Identification and antimicrobial susceptibility of Staphylococcus chromogenes isolates from intramammary infections of dairy cows

Staphylococcus chromogenes is a highly prevalent species in subclinical mastitis with a well-established impact on somatic cell count. Few data are available on its antimicrobial susceptibility. The objective of this study was three-fold: (1) to evaluate simple identification tests by comparing them with a genomic method; (2) to determine minimal inhibitory concentrations (MICs) of different antibiotics; (3) to search for the presence of important resistance mechanisms and resistance-determining genes.Seventy-three staphylococcal strains, all collected on different dairy farms, were tentatively identified as S. chromogenes based on their lack of hemolysis and their characteristic intermediate DNase activity. The identification of 70 strains was confirmed as S. chromogenes by tRNA intergenic spacer PCR (tRNA PCR). Three strains were identified as S. sciuri, a species that is naturally cloxacillin- and lincomycin-resistant.All 70 S. chromogenes strains were found to be normally susceptible to neomycin, gentamicin, erythromycin, enrofloxacin, and to penicillinase-stable penicillins and cephalosporins, represented in this study by cloxacillin. The latter result was confirmed by the absence of the mecA gene in each of 13 strains in which this gene was searched for. Twenty-seven (38%) strains were penicillinase producers. Three lincomycin-resistant S. chromogenes strains were found to carry the linA gene.It was concluded that S. chromogenes can be identified reliably in routine mastitis bacteriology, and that the only resistance of importance is against penicillinase-susceptible penicillins.     

Host-response patterns of intramammary infections in dairy cows

Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.     

Treatment of persistent intramammary infections with Streptococcus uberis in dairy cows

A survey was conducted of the prevalence of environmental pathogens, especially Streptococcus uberis, as causes of clinical mastitis in dairy cows. The response of intramammary infections with S uberis to conventional treatment was monitored by taking milk samples for bacteriology and somatic cell counting seven, 14 and 21 days after the treatment. The results showed that 51 per cent of the infections failed to respond, and the odds of cases failing to respond was significantly increased when the individual quarter somatic cell count seven days after the treatment was greater than 201,000 cells/ml. Ninety-six per cent of the suspected S uberis isolates identified by culture were confirmed as S uberis by using the api 20 Strep system. Restriction endonuclease fingerprinting was used to type the strains of S uberis isolated from 75 milk samples from 32 cows. Analysis showed that 96 per cent of the cases of S uberis that failed to respond to conventional treatment were persistent infections with one strain rather than reinfections with different strains. The persistent cases of S uberis were treated further with an extended course of intramammary preparations containing either procaine penicillin with dihydrostreptomycin or cefquinome. There was no significant difference between the cure rates achieved by the two preparations, and 55 per cent of the cases that had failed to respond to conventional treatment responded to the additional treatment.     

The epidemiology of Staphylococcus aureus infections from subclinical mastitis in dairy cows during a control programme

The objective was to investigate the spread of S. aureus in seven dairy herds. Milk samples were taken to determine mastitis pathogens and somatic cell count. An approved hygiene programme was established to control the spread of S. aureus from quarter to quarter. S. aureus isolates were differentiated by geno- and phenotyping to trace their spread within a herd.In two herds S. aureus showed a high prevalence, but were eliminated from the herds by the control programme. In these herds and a third herd typing results identified a particular type that was found much more frequently than all other types. The frequent types were repeatedly detectable during the study. In three other herds with a lower S. aureus prevalence and also in one herd with a long history of S. aureus and high prevalence subclinical mastitis was caused by several distinguishable S. aureus types. These types occurred simultaneously in the particular herd and showed little or no tendency to spread from quarter to quarter. They seemed to circumvent the control procedures resulting in a relatively high rate of new infected animals and therefore were hardly eliminated. The typing results and the clinical observations indicated that strains differed in their tendency to spread and their ability to infect udder quarters. In three herds typing methods identified a predominant type with the common epidemiological features of a contagious mastitis pathogen, while in the other herds the S. aureus type patterns were similar to that of environmental pathogens.     

Modelling the dynamics of intramammary E. coli infections in dairy cows: understanding mechanisms that distinguish transient from persistent infections

The majority of intramammary infections with Escherichia coli in dairy cows result in transient infections with duration of about 10 days or less, although more persistent infections (2 months or longer) have been identified. We apply a mathematical model to explore the role of an intracellular mammary epithelial cell reservoir in the dynamics of infection. We included biological knowledge of the bovine immune response and known characteristics of the bacterial population in both transient and persistent infections. The results indicate that varying the survival duration of the intracellular reservoir reproduces the data for both transient and persistent infections. Survival in an intracellular reservoir is the most likely mechanism that ensures persistence of E. coli infections in mammary glands. Knowledge of the pathogenesis of persistent infections is essential to develop preventive and treatment programmes for these important infections in dairy cows.     

