Frequency of non-aureus staphylococci and mammaliicocci species isolated from quarter clinical mastitis: a six-year retrospective study.

Non-aureus staphylococci and mammaliicocci (NASM) are the most frequently isolated bacterial group from bovine milk samples. Most studies focus on subclinical mastitis caused by NASM, however NASM can cause clinical mastitis (CM) as well. We evaluated retrospective data from 6 years (2017-2022) to determine the species and frequency of NASM isolated from quarter bovine CM. The data comprised of microbiological results from quarter CM samples routinely submitted to Quality Milk Production Services (QMPS), Cornell University, NY, US, for microbial identification by MALDI-TOF MS. A total of 9,909 microbiological results from 410 dairy herds were evaluated. Our results showed that 29 distinct NASM species were identified, with the 8 most prevalent NASM species being Staphylococcus chromogenes, S. haemolyticus, S. simulans, S. epidermidis, S. sciuri (now Mammaliicoccus sciuri), S. agnetis/S. hyicus, S. borealis, and S. xylosus. The NASM distribution remained similar among seasons, but the frequency of NASM CM cases was higher during the summer. Our results showed different patterns of variations in the isolation frequency over time, depending on the bacterial species: increasing or decreasing trends, cyclic fluctuations, and except for S. borealis, a significant seasonality effect for our study's most prevalent NASM was observed. This study showed that S. chromogenes remains the most frequent (43%) NASM species identified from bovine CM, followed by S. haemolyticus (18%), and S. simulans (12%).

Randomized noninferiority field trial evaluating a postmilking teat dip for the prevention of naturally occurring intramammary infections.

The aim of this study was to perform a positive-controlled field study under natural exposure conditions to test the efficacy of a newly developed chlorine dioxide-based postmilking teat disinfectant (experimental product, EX) for noninferiority compared with an already established chlorine dioxide-based teat disinfectant (positive control product, PC). After blocking by parity, approximately 200 Holstein cows in early to mid-lactation stages from a dairy farm near Padua, Italy, were randomly assigned to one of 2 groups. Over a 13-wk period between September and December 2021, the teats of cows were dipped with the EX or the PC after each milking. Milk samples were collected from individual quarters of enrolled cows for 13 wk to determine infection status. Teat condition was assessed at wk 1, 5, and 9. Mixed logistic regression was used to analyze the effect of treatment on the incidence of new intramammary infections. For the noninferiority analysis, the upper limit of the 95% confidence interval for the difference in new intramammary infection (NIMI) rate between the 2 treatments (EX - PC) had to be to the left of the critical value d (0.035) to conclude that EX was noninferior to PC in terms of the risk of NIMI. The results showed that the incidence of new infections in the quarters treated with EX (3.1%) was not different from that in the udder quarters treated with PC (2.6%). No overall difference was found between the treatments in terms of teat condition. As the upper limit of the 95% confidence interval of the NIMI rate difference was smaller than the predefined noninferiority limit, we concluded that the EX was noninferior compared with the PC.

Genomic Characterization of Mycoplasma arginini Isolated from a Housefly on a Dairy Farm and Comparison with Isolates from Bovine Milk and Lung Tissue.

Mycoplasma mastitis can be highly contagious, unresponsive to treatment, and cause severe economic problems in affected herds. Notable routes of Mycoplasma spp. transmissions are contaminated milking equipment and animal contact through respiratory secretions. Only a few studies report the environment as a possible source of infection. Our group studied the presence of pathogens in houseflies (Musca domestica) in a New York State dairy in the United States. Among others, a Mycoplasma spp. was found in the gut of a housefly captured in the sick pen and identified as M. arginini. Here, we characterized its genome and investigated its relatedness with eight isolates from milk, one isolate from lung tissue collected in the same dairy, and five other dairies in New York State. We applied whole-genome sequencing and phylogenetic analysis based on the sequences of the 16S rRNA gene and 76 conserved proteins. We also assessed an in silico virulence profile by considering a panel of 94 putative virulence genes. As a result of the genome analysis, the housefly M. arginini isolate was highly similar to the milk isolates; interestingly, the similarity was highest with M. arginini isolated from milk on the same dairy farm where the housefly was captured. The housefly and milk M. arginini isolates possessed 54 of the 94 pathogenicity genes considered. Our data support the hypothesis that houseflies are carriers of Mycoplasma spp. and can be considered within the possible roots of environmental transmission of infection in dairy cows. Nevertheless, M. arginini pathogenicity will need to be investigated with dedicated studies. IMPORTANCE It is critical to control the spread of bovine mastitis caused by Mycoplasma spp., as this disease can be highly contagious and have a severe economic impact on affected dairies. A better understanding of possible transmission routes is crucial for infection control and prevention. Based on our data, the composite milk isolates are genetically similar to the housefly isolate. This provides evidence that the same Mycoplasma species found in milk and associated with mastitis can also be isolated from houseflies captured in the dairy environment.

