogenomic analysis indicated that MPEC and commensal E. coli isolates formed clades based on common sequence types and O antigens, but did not cluster based on mammary pathogenicity. A comparative genomic analysis of MPEC and commensal isolates led to the identification of nine genes that were part of either the core or the soft-core MPEC genome, but were not found in any bovine commensal isolates. These apparent MPEC marker genes were genes involved with nutrient intake and metabolism [adeQ, adenine permease; nifJ, pyruvate-flavodoxin oxidoreductase; and yhjX, putative major facilitator superfamily (MFS)-type transporter], included fitness and virulence factors commonly seen in uropathogenic E. coli (pqqL, zinc metallopeptidase, and fdeC, intimin-like adhesin, respectively), and putative proteins [yfiE, uncharacterized helix-turn-helix-type transcriptional activator; ygjI, putative inner membrane transporter; and ygjJ, putative periplasmic protein]. Further characterization of these highly conserved MPEC genes may be critical to understanding the pathobiology of MPEC.

Prevalence, Isolation, Identification, and Risk Factors of Major Bacterial Cause of Camel Subclinical Mastitis.

Objective A crosssectional study was conducted between September 2015 and August 2016 in the district of Afar Regional State, Northeastern Ethiopia, to characterize the most prevalent bacterial pathogens and identify the associated risk factors of camel subclinical mastitis. California mastitis test (CMT) was used as a screening test, and standard bacteriological methods were carried out for isolation and identification of the pathogens. Results Among the total 96 lactating camels examined, 25 were found positive with the overall prevalence of 26%, with 25% and 1% subclinical and clinical mastitis cases, respectively. Totally, 384 quarters of udder were examined; of these, 10 of them were blind while the rest 374 were nonblind teats. The quarter level prevalence of subclinical mastitis was 8.9%. The analysis showed that statistically significant difference (P < 0.05) of tick infestation and subclinical mastitis. Additionally, among the bacteriologically tested 34 CMT positive milk samples, all of them showed growth on nutrient and blood agar plate. Out of these culture isolates, the major bacterial pathogens identified were Staphylococcus aureus (8.7%), Staphylococcus hyicus (6.52%), Staphylococcus intermedius (6.52), Coagulase-negative staphylococci (19.57%), Bacillus (19.57%), Escherichia coli (6.52%), and Pasteurella multocida (6.52%) species. Therefore, appropriate control measures and awareness creation to the community should be practiced.

Investigation of antimicrobial susceptibility and virulence factor genes in Trueperella pyogenes isolated from clinical mastitis cases of dairy cows.

Trueperella pyogenes is an opportunistic pathogen causing important diseases including mastitis and metritis in domestic animals such as dairy cows leading to prominent economic losses in food production industry. The aim of this study was to investigate bacterial species, antimicrobial susceptibility, and presence of virulence factor genes and genotyping of T. pyogenes isolates associated with summer mastitis cases from 22 different farms around Tehran, Iran. Fifty‐five percent of dairy cows with clinical mastitis symptoms was infected by T. pyogenesis indicated that this pathogen is the most important contributor to clinical mastitis in dairy cows in the present study. A significant correlation was illustrated between presence of virulence factor genes of isolated pathogen, biochemical patterns, and the utter infected types. Multidrug resistance susceptibility observed between isolates indicated the important need for prudent use of antimicrobials in treatment of mastitis caused by T. pyogenes and increased concerning of consumer health associated with recent problems of antimicrobial resistance. The categorization of isolates was implemented into seven different clonal related types by COX‐PCR at 80% of similarity cutoff with significance relationship to clonal types, CAMP test result and sampling time and biochemical profile. Regarding to the results obtained at the present study, T. pyogenes can be considered as an important typically cause of purulent and acute form of clinical bovine mastitis and loss of dairy productivity. Further studies with more sample size and high‐throughput omic methods in various sampling time and areas are suggested for study of this pathogen precisely.

Isolation of Escherichia Coli from Clinical Mastitis Cases of Cows in Nangarhar Province

Isolation of Escherichia Coli from Clinical Mastitis Cases of Cows in Nangarhar Province

Phagocytic activity and the phagocytic index of milk PMN’s as a marker for diagnosis and monitoring of the therapeutic and prophylactic efficacy of antioxidant formulation in bovine mastitis

