Treatment of clinically severe bovine mastitis - a scoping review.

Mastitis is a major health problem for bovines and can be categorized as non-severe or severe, based on clinical symptoms. A severe case of clinical mastitis is usually defined by the cow being affected systemically. It is important to consider how to handle severe cases because these cases can be fatal and cause high production losses. However, there are generally few detailed treatment guidelines. By conducting a scoping review on the topic, we aimed to synthesize the information that is available on treatment and outcomes, as reported from clinical trials and observational studies. This was facilitated by following the PRISMA-guidelines with a stepwise systematic screening of scientific literature on the subject, retrieved via Pubmed and Web of Science, using pre-defined selection criteria. The results yielded a total of 14 reports of treatment and outcomes in cases of naturally occurring severe clinical mastitis. Cross-trial comparison was difficult due to the different exclusion criteria and outcome definitions. Many studies focused on cases caused by gram-negative bacteria treated with intensive antibiotic protocols, often containing antibiotics that are categorized as critical for human health. Few focused on severe cases caused by gram-positive bacteria or on the relative use of non-antibiotic treatment. In general, only a small number of statistically significant differences were found in trials comparing different treatment protocols, with no obvious trends across trials. Our findings emphasize the need for more research into the treatment efficacy of antibiotic and non-antibiotic options for clinically severe mastitis. Furthermore, consideration of how trial conditions relate to the practical circumstances in a field setting could improve the applicability of reported results. This could help to provide practitioners with the information needed to make evidence-based treatment decisions in cases of clinically severe mastitis.

Treatment of mild to moderate clinical bovine mastitis caused by gram-positive bacteria: A noninferiority randomized trial of local penicillin treatment alone or combined with systemic treatment.

Bovine mastitis is one of the most important diseases in modern dairy farming, as it leads to reduced welfare and milk production and increased need for antibiotic use. Clinical mastitis in Denmark is most often treated with a combination of local and systemic treatment with penicillin. The objective of this randomized clinical trial was to assess whether worse results could be expected with local intramammary treatment with penicillin compared with a combination of local and systemic treatment with penicillin in terms of the bacteriological cure of mild and moderate clinical mastitis cases caused by gram-positive bacteria. We carried out a noninferiority trial with a noninferiority margin set to a relative reduction in bacteriological cure of 15% between these 2 treatment groups to assess the effect of reducing the total antibiotic use by a factor of 16 for each treated case. Clinical mastitis cases from 12 Danish dairy farms were considered for enrollment. On-farm selection of gram-positive cases was carried out by the farm personnel within the first 24 h after a clinical mastitis case was detected. A single farm used bacterial culture results from the on-farm veterinarian, whereas the other 11 farms were provided with an on-farm test to distinguish gram-positive bacteria from gram-negative or samples without bacterial growth. Cases with suspected gram-positive bacteria were allocated to a treatment group: either local or combination. Bacteriological cure was assessed based on the bacterial species identified in the milk sample from the clinical mastitis case and 2 follow-up samples collected approximately 2 and 3 wk after ended treatment. Identification of bacteria was carried out using MALDI-TOF on bacterial culture growth. Noninferiority was assessed using unadjusted cure rates and adjusted cure rates from a multivariable mixed logistic regression model. Of the 1,972 clinical mastitis cases registered, 345 (18%) met all criteria for inclusion (full data). The data set was further reduced to 265 cases for the multivariable analysis to include only complete registrations. Streptococcus uberis was the most commonly isolated pathogen. Noninferiority was demonstrated for both unadjusted and adjusted cure rates. The unadjusted cure rates were 76.8% and 83.1% for the local and combined treatments, respectively (full data). The pathogen and somatic cell count before the clinical case had an effect on the efficacy of treatment; thus efficient treatment protocols should be herd- and case-specific. The effect of pathogen and somatic cell count on treatment efficacy was similar irrespective of the treatment protocol. We conclude that bacteriological cure of local penicillin treatment for mild and moderate clinical mastitis cases was noninferior to the combination of local and systemic treatment using a 15% noninferiority margin. This suggests that a potential 16-fold reduction in antimicrobial use per mastitis treatment can be achieved with no adverse effect on cure rate.

Veterinary Treatment Approach and Antibiotic Usage for Clinical Mastitis in Danish Dairy Herds.