Observational study on dry period length and its associations with milk production, culling risk, and fertility in Italian dairy farms.

From an initial data set involving 84,189 lactations, this research evaluated the relationship between dry period length (DPL) and milk production, culling risk, and fertility. The data set included a total of 48,297 multiparous cow lactation records, with a calving event occurring in 2019 and 2020, belonging to 62 Italian herds with at least 150 cows. The DPL was classified into 5 categories (<40, 40-49, 50-60, 61-70, and >70 d) and these categories were used to establish the association between DPL and the outcome variables. All data obtained were assessed with simple and multiple linear regressions and Cox proportional hazard models. Cumulative milk production at 60 d in milk (DIM) was the highest in DPL categories of 61 to 70 d (2,480.29 kg/cow) and 50 to 60 d (2,474.39 kg/cow), and the lowest in <40 d (2,281.29 kg/cow). Similarly, DPL categories 61 to 70 d (10,830.94. kg/cow) and 50 to 60 d (10,817.48 kg/cow) had the highest 305-d milk production, whereas the <40 d (10,200.96 kg/cow) had the lowest one. The groups with a DPL of 40 to 49 d and >70 d had slightly, but significant, lower milk production both as cumulative 60 DIM and predicted 305-d milk production. Culling risk had a curvilinear behavior, with DPL <40 d and DPL >70 d showing significantly higher odds for culling during the first 60 DIM compared with DPL of 50 to 60 d [relative risk (RR): 1.53; RR: 1.46]. Within the same comparison, DPL of 61 to 70 d also had a slightly higher risk for culling (RR: 1.13). The DPL was associated also with fertility, with DPL of 40 to 49 d and 50 to 60 d having the greatest odds for pregnancy within the first 200 DIM. The DPL of <40, 61 to 70, and >70 d were negatively associated with fertility and showed pregnancy risks of 0.87, 0.95, and 0.94, respectively. This paper reinforces the importance of DPL as we demonstrated its association with milk production, culling, and fertility. Despite being attractive for high production dairy cows, very short dry periods are at the same time also associated with higher culling risk, lower milk production and fertility. Long DPL is detrimental, especially regarding culling and fertility. In summary, reducing variability in DPL and avoiding extremes by improving reproductive performance, maximizing late lactation milk production and making wise decisions on dry-off timing, may lead to better performances and lower early culling under Italian dairy conditions.

Pathogens associated with houseflies from different areas within a New York State dairy.

