Major Mastitis Pathogens Research Articles

Seasonal Prevalence of Major Mastitis Pathogens Isolated from Crossbred Cow Milk Samples in Sub-tropical India

The dairy industry suffers significant financial losses due to mastitis. In order to control and prevent mastitis, it is imperative to continuously monitor the bacteriology, prevalence, and incidence of major mastitis pathogens. Thus, the objective of this research was to find out how often mastitis causing bacteria were isolated from milk samples of crossbred cows over various seasons in India's sub-tropical environment. 240 milk samples from 80 different cows were collected over the course of three seasons [Hot Dry (April to June), Hot Humid (July to September), and Autumn (October to November)] in order to correlate the prevalence of four main mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli) with the season. The most prevalent isolate during all seasons was Staphylococcus aureus. Our findings demonstrated a discernible relationship between seasonal change and the isolation rate of the bacterium associated with bovine mastitis, with the hot-humid season having the greatest pathogen isolation rate.

Virulence Determinants of Buffalo Mastitis Originated Streptococcus agalactiae Isolates

Streptococci as a cause of mastitis have become the major concern to the dairy industry worldwide due to huge economic losses. Streptococcus agalactiae is a major contagious mastitis pathogen and continues to be a major cause of mastitis in dairy cattle and buffaloes. The aim of the study was to investigate the virulence determinants of S. agalactiae strains isolated from buffalo milk. Within the scope of the study, 24 S. agalactiae isolates from buffalo mastitis were examined. Biofilm production of isolates was investigated phenotypically by CRA method. It was observed that 18 (75%) isolates were positive for biofilm production. The presence of hylB, fnbB, scpB and spb1 virulence genes in S. agalactiae isolates were investigated by PCR. It was determined that 19 (79.17%) of the isolates were positive for scpB and 6 (25%) for fnbB virulence genes. None of the isolates were found to contain hylB and spb1 virulence genes. The antibiotic resistance profiles of the isolates among kanamycin, ampicillin, enrofloxacin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole antibiotic discs were determined by Kirby Bauer Disc Diffusion Method. Resistance were evaluated as % 41.7, % 45.9, % 25, % 12.5, % 20.9, and % 33.3, respectively. RAPD-PCR patterns of all isolates were determined using the ERIC-2 primer. The dendrograms of the RAPD patterns were plotted with the UPGMA method. It was determined that the isolates showed similarity between 59-95%. In conclusion, the research confirms the prevalence of various virulence genes in S. agalactiae isolated from buffalo mastitis. Further studies are therefore necessary to determine the molecular epidemiology and variability of S. agalactiae isolated from buffaloes, with the aim of improving mastitis control programs with regard to S. agalactiae.

A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.

Prevalence, Virulence, and Antimicrobial Resistance of Major Mastitis Pathogens Isolated from Taiwanese Dairy Farms.

Mastitis, a highly prevalent disease in dairy cows, is responsible for massive financial losses due to decreased milk yield, milk quality, and costly medication. This research paper investigates antimicrobial susceptibility in cows and the role played by both resistance and virulence gene distribution in bovine mastitis. A total of 984 raw milk samples were collected from five different dairy farms and cultured on sheep blood agar plates. Antimicrobial susceptibility was determined by disc diffusion, and corresponding resistance and virulence genes were detected by PCR. Among the collected milk samples, 73, 32, and 19 isolates of Streptococcus spp., Staphylococcus spp., and coliforms were identified, respectively. The antimicrobial susceptibility results showed that Streptococcus spp. were resistant to tetracycline (86.30%), neomycin (79.45%), and oxacillin (73.97%). Staphylococcus spp. were resistant to tetracycline (59.37%) and oxacillin (53.12%). Lastly, coliforms were resistant to oxacillin (100%) and bacitracin (68.42%). The genotyping results showed that Streptococcus spp. carried the resistance genes tetM (46.57%) against tetracycline, bcrB (41.09%) against bacitracin, and aph(3)-II (39.72%) against neomycin. Staphylococcus spp. carried the resistance genes bcrB (40.62%) and tetM (18.75%), and coliforms carried the resistance genes tetM (42.10%) and bcrB (57.89%). Moreover, 57.53%, 75.0%, and 63.15% of Streptococcus spp., Staphylococcus spp., and coliforms carried lmb, fib, and ompC virulence genes, respectively. All three tested bacterial genera showed no significant association between antimicrobial resistance genes and virulence factors, although they were negatively correlated (p > 0.05). The combination of resistance gene identification and susceptibility tests as components of the diagnosis of bovine mastitis can help in selecting effective antimicrobial agents to treat it.