Acute phase response in two consecutive experimentally induced E. coli intramammary infections in dairy cows

Background

Acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) have suggested to be suitable inflammatory markers for bovine mastitis. The aim of the study was to investigate acute phase markers along with clinical parameters in two consecutive intramammary challenges with Escherichia coli and to evaluate the possible carry-over effect when same animals are used in an experimental model.

Methods

Mastitis was induced with a dose of 1500 cfu of E. coli in one quarter of six cows and inoculation repeated in another quarter after an interval of 14 days. Concentrations of acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) were determined in serum and milk.

Results

In both challenges all cows became infected and developed clinical mastitis within 12 hours of inoculation. Clinical disease and acute phase response was generally milder in the second challenge. Concentrations of SAA in milk started to increase 12 hours after inoculation and peaked at 60 hours after the first challenge and at 44 hours after the second challenge. Concentrations of SAA in serum increased more slowly and peaked at the same times as in milk; concentrations in serum were about one third of those in milk. Hp started to increase in milk similarly and peaked at 36–44 hours. In serum, the concentration of Hp peaked at 60–68 hours and was twice as high as in milk. LBP concentrations in milk and serum started to increase after 12 hours and peaked at 36 hours, being higher in milk. The concentrations of acute phase proteins in serum and milk in the E. coli infection model were much higher than those recorded in experiments using Gram-positive pathogens, indicating the severe inflammation induced by E. coli.

Conclusion

Acute phase proteins would be useful parameters as mastitis indicators and to assess the severity of mastitis. If repeated experimental intramammary induction of the same animals with E. coli is used in cross-over studies, the interval between challenges should be longer than 2 weeks, due to the carry-over effect from the first infection.     

Effects of intramammary infusion of Bifidobacterium breve on mastitis pathogens and somatic cell response in quarters from dairy cows with chronic subclinical mastitis

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

Study of intramammary infections in dairy goats from mountainous regions in Italy

Extract

The manufacture of dairy products from the milk of small ruminants presents an opportunity to improve the economics of farming in remote areas such as the mountainous regions in northern Italy. Mastitis, particularly subclinical and chronic, is the most persistent and widespread problem affecting milk quality. Currently, there is a lack of information concerning the most common causal agents of mastitis in dairy goat herds in Italy. This study was undertaken to determine the aetiology and prevalence of intramammary infections (IMI), and to measure somatic cell counts (SCC) of dairy goats in mountainous regions of northern Italy.     

奶牛亚临床酮病检测

本研究采用试纸条法、紫外分光光度法和酮粉法检测奶牛亚临床酮病,结果表明：以紫外分光光度法定量检测结果为诊断标准,试纸条法具有较高的敏感性和特异性,分别为69％和94％,酮粉法的检测敏感性和特异性较低,分别为56％和90％.     

Approaches to modeling intramammary infections in dairy cattle

In this paper, three approaches (Markov processes, discrete-event simulation, and differential equations) to modeling intramammary infections (IMI; focusing on the dynamic changes between uninfected, subclinical, and clinical udder-health states) are described. The objectives were to describe the various approaches to modeling intramammary infections, determine if simulations of the examples of the three approaches yield stable prevalences, and discuss the approaches' limitations. The literature review showed that there is no agreement on the proportion of animals that change health states. The approach of discrete-event simulation modeling included the most cow-level risk factors and udder-health states (hence, was judged to replicated best the dynamics of the infection process) and yielded stable prevalences for all udder-health states. However, there remain parts of the dynamics that need further research. These include the pathogen-specific probabilities and times of occurrence for: regression of clinical IMI to subclinical IMI, flare-up of subclinical IMI to clinical IMI, and incidence of subclinical IMI. Also, the assumption in all current approaches of homogenous mixing is violated because the primary contact structure for contagious pathogens during milking is either between cows through residual infectious milk in the milking machine or within a cow by vacuum fluctuations or teat-cup liner slips. Better contact structures should be incorporated so that the effects of control strategies can be better-estimated. Moreover, the three modeling approaches discussed assumed that all non-infected quarters are susceptible to infection—which might be denied by work in genetic resistance.     

Haematology of subclinical fatty liver in dairy cows

The relationship between liver fat content and haematology was investigated in 369 cows from eight herds sampled in the second week after calving. High levels of fat in the liver were associated with a depression in total white cell count and in neutrophils, eosinophils and lymphocytes. There was no correlation between liver fat content and the percentage of E rosetting lymphocytes, packed cell volume or haemoglobin concentration. The changes in peripheral white cell counts may be related to the increased incidence of post parturient disease in cows with fatty liver.     

