Characterization of Streptococcus uberis with special consideration of supposed pathogenicity factors]

Streptococcus uberis, as one of the principal causes of bovine streptococcal mastitis, has been characterized serologically and biochemically. Serological grouping of S. uberis revealed polysaccharide antigens of groups E, G, P and U. The biochemical properties of S. uberis, determined with the Strep-Zym identification system, differed clearly from those of S. agalactiae and S. dysgalactiae. Some cultures of S. uberis produced the enzymes hyaluronidase and neuraminidase. In addition S. uberis partly demonstrated CAMP-like synergistic hemolytic activities on sheep blood agar, reacted specifically with the lectins from Helix pomatia and Dolichos biflorus and produced bacteriocin-like inhibitors. This reactions, possibly of importance as virulence factors, as well as "DNA-fingerprinting" of S. uberis, might serve as individual markers of the respective cultures in epidemiological studies.     

Streptococci from the bovine udder in dairy herds in Khartoum Province, Sudan

Five hundred and seventy-nine milk samples were collected from dairy cows on seven farms in Khartoum North area and one farm in Omdurman and examined by bacteriological cultures for the presence of streptococci. One hundred and ninety-three (33.33%) isolates were recovered and identified on the basis of bacteriological characteristics and biochemical reactions as: S. pyogenes, S. agalactiae, S. dysgalactiae, S. faecalis, S. faecium, S. bovis, S. equi, S. lactis and S. uberis. Fifty-seven isolates representing the preliminary identification were tested by the latex-agglutination test to determine the serological groups. It was found that 39 strains belonged to group B, 3 strains to group C. Four strains gave a weak reaction with group D sera and were identified by biochemical tests as S. uberis. Two isolates could not be identified by the available sera. The isolation of S. uberis, S. bovis, S. equi, S. lactis, S. faecalis, S. faecium and S. pyogenes from cows in the Sudan was reported for the first time.     

Polymerase chain reaction mediated identification of Streptococcus uberis and Streptococcus parauberis using species-specific sequences of the genes encoding superoxide dismutase A and chaperonin 60*

Streptococcus uberis, a well-known bacterial pathogen associated with bovine mastitis, appears to be biochemically and serologically almost indistinguishable from the closely related species Streptococcus parauberis. In the present study, species-specific oligonucleotide primers were designed using internal parts of the genes sodA, encoding superoxide dismutase A, and cpn60 encoding chaperonin 60 of S. uberis and S. parauberis, respectively. The two oligonucleotide primer pairs allowed a rapid and reliable PCR-mediated identification and differentiation of both species. These studies, performed with S. uberis and S. parauberis reference cultures and clinical isolates from routine diagnostics, revealed that the occurrence of S. parauberis as causative agent of bovine mastitis appears to be rare. In addition the sodA and cpn60 sequence data confirmed that both species could taxonomically be classified to the pyogenic group of genus Streptococcus.     

Rapid identification of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes by fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) has been reported to be an easy and rapid identification method for many human pathogens, but applications for common veterinary pathogens are lacking. Gene probes for FISH of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes were designed to provide probes for a specific identification of these bacteria from cultures. Specific FISH probes for these species have so far not been published. Both probes recognized all isolates of the target species correctly. With the S. uberis probe SUB 196 no false-positive results were obtained for reference strains as well as for clinical isolates. Probe APYO 183 for A. pyogenes produced false-positive reactions with so far rarely described Arcanobacterium species from animals at standard hybridization conditions. In order to avoid any incorrect classifications of microorganisms as A. pyogenes, two non-labelled competitor probes were designed and successfully evaluated.     

Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis

The antibacterial properties of bacteriophage lytic enzymes may be of importance in future mastitis control programs. A prophage was isolated from a strain of Streptococcus uberis (ATCC 700407) following exposure to mitomycin C. Partial sequencing of the phage DNA revealed a putative lysin based on sequence similarity to other streptococcal phage lysins. The putative lysin (Ply700) was recombinantly expressed in Escherichia coli, and chromatographically purified. Addition of the purified Ply700 to bacterial suspensions of S. uberis, Streptococcus pyogenes, and Streptococcus dysgalactiae caused a rapid, calcium-dependent lysis while there was little activity against Streptococcus agalactiae, Staphylococcus aureus, or E. coli. Killing of S. uberis in milk by Ply700 (50 microg/ml) was confirmed by plate count assay. Activity was related to the initial concentration of bacteria in that 31% killing (P<0.05) was observed with an inoculating dose of approximately 4500 cfu/ml, while 81% killing (P<0.01) was observed when the inoculum was reduced to approximately 600 cfu/ml. In contrast, complete sterilization was observed in parallel cultures suspended in assay buffer indicating that factors in milk are able to neutralize the lysin. Functional characterization of the C-terminal domain, as a component of a GFP fusion protein, revealed its calcium-dependent ability to bind to S. uberis. The C-terminal domain may have utility in targeting S. uberis while it remains to be determined if the lysin by itself has sufficient potency in milk for effective use in the control of S. uberis mastitis.     