Innovative Elastomers with Antimicrobial Activity May Decrease Infection Risks during Milking.

Contagious pathogens are very costly to dairy herds, and they may have zoonotic and reverse-zoonotic potentials and may contribute to the spread of antimicrobial resistance. One of the most important risk factors for spreading these infections is milking, when liner contamination may transfer the pathogens from infected to healthy cows. There is no effective protocol to prevent the transmission of infection without the segregation of infected cows. Recently, the availability of elastomers with patented antimicrobial components in their formulations has allowed the exploration of alternative methods to reduce the risk of infection. Two different types of elastomers (rubber and silicone) and nine different formulations were challenged with three major mastitis pathogens (S. aureus, S. agalactiae, and E. coli). The results that were obtained in this study were interesting and unexpected. Indeed, to our knowledge, this is the first study to show that basic rubber materials have intrinsic antimicrobial activity. Silicone elastomers did not exhibit the same levels of bactericidal activity, although they did exhibit some antibacterial capacity. A significant decrease in bacterial survival curves was observed for all the formulations tested when antimicrobial components were added. The different results observed for the various products are likely due to the different formulations and diverse manufacturing processes. The availability of these new materials that significantly reduce the bacterial load on the liner surface may reduce the risk of spreading intramammary infections during milking. This would be an important step forward in achieving global sustainability of dairy herds, consistent with the objectives of One Health, by reducing the risks of zoonotic diseases and antimicrobial treatments.

Expression, Regulation, and Function of β-Defensins in the Bovine Mammary Glands: Current Knowledge and Future Perspectives.

β-Defensins are cationic antimicrobial peptides (AMPs) that play an important role in the innate immune defense of bovines. They are constitutively expressed in mammary glands and induced differently in response to pathogens. Their expression is influenced by various factors, including hormones, plant-derived compounds, and dietary energy imbalance. The toll-like receptors (TLRs)/nuclear factor-kappa B (NF-κB) pathway plays a crucial role in β-defensin induction, while alternative pathways such as mitogen-activated protein kinase (MAPK) and epigenetic regulation also make substantial contributions. β-Defensins exhibit bactericidal activity against a wide range of pathogens, including two major mastitis pathogens, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), primarily through membrane disruption. β-Defensins have low cytotoxicity to host cells and demonstrate immunomodulatory properties, and pathogens also display minimal resistance to these AMPs. Given the increasing concern in antimicrobial resistance, the potential of β-defensins as natural antimicrobials has garnered considerable attention. This article provides an overview of the characteristics of bovine β-defensins, their expression pathways, their mode of action, and factors influencing their expression in the mammary glands of cattle. Additionally, it identifies the current gaps in research within this field and suggests areas that require further investigation. Understanding the regulation and function of β-defensins offers valuable insights to develop effective strategies for strengthening the immune system of mammary glands, reducing the reliance on synthetic antimicrobials, and explore novel natural antimicrobial alternatives.