Houseflies (Musca domestica) are nonbiting muscoids of importance because they can be mechanical vectors of many kinds of pathogens such as bacteria, protozoa, viruses, and helminth eggs. This study aimed to evaluate the bacterial communities associated with houseflies captured in 3 different areas on a dairy farm located in New York State. Variations in the bacterial community were also evaluated based on the flies' sex and external or internal location where the bacteria were isolated. A total of 101 flies were collected: 27 flies from the sick pen, 42 from calf hutches, and 32 from the milking parlor. A total of 485 organisms were isolated, 233 (48.0%) from 53 female flies and 252 (52.0%) from 48 male flies. Most (74%) bacteria were found in the internal parts of the flies, with only 26% isolated from the external surfaces. The number of isolates detected per fly ranged between 1 and 11. A total of 392 bacteria were identified at the species level. We isolated 26 species reported to be bovine contagious or environmental mastitis pathogens. Within the group of organisms considered contagious, we isolated Staphylococcus aureus and Mycoplasma arginini. This was the first time that a Mycoplasma species was isolated from houseflies. We identified 5 organisms considered foodborne pathogens that affect human health: Salmonella spp., Escherichia coli, Staph. aureus, Bacillus cereus, and Bacillus subtilis. Four of the organisms isolated in this study were also linked with milk spoilage, including Pseudomonas aeruginosa, Bacillus cereus, Bacillus licheniformis, and Paenibacillus lactis. This study confirmed that houseflies carry a high bacterial diversity, including organisms associated with animal infections, organisms that could be a concern for public health, or organisms that could negatively affect milk quality.

Short communication: Comparative performance of 3 on-farm culture systems for detection of mastitis pathogens interpreted by trained and untrained observers.

On-farm culture (OFC) systems facilitate pathogen-based mastitis management and can facilitate antimicrobial stewardship on dairy farms. Interpretation of the results, however, may present a challenge for those with limited microbiology experience. Here, we compared results of 3 OFC systems interpreted by trained and untrained observers against results of a standard laboratory reference method (aerobic culture and mass spectrometry). Milk samples (280 quarter and 60 composite) were selected from submissions for routine diagnostic testing to Quality Milk Production Services (Cornell University, Ithaca, NY) between August 2017 and January 2018. Samples were cultured simultaneously using the standard laboratory reference method and 3 commercially available OFC systems that varied in detail of pathogen identification (provided in parentheses) as follows: (1) Minnesota Easy Culture System II Bi-plate (University of Minnesota Laboratory for Udder Health, St. Paul; gram-positive, gram-negative), (2) Minnesota Easy Culture System II Tri-plate (gram-positive, gram-negative, some genus level), and (3) FERA Diagnostics and Biologicals AccuMast plate (Ithaca, NY; genus level, some species level). After 18 to 24 h of incubation, OFC plates were interpreted by 1 trained observer (>10 yr of experience in milk microbiology) and 6 untrained observers with no previous milk microbiology training, using only the manufacturers' instructions for guidance. Strength of agreement (κ) between observer groups and the reference method was determined for the available outcomes of each system. Interpreted by the trained observer, agreement was moderate for identifying gram-positive organisms (Bi-plate, κ = 0.56) and substantial for Streptococcus spp. (Tri-plate, κ = 0.64, AccuMast κ = 0.61). Interpretation by untrained observers resulted in fair agreement (κ = 0.29-0.37) for these organisms. Moderate agreement (κ = 0.43-0.59) was found across all 3 OFC for the identification of gram-negative organisms (Bi-plate), non-aureus staphylococci (Tri-plate and AccuMast), Lactococcus spp., and Enterococcus spp. (AccuMast) when interpreted by the trained observer, and fair to moderate agreement was found (κ = 0.31-0.53) among untrained observers. Across all 3 OFC, agreement was almost perfect (κ = 0.80-0.89) for Staphylococcus aureus for the trained observer, and moderate to substantial (κ = 0.56-0.61) for untrained observers. We concluded that all 3 OFC appeared suitable to support pathogen-based mastitis management when operated by trained observers. Training beyond the instruction manual is a prerequisite to make OFC systems useful for pathogen-based mastitis management.

Mycoplasma species isolated from bovine milk collected from US dairy herds between 2016 and 2019.