Determination of cloxacillin residues in dairy cows after intramammary administration

The aim of this study was to perform a comparative analysis of the characteristics of cloxacillin (CLO) (MRL of withdrawal in bovine milk is 30 ng/g) after a single intramammary (IMM) dose in the dry period (DP) and lactation (LP), and to establish a high‐performance liquid chromatography (HPLC) analytical method for CLO detection in milk. The research was conducted on a group of 10 cows in DP and 10 in LP. A single dose of 600 mg of CLO was administrated by the IMM route for a single quarter in DP and 500 mg for a single quarter in LP. CLO concentration was analyzed by HPLC. CLO was monitored at a wavelength of 206 nm. Pharmacokinetic calculations were performed using Phoenix^® WinNonlin^® 6.4 software. The calibration curve was linear over the range of 13.03–28 019.00 ng/g with the coefficient of determination R² > 0.999. CLO withdrawal in both the LP and DP group had a biphasic nature. The total CLO elimination in the DP and LP group was reached after 36 and 6.5 days, respectively. A quantitative and confirmatory method for the determination of CLO in fresh milk has been established. We have confirmed that the withdrawal of CLO in the DP group is not a linear process and has a stepwise character.     

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections

Background

Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC.A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC.

Results

The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster.

Conclusions

The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.     

Prevalence and incidence of intramammary infections in lactating dairy goats

AIM: To determine the prevalence of intramammary infection (IMI) of lactating dairy goats between 0 and 4 days postpartum, the prevalence and incidence rate of new IMI at Weeks 2, 14 and 27 of lactation, and the relationship between herd-level prevalence of IMI and bulk tank somatic cell count (BTSCC).

METHODS: Milk samples were collected from 8% of a herd (total 624 does) from 18 dairy goat herds in the Waikato region of New Zealand, for bacteriology and somatic cell count (SCC) determination, from both glands within 4 days of kidding (Week 0) and again at Weeks 2, 14 and 27. Prevalence of IMI was determined at each time point and incidence rate calculated for Weeks 0–2, 2–14, and 14–27. Greenwood and Reed-Frost models were compared for estimation of the transmission parameter for all pathogens, and for Staphylococcus aureus, coagulase negative staphylococci (CNS) and Corynebacterium spp. separately.

RESULTS: Bacteria were isolated from 1,122/4,814 (23.3%) glands, with CNS (13.4%) and Corynebacterium spp. (7.3%) being the most common isolates. Prevalence of any IMI increased with stage of lactation, varied among herds, and increased with age (all p<0.05). Incidence rate was 80, 24 and 7 new IMI/10,000 gland days for Weeks 0–2, 2–14 and 14–27, respectively. Incidence rate for any IMI increased with age and with the presence of an IMI in the contralateral gland, and varied among herds (p<0.001). The transmission of each pathogen was better modelled assuming contagiousness (Reed-Frost models), than not (Greenwood models).

At gland level, IMI increased SCC at all stages of lactation (p<0.001). The gland prevalence of IMI within herds was positively associated with ln BTSCC at Week 2 (p=0.02), but not Weeks 14 or 27 (p>0.05).

CONCLUSIONS: Prevalence of IMI increased with stage of lactation, but the highest incidence rate of new IMI occurred in early lactation. Models accounting for the contagious nature of infection fitted better than those not accounting for contagiousness. BTSCC was only associated with prevalence of IMI in early lactation.

CLINICAL RELEVANCE: Reduction of BTSCC in dairy goats may be best achieved by minimising the prevalence and incidence of new IMI in early lactation. Further studies are required to define management factors associated with the between herd, and stage of lactation, effects on prevalence and incidence rate in order to reduce BTSCC throughout lactation.     

Duration of bovine intramammary infections in commercial dairy herds

Data on the infection status of cows on seven commercial dairy farms were collected over 492 full lactations. Foremilk samples were taken at an average interval of five weeks. A total of 249 streptococcal and 433 staphylococcal infections were diagnosed. Spontaneous elimination occurred in 49 per cent of all streptococcal infections and in 54 per cent of Staphylococcus aureus infections. The average duration of spontaneously eliminated infections was 10.8 weeks for Streptococcus agalactiae, 9.9 weeks for Strep dysgalactiae, 10.4 weeks for Strep uberis and 12.8 weeks for Staph aureus. The average duration of infections persisting until drying off was 19.3 weeks for Strep agalactiae, 18.7 weeks for Strep dysgalactiae, 18.5 weeks for Strep uberis and 25.2 weeks for Staph aureus. The method and rate of elimination of infection as found in this analysis are of value for estimating new infection rates and selecting quarters for dry cow therapy.     

