Studies on Streptococcus agalactiae isolated from bovine mastitis in Indonesia

All 83 bacterial strains isolated from seven farms in three areas of the island of Java in Indonesia investigated in the present study could be identified as Streptococcus agalactiae. Identification was performed by cultural, biochemical and serological properties and by polymerase chain reaction amplification of species-specific parts of the gene encoding the 16S rRNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor (cfb) gene. All isolates were unpigmented. almost all of the isolates had the serotype pattern II/X. Despite these similarities a macrorestriction analysis of the chromosomal DNA of the bacteria revealed no significant homologies of the DNA-fingerprints of the S. agalactiae from the various areas. This last finding might possibly indicate that a single ancestral unpigmented serotype II/X S. agalactiae clone was responsible for the mastitis situation on Java and had evolved separately in the various farms and regions.     

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A–D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B respectively, but an unrelated restriction pattern for S. uberis ST-474 and ST-475 isolates from herds D and C respectively, were obtained. This signifies that the isolates of particular ST may exhibit related PFGE patterns suggesting detection of a faster molecular clock by PFGE than MLST. Since all the isolates of both the species belonged to novel sequence types, their epidemiological significance in global context could not be ascertained, however, evidence suggests that they have uniquely evolved in Indian conditions. Further research would be useful for understanding the role of these pathogens in bovine sub-clinical mastitis and implementing effective control strategies in India.     

Identification and characterization of Streptococcus agalactiae isolated from horses.

Seven group B streptococcal cultures isolated from three horses reacted with group B-specific antiserum, were CAMP positive, pigmented and showed the typical biochemical properties of Streptococcus agalactiae. The identification could be confirmed by PCR amplification of the 16S rRNA gene and a subsequent RsaI restriction pattern typical for S. agalactiae. In addition, the isolates were identified by amplification of species specific parts of the 16S rRNA gene, the 16S-23S rRNA intergenic spacer region and by amplification of the CAMP-factor (cfb) gene. Six isolates could be classified as serotype III/Rib, one isolate as serotype Ia/cbeta. The occurrence of the protein antigens Rib and cbeta could be confirmed by PCR amplification of the respective genes. The six isolates of serotype III/Rib were hyaluronidase negative, had a hylB gene with a size of 4.6 kb and an insertion element IS1548 of 0.98 kb. The isolate of serotype Ia/cbeta was hyaluronidase positive, had a hylB gene with a size of 3.3 kb and no insertion element IS1548. In addition, all seven isolates had the insertion element ISSag2 and the gene lmb encoding the laminin binding surface protein Lmb and the gene scpB encoding C5a peptidase. According to the present results the group B streptococci isolated from horses showed characteristics of human isolates of this species.     

奶牛乳房炎无乳链球菌的分离与鉴定

利用THB固体培养基和色素培养基初步筛选出奶牛乳房炎中无乳链球菌,以分离的12株疑似菌的基因组DNA为模板进行PCR扩增,对扩增产物进行分析,结合选择培养的生理生化特性对分离菌进行鉴定。结果表明,12株疑似菌中有8株为无乳链球菌.     

我国奶牛乳房炎无乳链球菌抗生素耐药性研究

为了查明我国奶牛乳房炎无乳链球菌对抗生素耐药情况,指导临床合理用药,从我国部分地区奶牛场采集的临床型奶牛乳房炎病乳中分离鉴定出无乳链球菌115株,采用K-B纸片法测定了这些菌株对抗生素的耐药情况。结果表明,无乳链球菌对目前临床上使用的大部分抗生素,如头孢唑啉、头孢噻肟、丁胺卡那霉素、卡那霉素、庆大霉素、四环素、强力霉素、麦迪霉素、林可霉素、氟苯尼考、多黏菌素B、环丙沙星、氟哌酸、氨苄青霉素/舒巴坦、头孢噻肟/棒酸和头孢他啶/棒酸均比较敏感;但对青霉素G、氨苄青霉素、链霉素、恩诺沙星、阿莫西林/棒酸和复方新诺明等有一定耐药性,其耐药率达50%～100%。     

Anti-microbial susceptibility of Streptococcus spp. isolated from bovine mastitis in Argentina

