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).     

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.     

Cause, occurrence, and clinical signs of mastitis and anorexia in cows in a Wisconsin study

Microbiologic culture revealed the following cause of mastitis and anorexia in 145 cows in Wisconsin to be Escherichia coli, 66 cows; Klebsiella spp, 3; Corynebacterium pyogenes, 27; streptococci, 21; staphylococci, 20; yeasts, 1; and no bacterial growth, 7. Mastitis was detected with approximately equal frequency throughout the year. Escherichia coli was isolated throughout the year, but was more common and was the predominant organism during the summer. Corynebacterium pyogenes was isolated most often in winter and spring; streptococci in fall, winter, and spring; and staphylococci throughout the year. Corynebacterium pyogenes caused most of the mastitis in nonlactating cows. Escherichia coli, C pyogenes, streptococci, and staphylococci were isolated with about equal frequency at parturition, whereas E coli was the predominant cause of mastitis in early and late lactation. Of cases of mastitis, 27% were seen 10 days before and after parturition. Local and systemic clinical signs of infection were similar for all causes, except that C pyogenes caused more (P less than 0.01) malodorous and purulent milk than did other organisms and was isolated more commonly from quarters with injured teats. Recovery was significantly (P less than 0.01) higher in cows with E coli infections, compared with recovery in cows with gram-positive organism infections. Cows with C pyogenes infections frequently had quarters that ultimately ceased lactation. A few cows were recumbent at initiation of antimicrobial therapy and a few were not eating 24 hours later; however, 50% of these cows recovered. Criteria such as season of year, stage of lactation, appearance of milk and udder, and appetite permitted the cause (gram-negative or gram-positive organisms) of the mastitis to be predicted with 77% accuracy.     

Comparative bacteriological studies on summer mastitis in grazing cattle and pyogenes mastitis in stabled cattle in Denmark.

A total of 143 secretions from clinical cases of summer mastitis (SM) in grazing cattle and from 89 cases of pyogenes mastitis (PM) in stabled cattle were examined bacteriologically. The typical bacteriological finding was a mixed flora in which the predominant organisms were Actinomyces pyogenes (SM-70%, PM-85%), Peptostreptococcus indolicus (54%, 54%), a microaerophilic coccus (Stuart-Schwan coccus) (26%, 25%), Fusobacterium necrophorum biovar B (22%, 12%), Bacteroides melaninogenicus (20%, 9%) and Streptococcus dysgalactiae (21%, 5%). All except six cases occurred in non-lactating animals or within three weeks after parturition. The majority of animals (about 90%) had only one quarter affected and no differences in quarter distribution were observed between the two groups.     

Antimicrobial resistance patterns to beta-lactams of gram-positive cocci isolated from bovine mastitis in Lithuania

The aim of the study was to isolate gram-positive cocci from cows with mastitis and to determine their resistance to beta-lactamic antibiotics. Eight hundred and nine strains were isolated and identified as staphylococci (n=516), streptococci (n=199) and enterococci (n=94) from sub-clinical and clinical cases of bovine mastitis in Lithuania. The most common causative agents of udder disease included: S. epidermidis (n=176), S. aureus (n=176), S. agalactiae (n=134), S. hyicus (136) and E. hirae (n=68). Isolates were analysed for antimicrobial resistance to penicillin, ampicillin, amoxicillin, cephalothin, cephalexin, amoxicillin + clavulanate. The susceptibility patterns were analysed using the agar disk diffusion method. S. aureus showed the highest level of resistance to amoxicillin (81.3%), penicillin (76.7%) and ampicillin (78.4%). The corresponding values for CNS strains were 59.7%, 59.7% and 50.6% against penicillin, ampicillin and amoxicillin respectively. Streptococci were the most frequently resistant to amoxicillin (29.3%), and enterococci to penicillin (27%), amoxicillin (27.5%) and amoxicillin/clavulanic acid (23.8%). The resistance of all tested mastitis pathogens to aminopenicillins and penicillin highly correlated (r=0.83). Compared with other antibiotics, amoxicillin and clavulanic acid combination tended to be more effective (p<0.05) against all tested bacteria in vitro. However, S. aureus, in 38.1% of cases, was resistant to this combination of antimicrobials. This study demonstrates that S. epidermidis, S. aureus, S. hyicus, S. agalactiae and E. hirae remain the most frequent mastitis causative agents on Lithuanian cattle farms. The highest resistance in vitro to penicillins was demonstrated by S. aureus, S. hyicus and S. intermedius. Resistance to cephalosporins remains low, irrespective of bacterial species of gram-positive cocci.     