A comparative randomized field trial on intramammary and intramuscular dry cow antibiotic treatment of subclinical Staphylococcus aureus mastitis in dairy cows

The efficacy of two dry cow treatment (DCT) regimens for subclinical Staphylococcus aureus mastitis was evaluated in naturally infected dairy cows. At dry-off, cows were assigned to two treatment groups by randomized blocks on the basis of parity and somatic cell count (SCC). Two antibiotic DCT regimens were used, namely: (1) a single intramammary infusion containing sodium nafcillin, procaine benzylpenicillin and dihydrostreptomycin; and (2) systemic cefquinome administered intramuscularly, twice at a 24-h interval. In the intramammary (IMM) treatment group, the S. aureus intramammary infection (IMI) rate was reduced from 40% (56/140 quarters) before dry-off to 20% (28/140) after calving. Seventy per cent (39/56) of the S. aureus-positive quarters were negative after calving, and 13% (11/84) of the negative quarters were positive after calving. In the systemic treatment group, the S. aureus IMI rate increased from 39% (29/74 quarters) before dry-off to 55% (41/74) after calving. Twenty-eight per cent (8/29) of the S. aureus-positive quarters were negative after calving and 45% (20/45) of the negative quarters were positive after calving. The odds ratio of an S. aureus-positive quarter being negative after calving in the IMM group relative to the systemic therapy group was 44.6 (95% confidence interval = 2.1-909.1, P < 0.01). Parity, quarter, milk SCC and N-acetyl-beta-D-glucosaminidase were tested in the model, and were found to have no significant effect on S. aureus cure rates or new IMI rates. The IMM treatment resulted in a higher cure rate compared with that observed in previous studies. The very low cure rate after systemic cefquinome treatment was comparable to the spontaneous cure rate observed in untreated controls in previous studies. The unfavourable results of the cefquinome systemic DCT might reflect inadequate pharmacokinetic properties of the drug regarding poor udder penetration in subclinical mastitis and short antimicrobial effect compared with the IMM treatment.     

Quarter,cow, and farm risk factors for intramammary infections with major pathogens relative to minor pathogens in Thai dairy cows

A cross-sectional study was carried out from May to September 2011 on 35 smallholder dairy farms in Chiang Mai, Thailand, to identify the quarter, cow, and farm factors that relate to intramammary infections (IMI) from major specified pathogens, compared to infections from minor pathogens. Data on general farm management, milking management, and dry cow management were recorded for each herd. Quarter milk samples were collected from either clinical or subclinical mastitis quarters. Dependent variables were binary data defining the specified major pathogens, including Streptococcus agalactiae (7.1 %), Streptococcus uberis (9.4 %), Streptococcus dysgalactiae (4.0 %), and other streptococci (16.7 %), as a case, and all minor pathogens as a control, in each dependent variable. The occurrence of S. agalactiae IMI was lower in first-parity cows and cows with short milking time. Cows with body condition score (BCS) <2.5 had higher occurrence of S. agalactiae IMI. The occurrence of S. uberis IMI was higher in quarters with California mastitis test (CMT) score 2, score 3, and having clinical mastitis and in farms with increasing age of vacuum system. Quarters with CMT score 3, having clinical mastitis, cow with manual milking after detaching milking cluster, and farms with high bulk milk somatic cell counts (BMSCC >500,000 cells/ml) had higher occurrence of S. dysgalactiae IMI. For other streptococci, quarters having clinical mastitis, BCS <2.5, and pulling down of milking cluster while milking increased occurrence of other streptococci IMI relative to minor pathogen IMI. These results highlight the importance of individual cow factors, milking characteristics, and BMSCC in determining the risk of IMI from major pathogens.     

奶牛乳房内灌注头孢噻呋钠的消除动力学研究

在奶牛乳房内灌注头孢噻呋钠后,采用超高效液相色谱一串联质谱法测定牛乳中头孢噻呋的浓度,对其消除动力学进行了研究。3头实验奶牛按每个乳房0．3g头孢噻呋灌注,牛乳中药物达到的最高浓度Cmax=107．89μg／mL,达峰时间Tmax=8h,药物半衰期T1/2=13．97h。在乳房内用药后的最初56h内,头孢噻呋浓度快速下降;最后一次给药88h后,所有乳腺中头孢噻呋的浓度都低于允许限量（0．1μg／mL）。故建议头孢噻呋在牛奶中的休药期为4d。