Estimation of the performance of two real-time polymerase chain reaction assays for detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae in pooled milk samples in a field study

The early detection of major mastitis pathogens is crucial for the udder health management of dairy herds. Testing of pooled milk samples, either individual test-day cow samples (TDCSs) or aseptically collected pre-milk quarter samples (PMQSs) may provide an easy to use and cost-effective group level screening tool. Therefore, the aim of this study was (1) to evaluate the sensitivity (Se) and specificity (Sp) of 2 commercial multiplex real-time PCR test kits applied to pooled milk samples using a Bayesian latent class analysis and (2) to estimate the probability of detection in relation to the pool size and the number of cows positively tested by BC within a pool. Pools of 10, 20 and 50 cows were assembled from 1,912 test-day samples and 7,336 PMQSs collected from a total of 2,045 cows from 2 commercial dairy farms. Two commercial quantitative real-time PCR kits were applied to detect Staphylococcus (Staph.) aureus, Streptococcus (Strep.) agalactiae and Strep. dysgalactiae in the pooled samples, and a bacteriological culture (BC) was applied to PMQSs yielding a cumulative pool result. A pool was considered BC-positive if it contained at least one BC-positive PMQS. Pathogens were more frequently detected in the PMQS pools than in the TDCS pools. Pools of 10 cows showed the highest probability of detection irrespective of sample type or type of PCR kit compared with larger pool sizes. Estimation with a Bayesian latent class analysis resulted in a median Se in PMQS pools of 10 cows for S. aureus of 63.3% for PCR kit I, 78.1% for PCR kit II and 95.5% for BC; the Sp values were 97.0, 97.6% and 89.1%, respectively. The estimated median Se for Strep. species for PCR kits ranged between 77.5 and 85.6% and for BC between 73.7 and 79.2%; the median Sp values ranged between 93.6 and 99.2% for PCR kits, and between 96.9 and 97.4% for BC. In addition, the probability of detection increased with an increasing number of BC-positive cows per pool. To achieve a probability of detection of 90%, the estimated number of positive cows in PMQS pools of 10 cows for Kit I was 4.1 for Staph. aureus, 1.5 for Strep. agalactiae and 1.3 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 6.9, 1.9 and 2.0 positive cows were required, respectively. For Kit II and PMQS pools, the number of positive cows required was 2.8 for Staph. aureus, 1.4 for Strep. agalactiae and 1.2 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 5.3, 1.8 and 2.0 positive cows were required, respectively. In conclusion, the type of samples used for pooling, the pool size and the number of infected cows per pool determine the probability of detecting an infection with major mastitis pathogens within a pool by PCR testing.

Colonization of Extramammary Sites with Mastitis-Associated S. aureus Strains in Dairy Goats.

Staphylococcus aureus (S. aureus), a major mastitis pathogen in dairy goats, is classified as a contagious pathogen. Although previous research has shown that extramammary body sites can be colonized with S. aureus, it is unknown whether these sites are reservoirs for intramammary infections. The aim of this research was to determine whether extramammary sites can be colonized with mastitis-associated S. aureus strains in dairy goats. Milk samples were collected from 207 primiparous goats and from 120 of these goats, extramammary site samples (hock, groin, nares, vulva and udder) were collected from a large commercial dairy goat herd in the Netherlands during four sampling visits. Extramammary site swabs and milk samples were (selectively) cultured and S. aureus isolates were spa genotyped. The prevalence of colonization of the extramammary sites at goat level was 51.7% and the prevalence of S. aureus intramammary infections was 7.2%. The nares were colonized most frequently (45%), while the groin area was colonized the least (2.5%). Six spa genotypes were identified in this herd and there was no significant difference in the distribution of spa genotypes between the milk or the extramammary sites (p = 0.141). Both in the extramammary sites and in the milk, spa genotypes t544 (82.3% and 53.3%) and t1236 (22.6% and 33.3%) were the dominant genotypes. These results show that in goats, extramammary sites, particularly the nares, are frequently colonized with mastitis-associated S. aureus strains. Extramammary sites may, thus, be a source of S. aureus intramammary infections that are not targeted by the intervention measures aimed at preventing transmission from infected udder glands.

Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.

