Comparative Studies on Corynebacter1Um Pyogenes Toxin Formation in Monocultures and Mixed Cultures: The Demonstration of a Stimulating Effect of Peptococcus Indolicus and Stuart-Schwan Cocci

The growth and the toxin (i.e. hemolysin) producing capacity of Corynebacterium pyogenes were studied in monocultures and in co-cultures with 1 or more of the organisms frequently accompanying it in summer mastitis in cattle (Peptococcus indolicus, Stuart-Schwan cocci, Bacteroides melaninogenicus subsp. levii, Fusobacterium necrophorum and Streptococcus dysgalactiae) or with organisms seldom associated with summer mastitis (Streptococcus uberis, Streptococcus agalactiae, non-toxic staphylococci and Escherichia coli).Pc. indolicus, and to some extent also Stuart-Schwan cocci, stimulated the growth as well as the hemolysin producing capacity of Gb. pyogenes (Table 1) while Str. dysgalactiae, Str. uberis, Str. agalactiae, E. coli and the majority of the staphylococci reduced these activities. Most F. necrophorum strains stimulated the growth, but not the hemolytic activity. With B. melaninogenicus the results were inconclusive.The effect of Pc. indolicus appeared to be associated with the production of a filterable factor (Tables 2 and 3).Mouse toxicity and hemolytic activity of culture filtrates were closely correlated (Table 4).     

Bovine mastitis and its association with selected risk factors in smallholder dairy farms in and around Bahir Dar,Ethiopia

Three hundred fifty one (195 local zebu and 156 Holstein x local zebu crosses) lactating cows of smallholder farms in Bahir Dar 'milk shed' were examined from September 2003 to March 2004 to determine mastitis prevalence, isolate pathogens and identify the role of some potential risk factors. Clinical prevalence was determined through examination of abnormalities of milk, udder or cow. California mastitis test (CMT) was used for determination of subclinical mastitis prevalence. Clinical prevalence at cow level was 3.9% in crossbreds and none in local zebu breeds. Subclinical mastitis at cow level based on CMT was high (34.4%) in crossbreds compared to indigenous zebu (17.9%) (p < 0.05). Quarter subclinical prevalence based on CMT was 17.9% and 4.9% in crossbreds and local zebu, respectively. The pathogens isolated from mastitic milk (CMT positive milk) were coagulase negative staphylococci (CNS), S. aureus, Str. agalactiae, Str. dysgalactiae, Str. uberis, Micrococcus species, C. bovis, A. pyogens, B. cereus, and S. intermedius. Among these, the most frequent isolates were CNS (50%), S. aureus (19%), Str. agalactiae (8%) and Str. dysgalactiae (7%). Among potential risk factors considered, stage of lactation, parity and breed were found to affect the occurrence of mastitis significantly (p < 0.05).     

Mannitol agar for microbiologic diagnosis of bovine mastitis

A medium containing mannitol (mannitol agar) was developed and evaluated as a tool for the microbiologic diagnosis of bovine mastitis. Mannitol agar supported growth of all important bacterial mastitis pathogens (staphylococci, streptococci, coliforms, and pseudomonads) except Corynebacterium pyogenes. Color change around colonies in the agar permitted the differentiation of pathogenic from nonpathogenic staphylococci. Most Staphylococcus aureus strains and some Staphylococcus epidermidis strains produced yellow zones. These yellow zone-producing strains (mannitol fermenters) of staphylococci were obtained from quarters with significantly elevated (P less than 0.05) somatic cell counts (SCC) in the milk, as compared with uninfected quarters and, therefore, would be considered pathogens. Mannitol-negative strains of S epidermidis (those with red zones) were obtained from quarters with SCC similar to those of uninfected quarters. The streptococci could be divided into 2 groups on the basis of color change around the colonies: Streptococcus agalactiae, Str dysgalactiae, and group G streptococci produced red zones; Str uberis, Str bovis, and enterococci produced yellow zones. Pathogenic streptococci (Str agalactiae, Str dysgalactiae, Str uberis, and group G streptococci) were obtained from quarters with SCC significantly higher (P less than 0.01) than those of uninfected quarters. Streptococcus bovis and enterococci were obtained from quarters with SCC similar to those of uninfected quarters and were considered nonpathogenic. Pathogenic streptococci were found in much higher concentration than nonpathogenic streptococci and could be differentiated on that basis.     