A clinical study was undertaken on bovine mastitis in the Kashmir valley to study the relationship between Phagocytic activity (PA) and the phagocytic index (PI) of milk neutrophils (PMN’s) and bovine mastitis. The PA and PI of milk PMN’s were assessed in mastitic animals and compared with normal, healthy, lactating animals. A significant decrease was found in the values of the PA and PI of milk neutrophils in clinical cases of mastitis as compared to the healthy control group. The utility of anti-oxidants in clinical management of mastitis was measured through the response to treatment with trace minerals such as Cu, Zn, Mn and Se in addition to conventional antibiotic therapy. Two groups of mastitic animals received two therapeutic regimens. Group I animals received antibiotics (Enrofloxacin 6.6 mg/kg BID for 5 days) and a self-formulated trace mineral anti-oxidant mixture at therapeutic doses for 7 days, while Group II animals only received the antibiotic (at the same dose rate and frequency). Therapeutic regimen I proved more efficacious than the therapeutic regimen II in the treatment of clinical mastitis, as evidenced by higher recovery rates, a lower number of mean days required for recovery, and higher PA and PI of the milk PMN’s on day 10 post treatment. For prophylactic studies, 24 healthy lactating cows were divided into two groups of 12 animals each. One group was supplemented with a self-formulated trace mineral anti-oxidant mixture at prophylactic doses for 30 days, which resulted in a significant improvement in udder health status in terms of the SCC, PA and PI of milk PMN’s. Supplementation with the antioxidant formulation played a significant role in early recovery and prophylaxis of bovine mastitis. Assessment of PA and PI of milk PMN’s could be utilised as an effective tool for diagnosing susceptibility to the occurrence of mastitis, as well as monitoring the therapeutic efficacy of different treatment regimes.

Outbreak of Pseudomonas aeruginosa mastitis in a dairy cow herd in northern Greece and its control with an autogenous vaccine.

In a dairy cow herd consisted of herd of 200 lactating Holstein-Friesian cows and heifers, clinical signs of mastitis in 40 out of 170 animals were observed. Treatments with antibiotics were proved ineffective. Milk bacterial cultures from 15 affected animals revealed Pseudomonas aeruginosa. An autogenous vaccine was administered subcutaneously, twice in a month period, to all adults. Cases of clinical mastitis declined significantly (p⟨0.0001) during next 3 months.

Antimicrobial susceptibility profile of Streptococcus agalactiae strains isolated from bovine mastitis

Bovine mastitis is the most important disease of dairy herds worldwide. Its main etiologic agents are bacteria, including Streptococcus agalactiae. The importance of this agent in bovine mastitis is because it is highly contagious and has a high impact on the occurrence of clinical mastitis cases and in the increase of the bulk milk somatic cell counts. The dry cow therapy and the treatment of the clinical mastitis cases stand out among the measures to control intramammary infections in cows. However, these strategies require knowledge about the antimicrobial susceptibility of the causal microorganisms. Thus, this study aimed to evaluate the antimicrobial susceptibility of 89 S. agalactiae strains isolated from bovine mastitis between the years 2004 and 2008 in dairy herds from Campo das Vertentes region, Minas Gerais State, Brazil. The disc diffusion technique was used and the antimicrobials currently used in mastitis therapy were tested. The isolates tested showed 100% susceptibility to chloramphenicol, ceftiofur, cefotaxime, enrofloxacin, and cefquinome. High frequencies of susceptibility (&gt;95%) were also observed for the beta-lactams (penicillin G, ampicillin, and oxacillin), cephalosporins (cephalotin, ceftiofur, cefotaxime, cefoperazone, and cefquinome), florfenicol, gentamicin, lincomycin, nitrofurantoin, and sulfamethoprim. The strains showed high frequencies of resistance to neomycin (15.74%), and tetracycline (21.35%). Multidrug resistance was detected in 2.25% of the tested isolates. The results pointed to variations in the antimicrobial susceptibility profiles of the studied strains and the importance of the use of the susceptibility tests to determine the correct antimicrobial to be applied in the treatment of bovine mastitis caused by S. agalactiae. The high frequencies of resistance observed to some antimicrobials, such as neomycin and tetracycline, commonly used in the treatment of mastitis and other pathologies, highlighted the need for more judicious use of antimicrobials on dairy farms.

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of β-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

Milk and serum proteomes in subclinical and clinical mastitis in Simmental cows

Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α1 acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595. SignificanceMastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis.

Amelioration of Oxidative Stress Through Supplementation of Self- formulated Anti-oxidant Mixture for Early Recovery and Prophylaxis of Bovine Mastitis in Kashmir Valley