Danish veterinarians' treatment approach and use of antibiotics for clinical mastitis were investigated through a web-based questionnaire. The objective of the study was to describe and evaluate how the clinical mastitis treatment practice in Danish dairy herds corresponds to evidence from the literature and legislative requirements, in order to suggest directions for improvements and approaches encouraging the prudent use of antibiotics. In total, 174 veterinarians working with cattle received the questionnaire and 85 (48.9%) completed it. Their answers suggested that the Danish treatment approach for clinical mastitis generally relies on combined systemic and intramammary antibiotic administration (92% would use this often or always) and almost always includes supportive treatment with nonsteroidal anti-inflammatory drugs (99% would use it often or always in combination with antibiotic therapy). While collecting milk samples in order to target treatment towards pathogens is a priority in the legislation and for veterinarians, the direct application seems hindered due to the waiting time with the currently used analysis practice. Consequently, 91% reported that they would start treatment immediately after clinical examination often or always. The results of this investigation show that there is a potential for improvement in targeting treatments towards the causative pathogen by encouraging methods that allow for a more rapid reliable pathogen determination. When this issue has been addressed, the available evidence on the best treatment practice of Gram-negative-caused mastitis cases can be applied properly, reducing the volume of antibiotic treatments with limited expected effect. Additionally, investigating the potential of reducing combined administration to only intramammary treatment in Gram-positive cases could be a further step towards a more prudent antibiotic strategy.

Effect of automatic cluster flushing on the concentration of Staphylococcus aureus in teat cup liners.

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

Comparison of phenotypic and genotypic antimicrobial resistance patterns associated with Staphylococcus aureus mastitis in German and Danish dairy cows.

Staphylococcus aureus is one of the most common pathogens associated with bovine mastitis in Germany and Denmark. Successful therapy is strongly linked to the susceptibility of the pathogen to the administered antimicrobial. An increase in resistant pathogens in human and veterinary medicine has become a concern worldwide and hampers therapy due to reduced susceptibility. In the present study, susceptibility testing was performed for 85 and 93 S. aureus isolates originating from mastitis cases on 12 German and 8 Danish dairy farms, respectively. Phenotypic examination was performed by detection of minimal inhibitory concentration (MIC) values using the broth microdilution method, followed by genotypic investigations of the blaZ and mecA resistance genes via PCR. The tested antimicrobials were the most frequently used ß-lactams in German and Danish dairy farms, including cefquinome, cefoperazone, cephapirin, penicillin, oxacillin, cloxacillin, amoxicillin-clavulanic acid, and cephalexin-kanamycin. Special attention was paid to varying therapy concepts because, in Germany, third- and fourth-generation cephalosporins have been predominantly used in mastitis therapy, whereas in Denmark, restrictive use of penicillin is followed by a general avoidance of cephalosporins. Differences in MIC values were mainly based on determined MIC90 values (MIC at which 90% of isolates are inhibited). In general, Danish S. aureus isolates were inhibited at comparatively lower MIC90 values than S. aureus isolated from German dairy farms for most ß-lactams. No differences were observed regarding cefquinome, because both German and Danish isolates exhibited MIC50 and MIC90 values of 0.5 and 1 µg/mL, respectively. In contrast, the MIC90 for penicillin against German and Danish S. aureus were 0.5 and ≤0.06 µg/mL, respectively. Resistance genes (blaZ, mecA) were only detected in German S. aureus isolates on 3 dairy farms in Germany. A total of 5 isolates tested positive for both blaZ and mecA, whereas 1 isolate carried the blaZ resistance gene only. A direct correlation between frequently used antimicrobials and reduced susceptibility could not be determined based on results of the present study. In addition to further research to determine factors associated with resistance development, we emphasize the urgent need for internationally standardized clinical breakpoints to assess resistance situations more accurately.

Expert evaluation of different infection types in dairy cow quarters naturally infected with Staphylococcus aureus or Streptococcus agalactiae.