MOLECULAR CHARACTERIZATION SOME BACTERIAL PATHOGENS CAUSING BOVINE MASTITIS WITH SPECIAL REFERENCE TO Mycoplasma bovis

In dairy industry, bovine mastitis is the most prevalent disease, which reduces milk production and causes economic losses. This study was conducted to estimate the prevalence of Mycoplasma bovis and some bacteria causing mastitis in dairy farms and partial sequencing of 16SrRNA target genes and Quinolones Resistance Determining Regions (QRDRs) (gyrA and parC) in M. bovis isolates. 370 milk samples were obtained from farms located in villages in Fayoum governorate, Egypt. The examined milk samples (8,91%) were positive for the California mastitis test (CMT). Multiplex RT-PCR was used for the recognition of microorganisms causing mastitis (Staphylococcus (S.) aureus, Streptococcus species (spp.), Escherichia (E.) coli, and Mycoplasma (M.) bovis) from mastitic milk. The results revealed that E. coli was the most predominant (84.8%) followed by S. aureus (81.8%) while M. bovis was the lowest one (51.5%). Mixed infection with two or more mastitic bacterial agents was also identified. All 33 examined mastitic milk samples were diagnosed with mixed infection with E. coli, S. aureus, Streptococcus spp. and M. bovis (36.36%), E. coli and S. aureus (21.21%), and rephrase E. coli, M. bovis, and Streptococcus spp. (6.06%). The sequence analysis of M. bovis 16SrRNA genes illustrated a high similarity of examined isolates to strains previously deposited in the GenBank recovered from the same locality. The gyrA amino acids showed no substitution but showed 100% similarity with M. bovis isolates worldwide. However, the amino acid sequence of parC, showed substitution at positions 2 (Gln to Arg) (CAG >>CGT), 75 (Ile to Ser) (ATT>>AGC), and 79 (Asn to Asp) (AAC>>GAT). Sequence results can lead to the creation of appropriate treatment and control measures for M. bovis, while multiplex RT-PCR, can be exploited as a standard diagnostic method for major mastitis pathogens.

Mastitis pathogens and antibiotic resistance in beef cows in Switzerland.

Mastitis in beef cows has not been studied as extensively as mastitis in dairy cows, and data from Switzerland are lacking. Various studies have shown a similar pathogen spectrum as in dairy cows, which could not be confirmed in this study. To gather initial data from Switzerland, milk samples from 297 lactating beef cows from 31 herds from the Engadin Valley in the Canton of Grisons were examined bacteriologically. At least one major or minor mastitis pathogen was recovered from at least one individual-quarter or composite sample from 33 % of all cows. The most common major mastitis pathogens were Staphylococcus aureus (8,4 % of cows), Pasteurella multocida (4,1 %), Streptococcus uberis (2 %) and Streptococcus dysgalactiae (1,7 %). Sixteen percent of the cows had at least one blind quarter, but only 32 % of these had been previously detected by the owners. In the second part of the study, milk samples from beef cows with mastitis were examined bacteriologically; the cows originated from various parts of Switzerland and had been presented for veterinary treatment. Pasteurella multocida (22 %) and Staphylococcus aureus (21 %) were the most common pathogens isolated. Antibiograms using microtitration and disk diffusion testing were generated for the Staphylococcus aureus, Pasteurella multocida and Streptococcus uberis strains from both parts of the study. Fifty-six percent of the Staphylococcus aureus strains were resistant to penicillin G. Our results showed that bacteriological examination of a milk sample aids in the diagnosis and allows specific treatment of mastitis in beef cows; this may be further improved with antibacterial susceptibility testing. Our preliminary data for the resistance patterns of mastitis pathogens in beef cows will facilitate evidence-based treatment strategies.