奶牛乳腺炎病原菌的分离鉴定及耐药性分析

2 0 0 2～ 2 0 0 3年采集上海地区 32个规模化奶牛场 433份奶样 ,共分离 2 54株细菌。其中包括金黄色葡萄球菌 48/2 54 (1 8 9% )、表皮葡萄球菌 34/2 54 (1 3 4% )、大肠杆菌 2 8/2 54 (1 1 0 % )、肺炎 /溶血巴氏杆菌 1 1 /2 54 (4 3 % )、无乳链球菌 2 5/2 54 (9 8% )、停乳链球菌 1 6/2 54(6 3 % )、乳房链球菌 1 5/2 54 (5 9% )及酵母菌 8/2 54 (5 9% )。表明葡萄球菌性乳腺炎仍占主导地位。药敏试验结果表明 ,分离菌株对临床常用的多种抗菌药物都产生了耐药性 ,耐药菌株的出现和抗生素的残留给乳腺炎的防治及食品安全带来了隐患     

Therapeutic effects of systemic or intramammary antimicrobial treatment of bovine subclinical mastitis during lactation

The short- and long-term treatment efficacy of administrating penicillin for bovine subclinical mastitis during lactation when using intramuscular (IM; 9.5 mg [15,000 IU]/kg bodyweight of benzyl penicillin potassium) injections twice daily for 5 days, or intramammary (IMM; 0.3g [300,000 IU] penethamate hydroiodide) administration once daily for 5 days was compared with a control group receiving no treatment. One hundred and twenty-six cows met the inclusion criteria, which were lack of clinical symptoms, no recent treatment with antimicrobials, and findings of penicillin-sensitive Staphylococcus aureus, Streptococcus dysgalactiae, or Streptococcus uberis in combination with an inflammatory reaction. At follow-up 42-58 days after treatment, the proportion of cows negative for the original infection was significantly higher in IM and IMM groups compared to controls, but the difference between antimicrobial treatment groups was not significant. The udder quarter milk somatic cell count (SCC) was significantly lower at follow-up in IM and IMM groups than in controls, but milk production did not differ between treatments. The culling rate during the 10-month period following treatment was significantly higher in the group treated with IMM penicillin than in the other two groups, but the risk of new mastitis treatments within 10 months did not differ between the three groups. The cure rate was significantly affected by lactation number (lower in older cows), breed (lower in the Swedish Holstein breed), pathogen (lower for S. aureus), and pre-treatment SCC (higher for above average SCC). In conclusion, beneficial long-term effects of antimicrobial treatment during lactation of subclinical mastitis caused by S. aureus, Str. dysgalactiae or Str. uberis were not found in the present study.     

Summer mastitis in heifers: a bacteriological examination of secretions from clinical cases of summer mastitis in Denmark

A total of 166 samples of secretions collected from Danish heifers suffering from clinically diagnosed summer mastitis were examined bacteriologically. One hundred and sixty three samples yielded positive findings whereas no growth was obtained from 3 specimens. The majority of samples revealed a mixed flora in which the predominant components were Actinomyces pyogenes, Peptostreptococcus indolicus, a microaerophilic coccus (Stuart-Schwan coccus), Fusobacterium necrophorum, Bacteriodes melaninogenicus and Streptococcus dysgalactiae. Pure cultures were recovered in only 7% of cases. P. indolicus was isolated from 875 of the cases, a microaerophilic coccus from 84%, A. pyogenes from 72%, Fusobacterium necrophorum from 52%, Str. dysgalactiae from 37%, B. melaninogenicus from 33% and various unidentified obligate anaerobic Gram-negative rods from 31%. The results confirm the complex bacterial aetiology of summer mastitis.     