Determining the species of mycoplasma isolated from culture-positive milk samples is important for understanding the clinical significance of their detection. Between August 2016 and December 2019, 214,518 milk samples from 2,757 dairy herds were submitted to Quality Milk Production Services (QMPS) at Cornell University for mycoplasma culture. From these samples, 3,728 collected from 204 herds were culture positive. Based on the request of herd managers, owners, or veterinarians, 889 isolates from 98 herds were subjected to molecular identification by PCR and amplicon sequencing. The largest proportion of the identified isolates were from New York State (78.1%), while the others came from the eastern United States (17.8%), Texas (2.0%), and New Mexico (2.1%). As expected, Mycoplasma spp. were the most common (855 isolates, 96.2%) and Acholeplasma spp. accounted for the remainder (34 isolates, 3.8%). Mycoplasma bovis was the most prevalent Mycoplasma species (75.1%), followed by M. bovigenitalium (6.5%), M. canadense (5.9%), M. alkalescens (5%), M. arginini (1.7%), M. californicum (0.1%), and M. primatum (0.1%). A portion of the isolates were confirmed as Mycoplasma spp. other than M. bovis but were not identified at the species level (16 isolates, 1.8%) because further information was not requested by the manager, owner, or veterinarian. Mycoplasma bovis was the only species identified in 59 of the 98 herds. However, more than 1 Mycoplasma sp. was identified in 29 herds, suggesting that herd infection with 2 or more mycoplasmas is not uncommon. Moreover, a Mycoplasma sp. other than M. bovis was the only species identified in 8 herds. From the subset of 889 mycoplasma culture-positive isolates from 98 herds, we determined that over a third of the herds had either more than 1 Mycoplasma sp. or a Mycoplasma sp. other than M. bovis detected in their milk samples. In conclusion, we observed that M. bovis is the most common pathogenic Mycoplasma species found in mastitic milk, but other Mycoplasma species are not uncommon. Our results suggest that it is critical to test milk samples for mycoplasmas using diagnostic tests able to identify both the genus and the species.

Public perceptions of antibiotic use on dairy farms in the United States.

There has been a global push for improved antimicrobial stewardship, including in animal agriculture, due to growing concerns about antimicrobial resistance. However, little is known about the general public's perceptions of antimicrobial use in animal agriculture. The aim of this study was to explore the US public's perceptions of antibiotic use in dairy farming and how these perceptions influence purchasing decisions. Data from the 2017 Cornell National Social Survey developed in collaboration with the Cornell Survey Research Institute were used to assess the public's perceptions. The Survey Research Institute of Cornell University (Ithaca, NY) administered the survey by telephone to a random sample of 1,000 adults in the continental United States. The survey collected information about perceptions of threat to human health posed by antibiotic use in cows on dairy farms and willingness to pay more for milk from cows raised without antibiotics, as well as several presumed explanatory variables, including respondents' knowledge of antibiotics, beliefs regarding cattle treatment in dairy farming, and 18 sociodemographic characteristics. Data were analyzed using logistic regression. Among respondents, 90.7% (n = 892/983) reported that antibiotic use on dairy farms posed some level of threat to human health and 71.5% (n = 580/811) indicated they would be willing to pay more for milk produced from cows raised without antibiotics. Respondents who believed that antibiotic use in dairy farming posed a moderate to high threat to human health were more likely to be female and report willingness to pay more for milk or not purchase milk. Additionally, consumers' willingness to pay more for milk from cattle raised without antibiotics was associated with the belief that antibiotic use posed some threat to human health, the belief that cows are treated better on organic dairy farms, an annual household income of $50,000 or greater, being born outside the United States, having a liberal social ideology, and being currently or formerly married. These results suggest that the general public's decisions as consumers of dairy products are associated with demographic factors in addition to perceptions of antibiotic use and cattle treatment in dairy farming. The rationale behind such perceptions should be further explored to facilitate consumers' informed decision making about antibiotic use in agriculture, links to cattle treatment, and associated willingness-to-pay attitudes.

Heat treatment of bovine colostrum: I. Effects on bacterial and somatic cell counts, immunoglobulin, insulin, and IGF-I concentrations, as well as the colostrum proteome.