Tomada de decisão sobre terapia de vaca seca: Avaliação econômica usando dados de campo sob condições de produção argentinas

ABSTRACT The cow somatic cell count (CSCC) is an indicator of intramammary infection (IMI), and it has been used to make decisions at dry-off about selective dry cow therapy (SDCT). This study assessed ex-ante the economic impact of implementing SDCT under milk production conditions in Argentina, using CSCC to identify cows with IMI at dry-off. Eighty six cows were sampled at dry-off and considered being infected if at least one quarter was infected with major mastitis pathogens. The CSCC sensitivity and specificity were estimated using the CSCC recorded from the test-day prior to dry-off. A simulation model was then developed to look into the economic benefits of applying SDCT under two herd level IMI prevalence (low: 5-15%, and high: 16-25%) at dry-off. The input variables were obtained from the field study, scientific literature, and real-world prices. The output was the net economic difference (NED) between SDCT and blanket dry cow therapy at the herd level. The sensitivity and specificity estimated for identifying cows infected with major pathogens for a CSCC threshold of 200,000 cells/mL were 31.4 (95% CI, 14.6-48.2) and 64.7 (95% CI, 50.6-78.8), respectively; the value for NED was maximized to -634 US$ and -455 US$ in the low and high IMI prevalence, respectively. In general, CSCC specificity was the variable with the most impact on NED and, in high IMI prevalence, milk price had a similar relevance. Thus, SDCT based on CSCC is a cost-effective option, under the milk production conditions in Argentina.

Clinical and Subclinical Mastitis in Ruminants: A Review of Etiological agents, Diagnosis, Clinical Management and Risk factors

Ruminant mastitis is a welfare and economic problem of livestock and the dairy industry. It is a major animal health problem in countries where livestock are raised for the purpose of milk production. This Review focuses on ruminant mastitis, etiologic agents, diagnostic test and associated risk factors. In this narrative review, a comprehensive literature search of all relevant articles was performed to identify articles published in Science Direct, Web of Science, PubMed, Scopus, MEDLINE, Google Scholar, LISTA and AJOL. English articles published from 2010 to 2023 were reviewed and used. The findings revealed that even though Staphylococcus aureus, Non-aureus Staphylococci species, and Streptococci are the major mastitis pathogens. Others like Escherichia coil, Klebsiella, Citrobacter, Enterobacter, Pseudomonas, Proteus, Serratia, Bacillus, and Corynebacterium etc were isolated from milk samples. Diagnosis was observed to be based on observation of milk and cardinal signs of inflammation of the mammary glands, the use of diagnostic test, like bacterial culture, Strip cup test, White side test (WST), Surf field mastitis test (SFMT), and California Mastitis test (CMT). Molecular typing techniques including PCR, Sequencing, MALDI-TOF MS, MLST, PFGE, spa typing etc have also been observed important role in characterizing agents of mastitis. Major risk factor observed is age, parity and stage of lactations. Clinical management of this condition in ruminants is based on the understanding of the etiology, and treatment options.

Rapid On-Site Detection of Major Mastitis Pathogens in Ruminants Using a Colorimetric Loop-Mediated Isothermal Amplification Assay

Rapid On-Site Detection of Major Mastitis Pathogens in Ruminants Using a Colorimetric Loop-Mediated Isothermal Amplification Assay

Association of Staphylococcus aureus genotypes with milk or colonization of extramammary sites in Dutch dairy cattle indicates strain variation in reservoirs for intramammary infections

Staphylococcus aureus (S. aureus) is a major mastitis pathogen with a detrimental impact on udder health in dairy cattle. Although S. aureus is generally classified as a contagious mastitis pathogen, control measures aimed at preventing contagious transmission are not always effective. Previous studies showed that various extramammary sites can be colonized with S. aureus and could be a reservoir for S. aureus intramammary infections (IMI). The aim of this study was to determine the prevalence of S. aureus extramammary site colonization in Dutch dairy cattle and to compare the spa genotypes of S. aureus isolates from IMI to spa genotypes of isolates from extramammary sites. Six herds were visited and from cows with a composite milk somatic cell count ≥200,000 cells/mL quarter milk samples and swabs from various extramammary sites (hock, groin, udder cleft, nares, and feces) were taken. Extramammary site samples were processed by a two-step high salt selective culture and presence of S. aureus was confirmed by femA PCR. S. aureus isolates from milk and extramammary sites were compared by spa typing. The cow level colonization varied from 0% to 73%, and the prevalence of IMI in the sampled cows varied from 0% to 61% between herds. The extramammary site with the highest prevalence of colonization was the hock (23%) and the lowest prevalence of colonization was found for the nares (5%) and feces (5%). Spa typing of S. aureus isolates from either extramammary sites or milk showed that in most herds there were one or two predominant S. aureus spa genotypes present. Different S. aureus spa genotypes could be categorized into three groups based on the distribution between milk or extramammary sites: i) predominantly milk associated, ii) associated with both milk and extramammary sites, and iii) associated with extramammary sites. In conclusion, we showed that the prevalence of extramammary site colonization differed significantly between herds and extramammary sites and that specific S. aureus spa genotypes were associated with milk (IMI) or extramammary site colonization. Extramammary S. aureus reservoirs could be a source for IMI that cannot be eradicated by intervention measures aimed at contagious mastitis pathogens.