Adherence as a Prerequisite for Infection of the Bovine Mammary Gland by Bacteria

Using a simple in vitro test it was demonstrated that staphylococci, Streptococcus agalactiae, and micrococci, the species of bacteria which are commonly isolated from udder infections, adhered to mammary gland epithelial cells readily and in large numbers. Some strains of organisms which are associated with sporadic outbreaks or occur less commonly, like Str. dysgalactiae and Str. uberis, adhered moderately. Escherichia coli, Klebsiella spp., Corynebacterium pyogenes, C. bovis, Str. bovis, and Str. faecalis, species which are isolated occasionally, adhered poorly. From these studies, it appears that selective adherence of bacteria to the epithelial cells is a factor contributing to the ability of organisms to infect the mammary gland and may, therefore, be considered an important stage in the pathogenesis of bovine mastitis.     

多重PCR检测奶牛乳腺炎金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母菌方法的建立与应用

参照GenBank发表的序列，在金黄色葡萄球菌、无乳链球菌和停乳链球菌16SrRNA与23SrRNA之间的区域设计了3对引物，参照念珠菌和隐球菌的18SrRNA的序列设计1对引物，建立了检测金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母真菌4种乳腺炎主要致病菌的多重PCR方法。参照Skladny的方法制备模拟了细菌感染l临床标本。结果表明：本试验建立的多重PCR方法具有较好的特异性，多重PCR方法检测乳样中的金黄色葡萄球菌的细菌最小浓度为10^4CFU／mL，检测无乳链球菌、停乳链球菌和酵母真菌的细菌最小浓度分别为10^4CFU／mL、10^3CFU／mL和10^3CFU／mL。通过对采自临床型乳腺炎（46个）和隐性乳腺炎（167个）动物共计213个乳样分别用传统细菌学培养法和多重PCR方法进行检测，多重PCR对金黄色葡萄球菌和酵母真菌的检测具有更高的检出率（P〈0．01），但该方法对无乳链球菌和停乳链球菌的检出率与培养法差异不显著（P〉0．05）。     

THE NEW SOUTH WALES MASTITIS CONTROL PROGRAM

Bacteriological examinations were made on quarter samples from cows in 35 herds over a 3 year period to monitor the response in a mastitis control program. Initially, Staphylococcus aureus predominated in 32 of the herds and the mean herd prevalence was 26%. The control measures halved this rate but there was considerable variation in response between herds. The decline occurred rapidly and there was a significant reduction (P less than 0.01) by 3 months. Streptococcus agalactiae predominated in 3 herds and the overall infection rate was 4.9%. Control measures eliminated the infection completely from most herds but reinfection occurred in 2 herds. The greatest decline occurred in the first 6 months and was significant (P less than 0.05). The measures had little effect upon Str. uberis and Str. dysgalactiae which remained fairly consistently at low levels. Initially, strains of Staph. aureus resistant to penicillin were dominant in most herds. In a minority of herds strains resistant to streptomycin predominated and in these herds there was a concurrent resistance to penicillin. These patterns did not change greatly over the control period. Resistance by Str. agalactiae to streptomycin occurred in most herds at the start of the program.     

Etiology and antimicrobial susceptibility of udder pathogens from cases of subclinical mastitis in dairy cows in Sweden

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Effects of a mastitis control programme on the incidence of clinical mastitis

Summary

The results of a mastitis control field experiment in Utrecht over three and a half years, including sevens herds with a total of 225 lactating cows are presented. Every case of clinical mastitis was examined bacteriologically. Quarter samples were taken routinely at an average interval of five weeks.

The incidence of clinical mastitis in each herd at the start of the experiment varied from 10 to 104 quarter cases per 100 cows per year. The majority of cases of clinical mastitis (33.1 per cent) occurred during the first month of lactation.

In 74.4 per cent of the lactations marked by clinical mastitis only a single case was observed during that lactation period. If the animals were on pasture during the first month of lactation, the incidence of clinical mastitis was significantly lower during this month, compared with the first month when they were housed. Str. dysgalactiae was the most common isolate from clinical cases (21.5 per cent). A total proportion of 40.3 per cent of the clinical cases were caused by cocci, 20.3 per cent by Gram‐negative bacteria and 16.0 per cent were bacteriologically negative. Of the clinical cases caused by streptococci and staphylococci, 33.3 per cent were preceded by subclinical infection, compared with 11.8 per cent of the clinical cases due to E. coli. The overall incidence of clinical mastitis in this experiment decreased from 47 to 31 per 100 cows per year.     