IgG antibody response to Corynebacterium pyogenes,Peptococcus indolicus and microaerophilic cocci in natural and experimental mastitis

A mixed inoculum of C. pyogenes, P. indolicus and a microaerophilic coccus was used to induce experimental mastitis in two heifers. During the course of the infection, the IgG titres against these bacteria were monitored by the indirect immunofluorescent antibody technique. At the same time, the course of the infection was studied bacteriologically and by determination of clinical-chemical parameters. In both heifers, high titres of IgG against C. pyogenes, P. indolicus and the microaerophilic coccus were detected. The reactivity of the IgG towards surface-associated antigens of these bacteria seemed to reside mainly in the IgG2 subclass. Sera obtained from Denmark from heifers with experimental mastitis, which had been induced similarly, were also found to have significantly raised IgG titres against C. pyogenes and P. indolicus. Sera from naturally infected heifers and dry cows, from whose udder secretions C. pyogenes and P. indolicus had been isolated, were examined comparatively with sera from healthy heifers and dry cows, respectively, for the presence of IgG against these two bacteria. Statistically significant differences in IgG titres against both bacteria were demonstrated between pregnant heifers with mastitis and healthy pregnant heifers and between dry cows with mastitis and clinically healthy controls.     

The Corynebacterium pyogenes infection of cattle. 2. Tenacity of Corynebacterium pyogenes]

Some common agents were tested for their effectiveness against Corynebacterium pyogenes. The pathogen proved most susceptable to Wofasteril. All germs were killed within ten minutes by a 0.005% solution. Equally good action was recorded from all the other tested agents as well (lactic acid, Lugol's solution, formalin, cupric sulphate, alcohol, and aethacridine. Other studies were conducted with the view to testing the survival capacity of Corynebacterium pyogenes in different media and storage conditions. The pathogen survived three months in routine media and mastitis secretion at room temperature. Regrowth of 38 in 50 strains took place after nine months of refrigerator storage in slanting blood agar tubes with paraffin plugs. Germs sampled from mastitis secretion and stored in a refrigerator were cultivable even after one year had elapsed. The detectability rate of Corynebacterium pyogenes did not change over months by storage of wound infection material at 12 degrees C below zero. The pathogen remained detectable five days from artificial contamination of cattle skin.     

Antimicrobial susceptibility of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows

The high prevalence of the great resistance to antimicrobials shown by the aetiological agents of infectious bovine mastitis, often leads to chronic recurrent mastitis, one of the main causes of early culling of dairy cows. The purpose of this study was to determine the in vitro susceptibility pattern of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows, to different anti-microbials. A total of 45 Staphylococcus spp. strains [33 coagulase-negative Staphylococcus (CNS) and 12 Staphylococcus aureus (SA)] were used. These strains were tested with 12 different antimicrobials by the Kirby and Bauer standardized disc diffusion method; 84.44% and 86.66% of the 45 strains were resistant to ampicillin and penicillin, respectively. The highest sensitivity was to cephalothin (84.44%), gentamicin (80%) and to sulphazotrin (77.77%). CNS showed higher resistance (P < 0.05) than SA. The in vitro susceptibility pattern of Staphylococcus spp. strains isolated from mammary parenchymas studied in the present investigation was similar to that observed in recent studies of in vitro and in vivo susceptibility patterns of Staphylococcus spp. isolated from cases of bovine mastitis.     

The role of Hydrotaea irritans in the transmission of summer mastitis

A series of experiments to investigate the possible role of the sheep head fly, Hydrotaea irritans, in the transmission of summer mastitis is reported. These showed that the pathogens Actinomyces pyogenes and Peptococcus indolicus may be harboured in the gut of the fly for up to 96 h following a contaminated meal; A. pyogenes survived on the surface of the fly for a similar period. H. irritans fed on the pathogens can contaminate a subsequent meal by regurgitation. However, no infections resulted from repeated exposure of the teats of heifers or cows to H. irritans fed on blood containing A. pyogenes and P. indolicus or on pus from a case of summer mastitis. In these experiments the frequency of feeding of the flies on the teat was high but subsequent bacterial contamination of the teat skin was low. Artificial contamination of the teat skin with greater than 10(6) cfu A. pyogenes led to a low incidence of mastitis even in the absence of cutaneous damage. Repeated applications of lower doses (less than 10(4) cfu) did not produce infection.     