The in vitro susceptibility to penicillin G, erythromycin and clindamycin was determined by the disc diffusion test and by E-test for a total of 47 streptococcal strains (three Streptococcus uberis, 36 Streptococcus agalactiae, eight Streptococcus dysgalactiae spp. dysgalactiae) isolated from bovine intramammary infections in Argentina. Moreover, resistance phenotypes of erythromycin-resistant streptococcal isolates was characterized. MIC90 of penicillin G, erythromycin and clindamycin for S. agalactiae were 0.75, 8.0 and 12.0 microg/ml respectively. Resistance to erythromycin and clindamycin was detected in 13 (27.6%) and 12 (25.5%) isolates respectively. No isolate was resistant to penicillin G. Resistance against macrolides, lincosamides and streptogramin B (MLS(B)) represented by the constitutive MLS(B) phenotype was present in 11 (23.4%) erythromycin-resistant isolates and two isolates (4.3%) expressed the M phenotype. The inducible MLS(B) phenotype was not identified. Results suggest that beta-lactams are the first-line antibiotics when treating streptococcal udder infections; however, the continuous monitoring of the antibiotic resistance is essential, as the emergence of resistant strains has become a growing concern on the therapy of bovine mastitis.     

Streptococcus agalactiae mastitis: a review.

Streptococcus agalactiae continues to be a major cause of subclinical mastitis in dairy cattle and a source of economic loss for the industry. Veterinarians are often asked to provide information on herd level control and eradication of S. agalactiae mastitis. This review collects and collates relevant publications on the subject. The literature search was conducted in 1993 on the Agricola database. Articles related to S. agalactiae epidemiology, pathogen identification techniques, milk quality consequences, and control, prevention, and therapy were included. Streptococcus agalactiae is an oblique parasite of the bovine mammary gland and is susceptible to treatment with a variety of antibiotics. Despite this fact, where state or provincial census data are available, herd prevalence levels range from 11% (Alberta, 1991) to 47% (Vermont, 1985). Infection with S. agalactiae is associated with elevated somatic cell count and total bacteria count and a decrease in the quantity and quality of milk products produced. Bulk tank milk culture has, using traditional milk culture techniques, had a low sensitivity for identifying S. agalactiae at the herd level. New culture methods, using selective media and large inocula, have substantially improved the sensitivity of bulk tank culture. Efficacy of therapy on individual cows remains high. Protocols for therapy of all infected animals in a herd are generally successful in eradicating the pathogen from the herd, especially if they are followed up with good udder hygiene techniques.     

Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea

One hundred and sixty-six Staphylococcus aureus isolates from mastitic milk samples from different cows on 26 farms were investigated for staphylococcal enterotoxins(SEs) and toxic shock syndrome toxin-1(TSST-1) by polymerase chain reaction(PCR) and reverse passive latex agglutination assay(RPLA). SEs and the TSST-1 gene were detected in thirty-seven isolates based on a multiplex PCR; SEA was detected in 32 isolates, SEB in 3 isolates, SEC in 1 isolate, and SEA and the TSST-1 gene in 1 isolate. Of the 37 enterotoxigenic isolates, thirty-three isolates were enterotoxigenic according to RPLA, where 29 isolates produced SEA, 3 isolates produced SEB, and 1 isolate produced SEC. The enterotoxin-producing S. aureus isolates were further characterized by pulsed-field gel electrophoresis(PFGE). A macrorestriction analysis revealed 11 PFGE patterns. Among the 33 enterotoxigenic S. aureus isolates, 45.4% exhibited the same PFGE pattern I. Accordingly, although the enterotoxin-producing S. aureus isolates from bovine mastitis were genetically diverse, 1 common genotype prevailed on the farms, indicating that PFGE pattern I isolates may be the most disseminated in Korea.     

Molecular detection of Streptococcus agalactiae in bovine raw milk samples obtained directly from bulk tanks