Background: During lactation, mammary epithelial cells exhibit a high metabolic rate and thus produce large amounts of ROS and lipid peroxides in vivo (Jin et al. 2014; Ganguly et al., 2016). A surplus of ROS and the absence of optimal amounts of antioxidants (which neutralize these free radicals or ROS) results in oxidative stress (Lykkesfeldt and Svendsen, 2007). A clinical study was undertaken on Bovine Mastitis in Kashmir valley to study the relation between oxidative stress and clinical mastitis. An attempt was also made to see the effect of supplementation of self-formulated anti-oxidant trace mineral mixture on recovery and prophylaxis of Bovine mastitis through amelioration of oxidative stress.Methods: The oxidative stress was assessed through estimation of superoxide dismutase (SOD), catalase, reduced glutathione (GSH), malondialdehyde (MDA) and nitric oxide (NO). In addition, blood trace mineral status for copper (Cu), zinc (Zn), manganese (Mn) and selenium (Se) were also assessed in mastitic animals and compared with normal healthy lactating animals. The utility of anti-oxidants in clinical management of mastitis was measured through response to treatment with trace minerals like Cu, Zn, Mn and Se in addition to conventional antibiotic therapy. Two groups of mastitic animals received two therapeutic regimens. Group I animals received antibiotics and self formulated anti-oxidant mixture at therapeutic doses while as Group II animals received only antibiotics (at same dose rate and frequency). Clinical recovery was assessed on the basis of CMT point score, milk somatic cell count and milk biochemistry. For prophylactic study, forty recently parturated lactating animals having susceptibility to occurrence of mastitis were divided into two groups of twenty animals each. One group of animals was supplemented with self-formulated anti-oxidant mixture at prophylactic doses for a period of thirty days so as to see the effect of supplementation on oxidative stress parameters and occurrence of clinical mastitis.Result: A significant decrease was found in the values of SOD, catalase, GSH and Cu, Zn, Mn and Se but a significant increase was found in the values of MDA and NO in clinical cases of mastitis as compared to healthy control group. Therapeutic regimen I proved efficacious than the therapeutic regimen II in treatment of clinical mastitis with higher recovery rates and lesser number of mean days required for recovery in group I than group II animals. The efficacy of prophylactic treatment was monitored by occurrence of mastitis during the course of therapy and one month after therapy. Group I animals did not suffer from clinical mastitis and showed considerable improvement in oxidative stress parameters, milk SCC and blood trace mineral status as compared to group II.

Multi Locus Sequence Typing and spa Typing of Staphylococcus Aureus Isolated from the Milk of Cows with Subclinical Mastitis in Croatia.

Background: The bacterial species S. aureus is the most common causative agent of mastitis in cows in most countries with a dairy industry. The prevalence of infection caused by S. aureus ranges from 2% to more than 50%, and it causes 10–12% of all cases of clinical mastitis. Aim: The objective was to analyze 237 strains of S. aureus isolated from the milk of cows with subclinical mastitis regarding the spa, mecA, mecC and pvl genes and to perform spa and multi-locus sequence typing (MLST). Methods: Sequencing amplified gene sequences was conducted at Macrogen Europe. Ridom StaphType and BioNumerics software was used to analyze obtained sequences of spa and seven housekeeping genes. Results: The spa fragment was present in 204 (86.1%) of strains, while mecA and mecC gene were detected in 10 strains, and the pvl gene was not detected. Spa typing successfully analyzed 153 tested isolates (64.3%), confirming 53 spa types, four of which were new types. The most frequent spa type was t2678 (14%). MLST typed 198 (83.5%) tested strains and defined 32 different allele profiles, of which three were new. The most frequent allele profile was ST133 (20.7%). Six groups (G) and 15 singletons were defined. Conclusion: Taking the number of confirmed spa types and sequence types (STs) into account, it can be concluded that the strains of S. aureus isolated from the milk of cows with subclinical mastitis form a heterogenous group. To check the possible zoonotic potential of isolates it would be necessary to test the persons and other livestock on the farms.

Diagnosis of clinical and subclinical mastitis in a rural property in Carambeí, State of Paraná

The aim of the present study was to diagnose clinical and subclinical bovine mastitis in a property in the municipality of Carambeí-PR. 86 dairy cows were diagnosed in two evaluations carried out on July 23 and November 4, 2020. The detection of clinical mastitis was performed by testing the black-bottomed mug, whereas the detection of animals with sub-clinical mastitis by the California Mastitis Test (CMT) and laboratory analysis of bacteria and fungi present. In the first evaluation, 9.4% of the animals had mastitis, while in the second 5.16% of the herd. The milk of the animals that tested positive were submitted to laboratory analysis. In the first evaluation, they were diagnosed with an incidence of 15% Escherichia coli, 15% Prothoteca / yeast, 23% Staphlococcus aureus, 46% Streptococcus agalactiae. In the second evaluation, there was an incidence of 25% Enterococcus sp. and 25% S. agalactiae, the other animals showed 50% Staphlococcus non aureus. There was a higher occurrence of subclinical mastitis in the property, in both evaluations. There was a reduction from the first to the second evaluation in the incidence of cases of subclinical mastitis and the cases of clinical mastitis were resolved. Contributing to the lower disposal of milk caused by the high somatic cell count.