停乳链球菌内蒙分离株抗原基因MIG的克隆

停乳链球菌是引起奶牛乳房炎的主要病原菌,根据GenBank中登陆的停乳链球菌表面蛋白MIG基因序列,设计一对引物,采用PCR的方法从临床分离的停乳链球菌内蒙分离株基因组DNA中扩增出MIG基因,得到一条1900bp的片段。将其连入PMD-19T载体中,经酶切,PCR及序列测定法进行鉴定。经DNA测序分析,证实与GenBank中停乳链球菌MIG基因(AF354651)序列同源性为95%,具有高度保守性。     

Etiological agents of dairy cows' mastitis in western part of Poland

The purpose of the study was to determine the etiological mastitis agents in cows from herds located in the western part of Poland in years 2003-2005. Bacteriological examinations of 18,713 samples taken from California Mastitis Test (CMT) positive quarters were performed according to standard methods. Some Gram-negative bacilli and Gram-positive cocci were examined with the use of API tests (Analytical Profile Index). It was found that 32.7% of samples that were CMT positive were culture negative and 9.3% of samples were contaminated. Streptococcus species (15.7%), coagulase negative staphylococci (CNS) (14.6%), Staph. aureus (8.6%), Gram-negative bacilli (4.0%) and Corynebacterium species (3.8%) were most frequently isolated. Escherichia coli (52.3%) dominated among Gram-negative bacilli followed by Klebsiella pneumoniae (4.1%), Pseudomonas aeruginosa (3.6%), Enterobacter cloacae (3.6%), Serratia marcescens (3.1%), Pasteurella multocida (3.1%), Acinetobacter lwofii (3.1%), and 26 other bacteria species. Stahylococcus xylosus (31.5%), Staph. hyicus (12.2%), Staph. haemolyticus (12.2%), Staph. sciuri (11.6%), Staph. chromogenes (8.8%), Staph. epidermidis (8.3%) and Staph. simulans (6.1%) were the most frequent CNS. Streptococcus uberis (50%), Str. dysgalactiae (19.7%), Str. acidominimus (6.6%) and Enterococcus faecalis (5.3%) were mostly found among CAMP-negative streptococci. An increase in frequency of mastitis caused by non-agalactiae streptococci, Staph. aureus, A. pyogenes and yeast-like fungi and a decrease in that produced by Str. agalactiae in 2005 as compared to years 2003 or 2004 were observed.     

Intracellular killing of mastitis pathogens by penethamate hydriodide following internalization into mammary epithelial cells

Penethamate hydriodide was highly effective in killing Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Staphylococcus aureus that internalized mammary epithelial cells. At higher concentrations (32 microg/mL to 32 mg/mL), killing rates ranged from 85% to 100%. At lower concentrations (0.032 microg/mL to 3.2 microg/mL), killing rates ranged from 0 to 80%. Results of this proof-of-concept study demonstrated that: (1) penethamate hydriodide is capable of entering mammary epithelial cells and killing intracellular mastitis pathogens without affecting mammary epithelial cell viability, (2) the in vitro model used is capable of quantifying the fate of mastitis pathogens internalized into mammary epithelial cells, and (3) this in vitro model can be used to determine the effectiveness of antibiotics at killing bacteria within the cytoplasm of mammary epithelial cells.     

奶牛急性乳房炎病原菌的分离与鉴定

[目的]为了掌握奶牛急性乳房炎致病菌的种类.[方法]通过采集病牛乳汁样品3份,分别对其病原茵进行分离与鉴定.检测[结果]表明:检出4种共13株细菌,其中致病性金黄色葡萄球菌6株;停乳链球菌5株;大肠埃希茵1株,乳房链球茵1株.[结论]该场奶牛的急性乳房炎是由致病性金黄色葡萄球菌、停乳链球菌、乳房链球菌和大肠杆菌共同引起...     