Mastitis is the most common infectious disease affecting dairy cattle; in addition, it remains the most economically important disease of dairy industries around the world. Streptococcus agalactiae, a contagious pathogen associated with subclinical mastitis, is highly infectious. This bacterium can cause an increase in bulk tank bacterial counts (BTBC) and bulk tank somatic cell counts (BTSCC). The microbiological identification of S. agalactiae in samples from bulk tanks is an auxiliary method to control contagious mastitis. Thus, there are some limitations for time-consuming cultures or identification methods and additional concerns about the conservation and transport of samples. Bulk tank samples from 247 dairy farms were cultured and compared through polymerase chain reaction (PCR), directed to 16S rRNA genes of S. agalactiae, followed by BTBC and S. agalactiae isolation. The mean value of BTBC was 1.08×10(6) CFU mL(-1) and the bacterium was identified through the microbiological method in 98 (39.7%; CI(95%)=33.8-45.9%) and through PCR in 110 (44.5%; CI(95%)=38.5-50.8%) samples. Results indicated sensitivity of 0.8571±0.0353 (CI(95%)=0.7719-0.9196) and specificity of 0.8255±0.0311 (CI(95%)=0.7549-0.8827). The lack of significant difference between microbiological and molecular results (κ=0.6686±0.0477 and CI(95%)=0.5752-0.7620) indicated substantial agreement between the methods. This suggests that PCR can be used for bulk tank samples to detect contagious mastitis caused by S. agalactiae.     

The kinetics of inflammation and phagocytosis during bovine mastitis induced by Streptococcus agalactiae bearing the protein X

The protein X of Streptococcus agalactiae is a surface antigen borne by a high proportion of strains isolated from bovine mastitis. We have tested the capacity of two strains of X-bearing Streptococcus agalactiae to induce mastitis in dairy cows. The reference X-strain (411.07) produced an intramammary infection with local clinical signs in the three inoculated quarters. Another X-bearing strain (443.31) of bovine origin produced infection in all 11 quarters inoculated with only 25 or 85 colony-forming units. In naive cows, strain 433.31 induced less exudation of plasma into the milk, shedding of bacteria, macroscopic alteration, and a lower somatic cell count (SCC) than did the reference strain. Only one quarter spontaneously eliminated the infection before antibiotic treatment 9 days after inoculation.The serum of all the cows contained naturally acquired or induced antibodies to the challenge strain (443.31) and possessed opsonic activity. Before inflammation occurred, the milk was almost devoid of antibody or opsonic activities. The early phase of infection was characterized by rapid multiplication of streptococci in the milk, followed by a sharp drop in bacterial counts concomitant with the onset of inflammation.Three cows immunized with protein X displayed higher SCC and bactericidal activity in milk from the inoculated quarter at the onset of inflammation than non-immunized cows. Two of the three immunized cows underwent an early and transient febrile episode and eliminated the infection.     

Herd benefit-to-cost ratio and effects of a bovine mastitis control program that includes blitz treatment of Streptococcus agalactiae

Twelve dairy herds that had participated in the Pennsylvania Dairy Herd Improvement Association (DHIA) program for at least 12 months, that had a 12-month mean DHIA somatic cell count greater than 700,000 cells/ml, and that had greater than 25% of lactating cows infected with Streptococcus agalactiae participated in a herd blitz treatment program. Initially, quarter milk samples for bacteriologic culturing were collected from all lactating cows. Subsequently, all cows identified as infected with Str agalactiae were treated, using a commercial penicillin-novobiocin intramammary infusion product. In addition, a herd mastitis management program of postmilking teat dipping and treatment of all cows at the start of the nonlactating period was instituted. Thirty days after the initial herd visit, samples from all lactating cows were again cultured, and cows infected at that time were treated. Twelve months after the initial herd visit, samples from all lactating cows were again cultured. Mean prevalence of infection with Str agalactiae decreased (P less than 0.05) from 23.0% of quarters and 41.6% of cows initially to 3.4% of quarters and 9.3% of cows at 30 days and 1.6% of quarters and 4.2% of cows at 1 year. Mean herd DHIA somatic cell count decreased (P less than 0.05) from 918,000 cells/ml initially to 439,000 cells/ml at 30 days and 268,000 cells/ml at 1 year.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genotyping of Staphylococcus aureus isolated from bovine mastitis.

The present study was designed to comparatively investigate 25 Staphylococcus aureus strains isolated from bovine subclinical mastitis. The S. aureus strains, obtained from six different farms at five locations in one region of Germany, were characterized by phenotypic and genotypic methods. The S. aureus could be identified and further characterized by their cultural, biochemical and hemolytic properties. To analyze the epidemiological relationship the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNA, by PCR amplification of the gene encoding the 16S-23S rRNA intergenic spacer, by PCR amplification of the gene encoding the IgG binding region and the X region of protein A and by amplifying, and subsequent, digestion of the gene encoding staphylococcal coagulase. The macrorestriction analysis revealed five DNA restriction patterns with DNA patterns I, III and IV occurring in three, four, and three different farms, respectively. In addition, clones with different DNA patterns could be found within one herd. The PCR products for the spacer DNA, the spa gene encoding the X region of protein A and the coa gene encoding coagulase corresponded mostly to the pattern observed by DNA fingerprinting. Amplification of the gene encoding the IgG binding region revealed sizes of 620 bp for 20 of the isolates and 280 bp for four isolates indicating, for the latter, a deletion of segments in this region. These findings show, that single, widely distributed clones seemed to be responsible for cases of bovine subclinical mastitis found in one region of Germany.     