Staphylococcus aureus-Cure-Associated Antigens Elicit Type 3 Immune Memory T Cells.

Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work. Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus. Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A+ cells among CD8+ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment. The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.

Ascorbic Acid-Mediated Modulation of Antibiotic Susceptibility of Major Bovine Mastitis Pathogens.

This study aimed to investigate the effects of ascorbic acid on antibiotic susceptibility of major bovine mastitis pathogens, including Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus agalactiae, and Escherichia coli. Minimum inhibitory concentrations (MICs) were determined by E-test method. The presence of 10 mM ascorbic acid decreased the MICs of penicillin and ampicillin while increased the MICs of erythromycin, kanamycin, streptomycin, and ciprofloxacin for all tested strains. Besides, ascorbic acid specifically reduced the MICs of tetracycline for gram-positive bacteria and chloramphenicol for gram-negative bacteria. This study highlights that ascorbic acid is a potential modulator of antibiotic activity against the major bovine mastitis pathogens.

22 Young Scholar Talk PHD: Investigating Ovine Mastitis: Microbial Sources and Management Methods to Reduce the Prevalence

Abstract Subclinical mastitis, a common intramammary disease in sheep production, can be costly to producers due to compromised performance and decreased longevity. Our research focused on preventative and therapeutic measures on mammary health where the objectives were to identify (1) the impact of Zn supplementation on the milk microbiome and (2) the impact of management practices at lambing and weaning on prevalence and cure rate of subclinical mastitis, the milk microbiome, and levels of antimicrobial resistance in milk bacteria. In study 1, the milk microbiome was characterized during the first 3 wk of lactation and at weaning in ewes divergent in dietary Zn supplementation (1× = CON or 3× recommendations = ZnTRT). Mammary health was assessed using somatic cell count (SCC) class. Amplicon sequencing revealed the presence of Staphylococcus, Mannheimia, Corynebacterium, and Pseudomonas, all major mastitis pathogens. Effects of breed, dietary Zn concentration, and SCC class on diversity were limited to early lactation, where CON milk had greater composition stability than ZnTRT milk (P = 0.02). In study 2, milk bacteria were characterized by culture; Staphylococcus spp. and Streptococcus spp. were tested for antimicrobial susceptibility. Subclinical mastitis prevalence ranged from 22–66% where coagulase-negative staphylococci (CoNS; 59%), bacilli (35%), Mannheimia haemolytica (10%), S. aureus (8%), streptococci (5%), and Corynebacterium spp. (5%) were common. Milk samples were compared by jug bedding treatment: jugs were cleaned and dried with barn lime before adding fresh straw (CLEAN) or fresh straw was added atop soiled bedding (SOILED). Bedding had no effect on subclinical mastitis (P = 0.57), but CoNS had greater sulfadimethoxine resistance in SOILED isolates (P = 0.03). Additional mastitis control methods at weaning were evaluated: penicillin injection (PENN), restricted feed access (FAST), or their combination (COMBO). Weaning treatment did not affect cure rate from weaning to 3-d post- weaning (P = 0.21). Further characterization of these samples via microbial sequencing are underway with source tracking analyses to investigate an exogenous route via retrograde transfer and environmental bacteria or endogenous route via an entero-mammary pathway. Results support that breed, animal husbandry, and mammary health variably affect the milk microbiome and provide novel insight to the impact of management practices in a milk microbiome longitudinal study.