The purpose of this study was to improve the diagnostic recommendations for Staphylococcus aureus and Streptococcus agalactiae control using bacterial culture (BC), polymerase chain reaction (PCR) and somatic cell count (SCC) as diagnostic methods. The study was carried out in three steps: firstly, diagnostic test patterns for naturally infected quarters with Staph. aureus (24 quarters) and Strep. agalactiae (16 quarters) were created by sampling the quarters each day for 21 days and analysing the daily quarter milk samples using BC, PCR and SCC. Secondly, 30 mastitis experts were asked to group and describe the diagnostic test patterns and to establish a diagnosis for each group. The experts' statements regarding the groups they established were subsequently examined using qualitative content analysis to assign "infection types" to the statements. Lastly, the test performance was estimated for BC, PCR and SCC using generalised logistic regression models with the interpreted statements as a reference for infection. The experts mainly identified the Staph. aureus quarter-patterns as persistent infections, while some had more dynamic patterns. Strep. agalactiae quarter-patterns mainly involved persistent infection, yet some appeared hard to diagnose and were assigned to almost all different infection types, while experts did not agree on the interpretation. Estimates of Se for detection of Staph. aureus infection were 95.9% [93.7; 97.3] for BC, 99.5% [98.3; 99.8] for PCR, and 96.1% [94.0; 97.5] for SCC. The corresponding Sp estimates were 74.5% [65.7; 81.7], 66% [57.2; 73.8] and 43.7% [36.2; 51.5] for BC, PCR and SCC, respectively. The Se estimates of BC and PCR for Strep. agalactiae infection were 100% [83.5; 100] and 99.9% [99.6; 100], respectively, whereas the Se of SCC detecting Strep. agalactiae infection was only 34.3% [26.4; 43.3]. This indicated that Strep. agalactiae-positive BC and PCR test results were more important than SCC results to the experts when diagnosing a quarter as infected. The Sp estimates of BC, PCR and SCC for Strep. agalactiae infection were 99% [72.8; 100], 97.7% [62.1; 99.9], and 65.7% [56.7; 73.7], respectively. We conclude that PCR and BC are highly sensitive in the detection of persistent and new infections as defined by the experts, although the Se was not always 100%. An accepted lower Sp suggests that experts place less emphasis on false-positive results. We recommend that efforts are made to develop consistent terminology to characterise intramammary infections over time so that the course of infection can be taken into account at diagnosis.

Bovine mastitis bacteria resolved by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method to identify the most common pathogenic bacteria in humans and animals. The goals of this study were to amend a commercial database with additional species, evaluate the amended database for identification of bacterial genera and species causing bovine mastitis, and describe the plethora of species involved. In total, 500 udder pathogenic isolates were subjected to MALDI-TOF MS using bacterial or fungal colony material; 93.5% could be identified to the species level, and 6.5% were identified only to the genus level. Isolates identified to the genus level required further identification to the species level by conventional methods or 16S rDNA sequencing. Mass spectra from verified species were used to expand the MALDI-TOF MS database to improve future identification ability. A total of 24 genera and 61 species were identified in this study. Identified isolates were mainly staphylococci, streptococci, Enterobacteriaceae, and coryneforme bacteria. In conclusion, MALDI-TOF MS is a powerful, rapid, and reliable technique to identify the most common microorganisms causing bovine mastitis, and the database can be continuously expanded and improved with additional species.

Association between teat skin colonization and intramammary infection with Staphylococcus aureus and Streptococcus agalactiae in herds with automatic milking systems.

The objective of this study was to investigate the association between teat skin colonization and intramammary infection (IMI) with Staphylococcus aureus or Streptococcus agalactiae at the quarter level in herds with automatic milking systems. Milk and teat skin samples from 1,142 quarters were collected from 300 cows with somatic cell count >200,000 cells/mL from 8 herds positive for Strep. agalactiae. All milk and teat skin samples were cultured on calf blood agar and selective media. A subset of samples from 287 quarters was further analyzed using a PCR assay (Mastit4 PCR; DNA Diagnostic A/S, Risskov, Denmark). Bacterial culture detected Staph. aureus in 93 (8.1%) of the milk samples and 75 (6.6%) of the teat skin samples. Of these, 15 (1.3%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was cultured in 84 (7.4%) of the milk samples and 4 (0.35%) of the teat skin samples. Of these, 3 (0.26%) quarters were positive in both the teat skin and milk samples. The PCR detected Staph. aureus in 29 (10%) of the milk samples and 45 (16%) of the teat skin samples. Of these, 2 (0.7%) quarters were positive in both the teat skin and milk samples. Streptococcus agalactiae was detected in 40 (14%) of the milk samples and 51 (18%) of the teat skin samples. Of these, 16 (5.6%) quarters were positive in both the teat skin and milk samples. Logistic regression was used to investigate the association between teat skin colonization and IMI at the quarter level. Based on bacterial culture results, teat skin colonization with Staph. aureus resulted in 7.8 (95% confidence interval: 2.9; 20.6) times higher odds of Staph. aureus IMI, whereas herd was observed as a major confounder. However, results from the PCR analyses did not support this association. Streptococcus agalactiae was isolated from the teat skin with both PCR and bacterial culture, but the number of positive teat skin samples detected by culture was too low to proceed with further analysis. Based on the PCR results, Strep. agalactiae on teat skin resulted in 3.8 (1.4; 10.1) times higher odds of Strep. agalactiae IMI. Our results suggest that Staph. aureus and Strep. agalactiae on teat skin may be a risk factor for IMI with the same pathogens. Focus on proper teat skin hygiene is therefore recommended also in AMS.