The objective of this study was to investigate the effects of heat treatment on colostral low-abundant proteins, IgG and IgA, insulin, and insulin-like growth factor I (IGF-I), as well as bacteria and somatic cells. First-milking colostrum samples >8 L and Brix % > 22.0 were harvested from 11 Holstein cows on a commercial dairy in New York State and split into 2 aliquots using single-use colostrum bags. One aliquot of each pair was cooled on ice immediately after harvest (raw, R; n = 11), and the other was heat-treated for 60 min at 60°C (heat, H; n = 11). All samples were analyzed for IgG and IgA via radial immunodiffusion assay and insulin and IGF-I concentrations by radioimmunoassay. Total bacterial counts and somatic cell counts (SCC) were determined using standard plate culture techniques and flow cytometry, respectively. Samples from a subset of 5 pairs (n = 10) were further analyzed by nano liquid chromatography-tandem mass spectroscopy, after ultracentrifugation at 100,000 × g for 60 min at 4°C to enrich the low-abundant protein whey fraction. Data were analyzed using either paired t-test or Wilcoxon signed-rank test or using an online software package to analyze proteomics data. Outcomes of proteomics analysis were fold change ≥1.5 between pairs, and paired t-tests with false discovery rate-adjusted P-value < 0.05. The median reduction of IgA concentrations was 8.5% (range: 0-38.0%) due to heat treatment, whereas IgG concentrations did not change due to treatment. Insulin concentrations decreased by a median of 22% (7-45%), and IGF-I decreased by 10% (0-18%) in H samples. Heat treatment was associated with a median reduction of SCC of 36% (0-90%) in paired samples, as well as a median reduction in total bacterial count of 93% (45-100%) in H versus R samples. Proteomics analysis identified a total of 328 unique proteins that were present in all 10 samples. Nine of the 25 proteins that decreased by at least 1.5-fold in H compared with R were identified as complement proteins. We conclude that heat treatment of colostrum is associated with a reduction in the concentration of bacterial counts and SCC, IgA, insulin, and IGF-I. In addition, proteomics analysis of colostral whey identified several complement components and other proteins that decreased in abundance due to heat treatment. Although IgG concentrations were unaffected and a reduction in bacterial counts was achieved, the change in several immunologically active proteins and growth factors may have biologically important effects on the developing immune system of the neonate fed heat-treated colostrum.

Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries.

Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.

Distribution of Lactococcus spp. in New York State dairy farms and the association of somatic cell count resolution and bacteriological cure in clinical mastitis samples.

We investigated the distribution of pathogenic non-agalactiae gram-positive, catalase-negative cocci (GPCN) in a convenience sample of New York State dairy farms. Our primary objective with the clinical mastitis (CM) GPCN samples was to evaluate somatic cell count (SCC) resolution and bacteriological cure of Streptococcus dysgalactiae or Streptococcus uberis versus Lactococcus lactis or Lactococcus garvieae in cows that received an approved intramammary treatment. In phase I, we assessed the distribution of the GPCN and SCC resolution. In phase II, we evaluated the SCC resolution and bacteriological cure in CM samples from the 4 farms with the highest prevalence of L. lactis or L. garvieae in phase I. In phase I, 8,868 CM and subclinical mastitis (SCM) milk samples were received from 143 farms. The GPCN samples identified by culture were confirmed with MALDI-TOF. From the 473 MALDI-TOF-confirmed GPCN samples, 155 were S. dysgalactiae (33%); 150, S. uberis (32%); 112, L. lactis (24%); 16, L. garvieae (3%); and 40, other GPCN (8%). From these, 277 were CM samples and 127 were eligible for the evaluation of SCC resolution, which was defined as SCC ≤200,000 cells/mL in a composite sample 15 to 60 d post-diagnosis. The odds of SCC resolution in CM samples was evaluated with multivariable logistic regression, and the odds were 6.1 [95% confidence interval (CI):2.7-13.9] times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. In phase II, a total of 1,662 CM and SCM samples were evaluated with microbiological methods as in phase I, of which 211 samples were confirmed by MALDI-TOF: 39% were S. dysgalactiae (n = 61) and S. uberis (n = 21); 55%, L. lactis (n = 114) and L. garvieae (n = 2); and 6%, other GPCN (n = 13). In total, 168 CM samples were eligible for analysis and 118 were included in the final SCC resolution model. Similar statistical methods as in phase I were performed, and the odds of SCC resolution were 2.4 (95% CI: 1.1-5.5) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Bacteriological cure was defined as having a different or negative culture on a quarter sample taken 14 to 28 d after initial diagnosis. The odds of bacteriological cure (n = 121) were 8.0 (95% CI: 2.5-25.6) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Differences in SCC resolution and bacteriological cure between these groups may dictate a different management approach.