Synergistic Antibacterial and Anti-inflammatory Activities of Ocimum tenuiflorum Ethanolic Extract against Major Bacterial Mastitis Pathogens.

Mastitis is the most prevalent global illness affecting dairy cows. This bacterial infection damages and inflames the udder tissues. Several plant extracts have demonstrated synergistic antibacterial activities with standard drugs in mastitis treatment. Scant information exists on Ocimum tenuiflorum L. This study evaluated the antibacterial activity of O. tenuiflorum extract and its interaction with antibacterial drugs against common mastitis pathogens including Staphylococcus aureus, coagulase-negative Staphylococci (CNS), Streptococcus agalactiae, and Escherichia coli. Anti-inflammatory activities in LPS-stimulated RAW264.7 macrophage cells were also studied. The O. tenuiflorum extract exhibited antibacterial activities against S. aureus, CNS, and S. agalactiae with minimum inhibitory concentration (MIC) ranging from 3.9 to 31.2 µg/mL and minimum bactericidal concentration (MBC) ranging from 15.6 to 500 µg/mL. Combinations of O. tenuiflorum with penicillin or amoxicillin-clavulanic acid showed synergistic effects against all tested strains but an additive effect with cefazolin and gentamicin. Pretreatment of the extract significantly decreased the expression of inflammatory molecules (IL-6, TNF-α, IL-1β, iNOS, COX-2, and PGE2) generated by LPS in macrophages. Results suggested O. tenuiflorum effectiveness against various Gram-positive mastitis bacteria, with the potential to reduce antibacterial doses and combat inflammation.

Spatio-Temporal Variation in the Prevalence of Major Mastitis Pathogens Isolated From Bovine Milk Samples Between 2008 and 2017 in Ontario, Canada.

An understanding of the spatio-temporal distribution of several groups of mastitis pathogens can help to inform programs for the successful control and management of mastitis. However, in the absence of an active surveillance program such information is not readily available. In this retrospective study we analyzed passive surveillance data from a diagnostic laboratory with an aim to describe the spatio-temporal trend of major mastitis pathogens between 2008 and 2017 in Ontario dairy cattle. Data for all milk culture samples submitted to the Animal Health Laboratory (AHL) at the University of Guelph between 2008 and 2017 was accessed. Descriptive analyses were conducted to identify the major pathogens and Chi-square goodness-of-fit tests were used to compare between multiple proportions. Likewise, univariable logistic regression analysis was performed to determine if there was a change in the probability of isolating the major mastitis pathogens depending on geography or time. Seasonality was assessed by calculating the seasonal relative risk (RR). Of a total of 85,979 milk samples examined, more than half of the samples (61.07%) showed no growth and the proportion of samples that showed no growth almost halved during the study period. Of the samples (36.21%, n = 31,133) that showed any growth, the major bacterial pathogens were Staphylococcus aureus (15.60%), Non-aureus Staphylococci (NAS) (5.04%), Corynebacterium spp. (2.96%), and Escherichia coli (2.00%). Of the NAS, the major species reported were Staphylococcus chromogenes (69.02%), Staphylococcus simulans (14.45%), Staphylococcus epidermidis (12.99%), and Staphylococcus hyicus (2.13%). A temporal change in the prevalence of contagious pathogens like S. aureus and Corynebacterium spp. was observed with an increasing odds of 1.06 and 1.62, respectively. Likewise, except for Trueperella pyogenes, the prevalence of all the major environmental mastitis pathogens increased during the study period. The isolation of most of the pathogens peaked in summer, except for S. aureus, T. pyogenes, and Streptococcus dysgalactiae which peaked in spring months. Interestingly, a regional pattern of isolation of some bacterial pathogens within Ontario was also observed. This study showed a marked spatio-temporal change in the prevalence of major mastitis pathogens and suggests that a regional and seasonal approach to mastitis control could be of value.