Changes in immunoglobulin levels in whey during experimental Streptococcus agalactiae mastitis

Total protein and immunoglobulin levels in the wheys of eight first lactation heifers, four vaccinated and four unvaccinated, were measured during three consecutive experimental intramammary infections with Streptococcus agalactiae. There were no significant differences between infections 1, 2 and 3 in the protein or immunoglobulin content of the uninfected quarters. Peak whey total protein of the infected quarters came earlier with each infection, until by the third they were seen after eight hours. During this acute phase a reversal of the normal milk IgG1/IgG2 ratio in all infected quarters was measured. Increases in whey IgA and IgM in the infected quarters of the vaccinates were also noted. A similar response only occurred following the third infection of the unvaccinated animals. All whey immunoglobulin levels returned to normal by 48 hours after infection, after which only IgG1 levels increased in infected quarters.     

屠宰场猪链球菌分离菌株分型及其致病性和耐药性分析

为了解吉林省猪链球菌的流行情况,从屠宰场采集的猪咽拭子和鼻拭子样品中分离鉴定猪链球菌,并进行菌株血清型、基因型和毒力表型的鉴定,以及致病性和耐药性的分析。结果表明,从100份样品中共分离鉴定猪链球菌104株,其中29株鉴定为血清2型、9型和1型等几种常见的致病性血清型,其他75株不属于常见的致病性血清型。血清2型的菌株中,2株经鉴定为ST1基因型和mrp+epf+sly+毒力型,并经动物试验鉴定为强毒菌株;其他菌株均为ST28基因型和mrp+epf-sly-毒力型,具有中等毒力。血清9型的菌株,均为mrp-epf-sly-毒力型,动物试验鉴定均为强毒菌株。根据Kirby-Bauer纸片扩散法的药敏试验结果,98%的猪链球菌分离株对四环素耐药;对大环内酯类、克林霉素和链霉素的耐药率都在50%以上;对β-内酰胺类、氯霉素和喹诺酮类抗生素的耐药率小于20%。总体分析,从屠宰场分离的猪链球菌强毒菌株所占的比例并不高,但是菌株多重耐药的情况非常严重。     

Sequestration of N-acetyl-beta-D-glucosaminidase in somatic cells during experimental bovine mastitis induced by endotoxin, Staphylococcus aureus or Streptococcus agalactiae

Experimental mastitis was induced in cows by intramammary infusion of endotoxin, Staphylococcus aureus or Streptococcus agalactiae. The inflammatory response was monitored by somatic cell counting and N-acetyl-beta-D-glucosaminidase (NAGase). NAGase activity was analysed in fresh milk samples in parallel with samples treated by a cycle of freezing and thawing combined with detergent treatment to release the cell-bound NAGase. Before the udder reacted by inflammation, the total NAGase activity consisted of free extracellular activity. Later on when the inflammation was established, much of the milk NAGase remained sequestered intracellularly. S agalactiae was linked with a high degree of cellular NAGase sequestration indicating a blockage of the lysosomal release function from the phagocytes. S aureus delayed the inflammatory response.     

Recovery of Streptococcus agalactiae and Staphylococcus aureus from fresh and frozen bovine milk

Three hundred seventy-three milk samples were screened for Streptococcus agalactiae and Staphylococcus aureus. After sample storage at -20 C for 23 days, the frequency of Str agalactiae isolation increased 2.50 times. The frequency of S aureus isolation increased 1.48 times in the same interval. Increases in the proportion of these isolates were highly significant (P = 0.000006 and 0.0001, respectively). Results of the study indicate that optimal procedures for microbiological testing for these mastitis pathogens may include preculture freezing. The magnitude of the increase in the proportion of isolates indicates the existence of an important population of infected cattle shedding bacteria at concentrations not detected by use of standard microbiological techniques.