Staphylococcus aureus intra-mammary infection affects the expression pattern of IL-R8 in goat.

IL-1R8 is a member of Interleukin-1 receptor family acting as a negative regulator of inflammation reliant on ILRs and TLRs activation. IL-1R8 role has never been evaluated in acute bacterial mastitis. We first investigated IL-1R8 sequence conservation among different species and its pattern of expression in a wide panel of organs from healthy goats. Then, modulation of IL-1R8 during natural and experimental mammary infection was evaluated and compared in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected goats. IL-1R8 has a highly conserved sequence among vertebrates. Goat IL-1R8 was ubiquitously expressed in epithelial and lymphoid tissues with highest levels in pancreas. IL-1R8 was down-regulated in epithelial mammary cells following S. aureus infection. Interestingly it was up-regulated in leukocytes infiltrating the infected mammary tissues suggesting that it could represent a target of S. aureus immune evasion.

Pentraxin 3 is up-regulated in epithelial mammary cells during Staphylococcus aureus intra-mammary infection in goat.

Pentraxin 3 is the prototypic long pentraxin and is produced by different cell populations (dendritic cells, monocytes/macrophages, endothelial cells, and fibroblasts) after pro-inflammatory stimulation. Different studies demonstrated the up-regulation of PTX3 during mastitis in ruminants, but its role is still unknown. We first investigated the conservation of PTX3 sequence among different species and its pattern of expression in a wide panel of organs from healthy goats. We studied the modulation of PTX3 during natural and experimental mammary infection, comparing its expression in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected animals. We confirmed the high conservation of the molecule among different species. Goat PTX3 was expressed at high levels in bone marrow, mammary gland, aorta, rectum, pancreas, skin and lungs. PTX3 was up-regulated in epithelial mammary cells and in milk cells after S. aureus infection, suggesting that it represents a first line of defense in goat udder.

Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis.

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC <100,000 cells/mL and culture-negative) were also negative for cathelicidin, corresponding to 100% specificity in the evaluated sample cohort. This study confirmed the value of the milk cathelicidin ELISA for detecting bovine mastitis, and highlighted the influence of mastitis-causing microorganisms on cathelicidin abundance. This influence did not compromise diagnostic performance; instead, it may have better reflected disease severity and evolution than SCC.

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds, related swine farms, and humans in contact with herds.

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms' owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval=0-10.2%) and 60% (33/55; 95% confidence interval=47.05-72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.

Evaluation of milk cathelicidin for detection of bovine mastitis.

Mastitis due to intramammary infection is one of the most economically relevant diseases in dairy cows, causing reductions in milk quality and quantity. Currently, mastitis monitoring is based on somatic cell count (SCC) and bacteriologic culture (BC) of milk. Nevertheless, inflammation-specific protein markers might provide more sensitive and reliable assays, enabling immunoassay-based screening strategies. Cathelicidin is an inflammatory protein released in milk that has recently demonstrated fair reliability and diagnostic potential for ewe mastitis. To assess its performance in cows, 531 quarter milk samples from 2 herds were tested using cathelicidin ELISA, SCC, and BC. We found that 29.0% of samples were positive for cathelicidin, 18.8% had SCC >200,000 cells/mL, and 13.7% were BC-positive. Cathelicidin showed a strong positive correlation with SCC as demonstrated by receiver operating characteristics curve analysis and by the clustering of cathelicidin-negative and cathelicidin-positive samples in association with low and high SCC values, respectively. For evaluating the diagnostic performance of a novel test, BC cannot be considered a reliable gold standard for true disease status because of its known limitations. Therefore, we assessed the sensitivity (Se) and specificity (Sp) of the milk cathelicidin ELISA using a latent class analysis approach together with BC and SCC by considering different diagnostic thresholds to identify the preferred Se/Sp combination. We modeled conditional dependence of cathelicidin and SCC to account for their close association. The cathelicidin ELISA showed higher Se than SCC and BC for almost all threshold combinations. In fact, at the best-performing threshold combination, the Se of cathelicidin was 80.6%, 6.2 percentage points higher than that of SCC >200,000 cells/mL (74.4%) and similar to that of SCC >100,000 cells/mL (80.2%). Most importantly, this Se was obtained with a loss in Sp of only 1.4 percentage points compared with SCC >200,000 cells/mL (94.9% Sp for cathelicidin vs. 96.3% for SCC >200,000). The limited Se of BC (38.8%) was also confirmed in this study, and BC showed a slightly lower Sp than both cathelicidin and SCC for most of threshold combinations. This study confirmed that cathelicidin is released in the milk of cows with mastitis and that its presence is highly correlated with SCC. The measurement of cathelicidin by ELISA may hold significant potential for improving the sensitivity of mastitis detection in dairy cows while maintaining high specificity.