牛源性停乳链球菌最佳培养基的筛选及影响因素研究

为了提高奶牛乳房炎多联苗的免疫效果,对停乳链球菌进行了最佳培养基的筛选及生长过程中影响因素的研究。结果表明,停乳链球菌在改良肉汤中生长缓慢,不适宜其生长,但在优化培养基和THB培养基中长势良好,尤其是在优化培养基中,停乳链球菌生长延迟期大大缩短,培养4 h即进入对数生长期,约12 h达到生长高峰期,生长周期比改良肉汤缩短近约50%,比THB缩短约2 h,且生长高峰期的细菌数量比THB和改良肉汤亦明显提高。培养过程中影响因素试验结果表明,溶氧量对停乳链球菌生长无影响;在优化培养基中,葡萄糖较适宜的浓度为10 g/L;在对数生长期内补加NaOH调节pH值,能明显提高细菌的产量,并能延长其对数生长期。     

Mycoplasma mastitis: a review of transmission and control

The Mycoplasma sp. that cause mastitis are simple, cell wall-less, bacteria that can colonize and cause diseases in other extramammary sites in the bovine. Prevalence of mycoplasma mastitis appears to be increasing in many locations throughout the world. The best method to identify this group of pathogens is through direct culture on mycoplasma agar media. However, limitations with this culture procedure are the duration of culture, 10 days, special conditions required and thus added expense, and the lack of primary specificity to distinguish between true pathogens and commensal organisms. Thus culture of bulk tank milk samples has been advocated as a primary screening method to determine the mycoplasma status of a herd. This monitoring system is reasonably successful but the sensitivity of detection of Mycoplasma sp. in bulk tank milk is affected by a significant minority of cows that might shed the organism at levels below the threshold of detection. Contagious mastitis control procedures have been effective in controlling outbreaks of mycoplasma mastitis. Yet new methods of control might be warranted, methods that may prevent the outbreak. Current data suggests that a significant number of new outbreaks may occur via internal or animal-to-animal transmission of mycoplasma mastitis pathogens from asymptomatic carriers.     

Intramammary infections in a dairy herd with a low incidence of Streptococcus agalactiae and Staphylococcus aureus infections.

In a dairy herd with a low incidence of intrammary infections due to Streptococcus agalactiae and Staphylococcus aureus, clinical mastitis remained a serious problem despite good control of nonclinical mastitis through postmilking teat disinfection and antibiotic therapy of known infected quarters at the end of lactation. During the 2-year study, the incidence of clinical mastitis was 0.88 cases/cow-year; 32.2% were caused by streptococcal species other than Str agalactiae and 33.5% by gram-negative organisms. Among all new infections detected, 54.1% were caused by streptococcal species other than Str agalactiae and 25.7% by gram-negative bacteria. Among new infections, 41.6% occurred during the nonlactating period or within a few days of calving. Incidence of clinical mastitis was highest in the 1st month of lactation. Among 84 gram-negative infections, 42.8% were caused by Klebsiella pneumoniae, 20.2% by Escherichia coli, and 23.8% by Enterobacter spp. Among the many serotypes of K pneumoniae and E coli, none was predominant.     

Time to clearance of mycoplasma mastitis: The effect of management factors including milking time hygiene and preferential culling

Factors associated with time to clearance of mycoplasma mastitis were studied in 18 dairy cattle herds. Most herds cleared mycoplasma mastitis within 1 month; < 50% of the herds culled diseased cows preferentially, yet culling was not associated with hastened clearance. Other known mastitis biosecurity and management practices were not associated with clearance time.     

Bulk tank milk analysis: factors associated with appearance of Mycoplasma sp. in milk

Factors associated with the presence of Mycoplasma sp. in bulk tank milk samples were evaluated from 664 herds during 2.25 years. Milk quality components were not strongly related to the presence of Mycoplasma sp. in bulk tank milk. The presence of other contagious mastitis pathogens, Staphylococcus aureus and Streptococcus agalactiae, was also not related to the presence of mycoplasma, suggesting that the aetiology and transmission of mycoplasma mastitis were different from transmission of other contagious mastitis pathogens. The occurrence of the first isolation of mycoplasma from a bulk tank was not correlated to season of the year. Mycoplasma in bulk tank milk samples were more likely to be found in herds shipping more milk, an indirect measure of herd size. This suggests that larger herds are more likely to have mycoplasma mastitis. However, the first appearance of mycoplasma mastitis in a bulk tank sample was followed by a sample without this pathogen in more than 60% of herds. Mycoplasma sp. was not detected in any herd a year after first isolation. These findings suggest that this pathogen could be controlled and eliminated from herds.