Accuracy of qPCR and bacterial culture for the diagnosis of bovine intramammary infections and teat skin colonisation with Streptococcus agalactiae and Staphylococcus aureus using Bayesian analysis.

Streptococcus agalactiae (Strep. agalactiae) and Staphylococcus aureus (Staph. aureus) are originally regarded as contagious mastitis pathogens, however, both pathogens have recently been isolated from extramammary and environmental sites, indicating that other sites than the udder might contribute to the spread of these pathogens potentially causing intramammary infections. Diagnostic tools to identify pathogens at extramammary sites are available but still needs to be validated. The objective of this cross-sectional field study was to estimate the diagnostic sensitivity (Se) and specificity (Sp) of the commercially available Mastit4 qPCR assay and bacterial culture (BC) in identifying Strep. agalactiae and Staph. aureus from milk and teat skin samples. We randomly selected 30-40 cows with high somatic cell counts from eight Danish Strep. agalactiae-positive dairy herds with automatic milking systems. Teat skin samples and aseptic milk samples were collected from right rear quarters (n = 287) for BC and PCR analysis. Se and Sp were estimated in a Bayesian latent class analysis. For milk samples, the Se and Sp of qPCR for Strep. agalactiae were estimated to 0.97 and 0.99, respectively, whereas the Se and Sp of BC were 0.41 and 1.00, respectively. The Se and Sp of qPCR for Staph. aureus were estimated to 0.95 and 0.99, respectively, whereas the Se and Sp of BC were 0.54 and 0.77, respectively. For teat skin samples, the Se and Sp of qPCR for Strep. agalactiae were estimated to be 0.97 and 0.96, respectively, whereas the Se and Sp of BC were 0.33 and 1.00, respectively. The Se and Sp of qPCR for Staph. aureus were estimated to 0.94 and 0.98, respectively, whereas the Se and Sp of BC were 0.44 and 0.74, respectively. In conclusion, the Se for diagnosing Strep. agalactiae and Staph. aureus IMI was higher for qPCR than BC, suggesting that qPCR is a valuable method for detecting both pathogens from quarter-level milk samples. The performance of BC in the detection of Strep. agalactiae and Staph. aureus on teat skin was poor compared to qPCR, indicating that differences in the target condition of the two methods should be considered when implementing them as routine diagnostic tests for detecting teat skin colonisers. The low Se of BC may preclude the use of BC for skin testing, and qPCR is better for this task.

Typeability of MALDI-TOF assay for identification of non-aureus staphylococci associated with bovine intramammary infections and teat apex colonization.

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), a culture-dependent assay, has recently been implemented for routine identification of non-aureus staphylococci (NAS) species from milk, but the assay has never been investigated for NAS from nonmilk or environmental samples. The objective of this study was to evaluate the typeability of the MALDI-TOF assay for the identification and differentiation of bovine-associated NAS species on aseptically collected quarter milk and teat skin samples in dairy herds. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabs and foremilk samples from right hind and left front quarters. Teat skin swabs and milk samples were collected aseptically for preliminary identification using bacterial culture on chromogenic and calf blood agars. Colonies from milk and teat skin samples with suspicion of having NAS were identified to species-level by MALDI-TOF assay. Out of 511 isolates from 284 quarters (142 cows), 78% (n = 399) were identified by MALDI-TOF. The percentage of correctly identified NAS from milk (91%, 105/115) using MALDI-TOF was higher than the percentage from teat skin (68%, 268/396). Out of the identified isolates, 93% (n = 373) were successfully identified as NAS, whereas the remaining 26 (7%) were shown to be other bacterial species. Out of 26 NAS isolates, 1 originated from milk (Corynebacterium stationis), whereas 25 originated from teat skin representing Aerococcus viridans (n = 7), Bacillus pumilus (n = 13), Enterococcus saccharolyticus (n = 1), Clostridium septicum (n = 1), Corynebacterium stationis (n = 2), and Corynebacterium casei (n = 1). The MALDI-TOF identified 85 (98/115) and 62% (245/396) of the isolates in the first test. Isolates that were not identified to species-level at first test were subjected to a second test, and 47 (8/17) and 32% (48/151) from milk and teat skin, respectively, were identified. After 2 rounds of MALDI-TOF, 22% (n = 112) of the isolates were not identified, representing 103 from teat skin and 9 from milk. Eighteen isolates without identification by MALDI-TOF were successfully identified to species-level using sequencing, where 16 were correctly identified as NAS, whereas the other 2 were Corynebacterium stationis. In conclusion, MALDI-TOF is a reliable assay for identification and typeability of NAS species from aseptically collected quarter milk samples. The assay may be used for identification of NAS species from teat skin swabs. However, confirmation using nucleic acid-based tools is vital for accurate species identification of some species and strains.

Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization.

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with automatic milking systems, and (2) examine if the isolated NAS influences the expression of S. aureus virulence factors controlled by the accessory gene regulator (agr) quorum sensing system. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabbing and aseptic quarter foremilk samples from right hind and left front quarters. Teat skin swabs were collected using the modified wet-dry method and milk samples were taken aseptically for bacterial culture. Colonies from quarters with suspicion of having NAS in milk or teat skin samples (or both) were subjected to MALDI-TOF assay for species identification. To investigate the interaction between S. aureus and NAS, 81 isolates NAS were subjected to a qualitative ß-galactosidase reporter plate assay. In total, 373 NAS isolates were identified representing 105 from milk and 268 from teat skin of 284 quarters (= 142 cows). Sixteen different NAS species were identified, 15 species from teat skin and 10 species from milk. The most prevalent NAS species identified from milk were Staphylococcus epidermidis (50%), Staphylococcus haemolyticus (15%), and Staphylococcus chromogenes (11%), accounting for 76%. Meanwhile, the most prevalent NAS species from teat skin were Staphylococcus equorum (43%), S. haemolyticus (16%), and Staphylococcus cohnii (14%), accounting for 73%. Using reporter gene fusions monitoring transcriptional activity of key virulence factors and regulators, we found that out of 81 supernatants of NAS isolates, 77% reduced expression of hla, encoding a-hemolysin, 70% reduced expression of RNAIII, the key effector molecule of agr, and 61% reduced expression of spa encoding protein A of S. aureus, respectively. Our NAS isolates showed 3 main patterns: (1) downregulation effect such as S. chromogenes (milk) and Staphylococcus xylosus (milk and teat), (2) no effect such as Staphylococcus sciuri (teat) and S. vitulinus (teat), and the third pattern (c) variable effect such as S. epidermidis (milk and teat) and S. equorum (milk and teat). The pattern of cross-talk between NAS species and S. aureus virulence genes varied according to the involved NAS species, habitat type, and herd factors. The knowledge of how NAS influences S. aureus virulence factor expression could explain the varying protective effect of NAS on S. aureus intramammary infections.

Genomic investigation of Staphylococcus aureus isolates from bulk tank milk and dairy cows with clinical mastitis.

Staphylococcus aureus is one of the most common pathogens that cause mastitis in dairy cows. Various subtypes, virulence genes and mobile genetic elements have been associated with isolates from bulk tank milk and clinical mastitis. So far, no Danish cattle associated S. aureus isolates have been whole-genome sequenced and further analyzed. Thus, the main objective was to investigate the population structure and genomic content of isolates from bulk tank milk and clinical mastitis, using whole-genome sequencing. This may reveal the origin of strains that cause clinical mastitis. S. aureus isolates from bulk tank milk (n = 94) and clinical mastitis (n = 63) were collected from 91 and 24 different farms, respectively and whole-genome sequenced. The genomic content was analyzed and a phylogenetic tree based on single nucleotide polymorphisms was constructed. In general, the isolates from both bulk tank milk and clinical mastitis were of similar genetic background. This suggests that dairy cows are natural carriers of the S. aureus subtypes that cause clinical mastitis if the right conditions are present and that a broad range of subtypes cause mastitis. A phylogenetic cluster that mostly consisted of ST151 isolates carried three mobile genetic elements that were primarily found in this group. The prevalence of resistance genes was generally low. However, the first ST398 methicillin resistant S. aureus isolate from a Danish dairy cow with clinical mastitis was detected.