Randomized noninferiority field trial comparing 2 first-generation cephalosporin products at dry off in quarters receiving an internal teat sealant in dairy cows.

The study objective was to compare 2 commercial dry cow mastitis products at the quarter level, with concurrent internal teat sealant application, evaluating the cure risk difference, odds of a cure, odds of a new intramammary infection (NIMI) during the dry period, and risk for a clinical mastitis (CM) case between calving and 60d in milk (DIM). A total of 590 cows (2,360 quarters) from 8 commercial dairy herds in Italy were enrolled and randomized to 1 of the 2 treatments at dry off: Cefovet A (CF; 250mg of cephazoline; Merial Italia SpA, Milan, Italy), and Cepravin (CP; 250mg of cephalonium dehydrate MSD Animal Health Srl, Segrate, Italy). Quarter milk samples were collected before dry cow therapy treatment at dry off, 2 to 9 DIM, and 10 to 17 DIM. Quarter milk samples from CM cases were collected during the first 60 DIM. Noninferiority analysis was used to evaluate the effect of treatment on the risk difference of a bacteriological cure during the dry period, the primary outcome. The odds of cure, developing a NIMI during the dry period, and the risk of a CM event within 60 DIM were evaluated with multivariable logistic regression and hazard analysis, respectively. The overall crude quarter-level prevalence of NIMI at dry off was 15.3%. The most common pathogen isolated from milk samples at dry-off was coagulase-negative staphylococci. Noninferiority analysis showed no effect of treatment on the risk difference for a cure between dry off and both postpartum samples, difference was 0.013. The least squares means from the multivariable model evaluating the odds of cure was 94% for CF and 95%for CP. We observed no effect of treatment on the odds for the presence of a NIMI at 2 to 9 DIM (least squares means: CF=0.09 and CP=0.07), nor did we note a difference in risk of experiencing a CM event between calving and 60 DIM (hazard ratio=0.8). In conclusion, no difference was observed between the 2 products evaluated when assessing the aforementioned outcomes in quarters also receiving an internal teat sealant.

The bovine milk microbiota: insights and perspectives from -omics studies.

Recent significant progress in culture-independent techniques, together with the parallel development of -omics technologies and data analysis capabilities, have led to a new perception of the milk microbiota as a complex microbial community with great diversity and multifaceted biological roles, living in an environment that was until recently believed to be sterile. In this review, we summarize and discuss the latest findings on the milk microbiota in dairy cows, with a focus on the role it plays in bovine physiology and health. Following an introduction on microbial communities and the importance of their study, we present an overview of the -omics methods currently available for their characterization, and outline the potential offered by a systems biology approach encompassing metatranscriptomics, metaproteomics, and metametabolomics. Then, we review the recent discoveries on the dairy cow milk microbiome enabled by the application of -omics approaches. Learning from studies in humans and in the mouse model, and after a description of the endogenous route hypothesis, we discuss the role of the milk microbiota in the physiology and health of both the mother and the offspring, and report how it can be changed by farming practices and during infection. In conclusion, we shortly outline the impact of the milk microbiota on the quality of milk and of dairy products.