Heifer mastitis and dry-cow mastitis: A bacteriological survey in Sweden

A bacteriological investigation was performed on 145 heifers and 55 dry-cows with clinical mastitis during July 1976–June 1977. Areas in Sweden from which heifer and dry-cow mastitis has been reported as a problem were included. Mastitis occurred more frequently in the Swedish Fresian breed (SLB) than in the Swedish Red and White breed (SRB) of dairy cattle. C. pyogenes was isolated in pure culture from 15% and 4%, respectively, of the cases of heifer and dry-cow mastitis from which this bacterium could be isolated. Corresponding figures for pure culture of P. indolicus, micro-aerophilic cocci and S. dysgalactiae were 2% and 0%, 0% and 0% and 44% and 35%, respectively. The bacteria most frequently isolated together with C. pyogenes in the heifer material were P. indolicus (69%), the microaerophilic cocci (59%) and S. dysgalactiae (31%). Corresponding data for the dry-cow material were 92%, 35% and 50%, respectively. Thus, C. pyogenes was the causative agent of heifer and dry-cow mastitis in only about 40% of the mastitis cases investigated, and the most characteristic feature of these forms of mastitis involving C. pyogenes was mixed infection.     

奶牛乳房炎相关链球菌PCR检测方法的建立

利用编码3-磷酸甘油醛脱氢酶的gapC基因具有高度特异性的特点,建立PCR方法鉴定与奶牛乳房炎相关的链球菌。根据已有gapC基因序列设计1对引物,以分离自患乳房炎奶牛乳样的10株革兰阳性球菌、10株革兰阳性杆菌、10株革兰阴性杆菌作为待检菌株,进行PCR扩增。结果表明,在10株革兰阳性球菌中,有8株球菌可以扩增出约1011bp的目的条带,而其他2株革兰阳性球菌及20株杆菌均无相应PCR产物出现。通过传统的生化鉴定与16S rDNA序列分析相结合证实,能扩出gapC基因的8株革兰阳性球菌分别为乳房链球菌(Streptococcus uberis)、牛链球菌(S.bovis)与猪链球菌(S.suis)。说明基于gapC基因的PCR方法,用于鉴定奶牛乳房炎相关链球菌具有较强的特异性。     

Staphylococcal infections in rabbit does on two industrial farms

The main reasons for culling adult rabbit does on two Spanish rabbit farms were investigated for a year. The most important conditions were mastitis (33.3 per cent), followed by subcutaneous abscesses (9.9 per cent) and pyometra (8.7 per cent). Staphylococcus aureus infections were the most severe problem, the organism being isolated from 69.2 per cent of infected animals. Pasteurella species were more prevalent in cases of pyometra and pneumonia. Two strains of S aureus were identified by using polymorphism of the coagulase gene as the criterion. One of these strains was responsible for the majority of the staphylococcal infections and was isolated from several pathological processes.     

The detection of obligate anaerobic bacteria in udder secretions of dry cattle with mastitis during summer: a comparison between gas-liquid chromatography and bacteriological culturing methods

Udder secretions sampled during the summer in 1984 and 1985 from mastitic quarters of 51 non-lactating cattle, mainly heifers less than 2 years of age, were examined bacteriologically for the presence of (facultative) aerobic and obligate anaerobic bacteria (OAB) and by gas-liquid chromatography (GLC) in order to detect volatile fatty acids (VFA), metabolic end-products of OAB. Forty-nine samples yielded positive cultures and in 20 cases these were mixtures of (facultative) aerobes and OAB. Only two specimens appeared to be sterile and from one specimen only were OAB cultured. Corynebacterium pyogenes was isolated from 35% of the cases and Peptococcus indolicus and Fusobacterium necrophorum from 31 and 22%, respectively. In most specimens (19/21) which yielded OAB after culturing, VFA (C3-C6) could be detected by GLC. Detection of VFA in summer mastitis secretions appeared to be a useful technique to evaluate the importance and association of OAB with summer mastitis. Because samples can be easily collected and stored at -20 degrees C, this is especially advantageous in situations where adequate facilities for the isolation of OAB are not readily available.     

Isolation of Mycoplasma salivarium from swine

Mycoplasma salivarium, a common human oropharyngeal mycoplasma, was isolated from nasal and pharyngeal secretions of 14 of 284 swine in a barrier-maintained, disease-free herd. M. salivarium was recovered from one boar 6 times over a 26-month period and one time only from 13 other swine. Human isolates of M. salivarium were compared with the swine isolates by DNA-DNA hybridization and SDS-PAGE of the cell proteins and the strains were shown to be closely related. One of eight of the swine from which M. salivarium was isolated had complement-fixing antibodies and another culture-positive animal had metabolic-inhibiting antibodies to M. salivarium. Overt disease was not associated with the organism. These results support previous findings that mycoplasmas closely related to M. salivarium may be isolated from the nasopharynges of swine and they further indicate that the organism can establish persistently in swine without evidence of overt disease.     

Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences

Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.     

Acute phase proteins in milk in naturally acquired bovine mastitis caused by different pathogens

The concentrations of haptoglobin (Hp) and serum amyloid A (SAA) and the activity of N-acetyl-β-D-glucosaminidase (NAGase) in milk from 234 cows with spontaneous mastitis caused by different pathogens were measured to assess whether they corresponded with the clinical signs of mastitis and whether there were any differences between pathogens. Ninety-eight of the cows had clinical mastitis and 136 had subclinical mastitis. There were statistically significant positive correlations between the concentrations of SAA and Hp and the activity of NAGase. Significant differences in the concentrations of acute phase proteins and NAGase activity were found in milk from cows with mastitis caused by different pathogens. The highest concentrations of Hp and NAGase were found in cases of mastitis caused by Escherichia coli and Arcanobacterium pyogenes, and the lowest concentrations were from cases of mastitis caused by coagulase-negative staphylococci. Very low SAA concentrations were found in milk from the cases caused by A pyogenes, in contrast to cases caused by other major mastitis pathogens. The median concentration of SAA was over 10 times higher in cases of mastitis caused by E coli than in mastitis caused by other pathogens. There were significant differences in the mean Hp concentration and NAGase activity between clinical and subclinical mastitis. In approximately one-third of the samples, the Hp concentration was below the detection limit, potentially compromising the use of Hp as a mastitis marker.     

牛源化脓隐秘杆菌PLO蛋白的原核截短表达及其溶血活性检测

为研究牛源的化脓隐秘杆菌溶血素(PLO)生物学功能,本研究应用PCR方法扩增牛源化脓隐秘杆菌(A.pyogenes)PLO全基因序列,通过生物信息学方法分析其与猪源A.pyogenes的PLO蛋白差异,并构建了溶血功能区重组表达质粒pQE30-PLO585,在E.coli XL1Blue中用IPTG诱导表达。结果表明,扩增到PLO蛋白基因ORF为1 605 bp,编码535个氨基酸,与猪源PLO的核苷酸序列同源性为97.4%,氨基酸同源性为97.2%,在生物学活性功能区没有发生改变。表达的PLO蛋白溶血功能区重组蛋白能够被阳性血清识别,而且具有溶解绵羊红细胞的活性,产生β溶血现象。本研究获得了牛源A.pyogenes截短重组PLO蛋白,并证明PLO具有β溶血功能与较好的抗原性。     

Analysis of Clinical Mastitis Pathogenic Bacterial Species in Three Dairy Farms of Different Areas

In order to control mastitis,clinical mastitis pathogenic bacterial species of three dairy farms in different areas were isolated and identified.The types and infection condition of pathogenic bacteria,environmental conditions and management methods of the farms were analyzed.61 clinical mastitis milk samples from three different large-scale dairy farms in Fujian,Xinjiang,and Chongqing were collected for bacterial isolation and identification using traditional microbial identification methods and PCR technology.Among clinical mastitis milk samples collected in this trial,single infection sample was 44 (72.13%)and mixed infections sample was 15 (24.59%).80 bacteria were isolated,mainly E.coli,Staphylococcus and Streptococcus,including 13 strains of infectious microorganisms (16.25%),46 strains of environmental microorganisms (57.50%),11 strains of chance of microorganisms (13.75%),10 strains of other microorganisms (12.50%).Clinical mastitis in three dairy farms were still given priority to single infection,and mixed infection occupies certain proportion,with infectious pathogens and environmental pathogens as the main pathogenic.In the actual production the dairy cow mastitis infection and pathogen species could be carried out through the isolation and identification of mastitis pathogens in large-scale dairy farms.It was helpful to identify the herd management problems and make improvements to better control the occurrence of mastitis combining with the actual production conditions.     

Corynebacterium pyrogenes; a biochemical and serological study

The study comprises 136 strains of Corynebacterium pyogenes originating from cattle (105), swine (20), sheep (1), and insects (10). For comparison 2 strains of human origin and 1 strain of Gorynebacterium hemolyticum were examined.One of the bovine strains was atypical, being gelatinase-negative, otherwise the strains of Cb. pyogenes were found to be biochemically identical apart from minor deviations in fermentation patterns (Table 1). Neither were antigenic differences demonstrated (gel diffusion analyses, Figs. 1 and 2).Both of the human strains agreed biochemically with Cb. pyogenes (Table 1). By gel diffusion cross analyses one of them was found to be identical with Cb. pyogenes, the other not, though anti-genically related to it (Fig. 2).Gb. hemolyticum deviated biochemically as well as serologically from Gb. pyogenes, but the 2 organisms shared antigenic determinants (Fig. 3).     

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.