Role of collagen in adherence of Streptococcus uberis to bovine mammary epithelial cells

We reported previously that pre-incubation of Streptococcus uberis with collagen induced expression of S. uberis surface proteins. In a subsequent study, we showed that incubation of S. uberis with extracellular matrix proteins, particularly collagen, increased adherence and internalization of S. uberis to mammary epithelial cells. In the present report, the potential mechanism by which S. uberis exploits the presence of collagen to enhance adherence to bovine mammary epithelial cells was evaluated. Adherence assays were conducted with S. uberis pre-treated with and without collagen and co-cultured in medium supplemented with or without collagen. Pre-incubation with collagen followed by co-culture in medium containing collagen up-regulated ligands that enhanced adherence of S. uberis to mammary epithelial cells. Collagen-up-regulated ligand(s) also increased adherence of S. uberis to mammary epithelial cells in the absence of collagen, but adherence was lower than when collagen was present during the adherence assay. Chloramphenicol was added to the culture medium to inhibit bacterial protein synthesis. Adherence decreased significantly in chloramphenicol-treated S. uberis pre-treated or co-cultured in the presence of collagen. These results suggest that S. uberis expresses ligands with affinity for collagen that are up-regulated by collagen. We hypothesize that these ligands increase adherence by using collagen as a bridge between the bacterium and host cell and/or by direct interaction with host cell receptor(s).     

The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.     

Molecular characterization of the antimicrobial resistance of Riemerella anatipestifer isolated from ducks

Streptococcus uberis is a major environmental mastitis-causing pathogen. The infections are predominantly subclinical and are frequently undetected and untreated for extended periods of time. More information about the pathogenesis of S. uberis mastitis would be useful. To our knowledge, no experimental studies into the mastitis pathogenesis caused by S. uberis have been described in lactating goats. The aim of this study was to reproduce an experimentally induced S. uberis subclinical mastitis in lactating goats aimed to evaluate the inflammatory response, dynamics of infection and the pathological findings within the first hours of intramammary inoculation with S. uberis. Six Saanen goats in mid-lactation were inoculated with 1.7 × 10(8)cfu of S. uberis. Bacterial growth peaked in milk from challenged right mammary halves (RMH) at 4h PI. Shedding of viable bacteria showed a marked decrease at 20 h PI. Mean somatic cell counts in milk from the RMH peaked at 20 h PI. Inoculation with S. uberis was followed by a decrease in the mean total number of leukocytes. Signs and systemic symptoms were not evoked by intramammary inoculation. S. uberis could be isolated in tissue from all RMH. Histological examination of specimens of the RMH and lymph nodes of the goats showed an increased inflammatory response throughout the experiment. The histological findings correlated with the immunohistochemical detection of S. uberis in RMH. In conclusion, the experimental inoculation of S. uberis in lactating goats is capable of eliciting an inflammatory response and causing pathological changes, resulting in a subclinical mastitis. This investigation shows that goat might to represent a valuable model for the study of the mastitis pathogenesis caused by S. uberis.     

Ribotyping of Streptococcus uberis from a dairy's environment, bovine feces and milk

Streptococcus uberis is a major cause of bovine mastitis and infections commonly result from environmental exposure to the pathogen. To identify specific sources of mastitis-causing S. uberis strains, samples were collected monthly from the environment and feces of dry cows in a grazing herd. Environmental and fecal strains of S. uberis were compared to those found in milk. S. uberis was detected in 63% of 94 environmental samples, including water, soil, plant matter, bedding material, flies, and hay, in 23% of 107 fecal samples, and in 4% of 787 milk samples. Automated PvuII ribotyping revealed 48 ribotypes among 266 isolates. Per sample, up to five ribotypes were detected. The distribution of ribotypes did not differ significantly among environmental, fecal and milk samples. Specific environmental sources or strains of udder-pathogenic S. uberis were not identified. Fecal shedding was not persistent and did not differ between dry-off and calving. The proportion of fecal samples containing S. uberis was highest during the summer grazing season. S. uberis was common in farm soil (31 of 35 samples) but not in non-farm soil (0 of 11 samples). We hypothesize that fecal shedding of S. uberis may play a role in maintenance of S. uberis populations in the dairy ecosystem.     

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.     

Prevalence and associations between bacterial isolates from dry mammary glands of dairy cows

To assess the prevalence and patterns of bacterial isolates, cultures were made from the dry mammary glands of dairy cows in six commercial dairy herds in the UK. Milk samples were taken from all four quarters of 480 cows at drying off and at weekly intervals from 14 days before to seven days after calving. A major mastitis pathogen was isolated from at least one quarter of 220 (45.8 per cent) of the cows and from more than one quarter of 90 (18.8 per cent) of them. During the late dry to calving period, of the 957 quarters with three culture results, a major mastitis pathogen was cultured from 236 (24.7 per cent) quarters of 186 (38.8 [corrected] per cent) cows. The most commonly isolated major pathogen was Escherichia coli, followed by Streptococcus uberis and coagulase-positive staphylococci. There were significant differences between the patterns of isolates from different farms and in different calving months, suggesting that the rate of infection was partially dependent on external conditions. The isolation of E. coli, S. uberis or coagulase-positive staphylococci from a cow during the late dry/periparturient period was associated with an increased risk of that cow being culled in the next lactation. Bayesian general linear mixed models were used to assess the associations between the different bacterial species. The probability of isolating either E. coli or S. uberis was significantly greater when the other organism was cultured in a milk sample; this was also true of coagulase-positive staphylococci and S. uberis. When Corynebacterium species were isolated from a milk sample, the probability of isolating coagulase-positive staphylococci or S. uberis decreased significantly, and when coagulase-negative staphylococci were isolated the probability of isolating coagulase-positive staphylococci was reduced.     

Comparative evaluation of Vitek gram-positive identification system and API Rapid Strep system for identification of Streptococcus species of bovine origin

The Vitek Gram-positive identification system (GPI, Vitek Systems, Inc., Hazelwood, MO) and the API Rapid Strep system (Analytab Products, Plainview, NY) were evaluated for species identification of streptococci isolated from bovine mammary glands and compared to conventional biochemical methods. A total of 144 strains including Streptococcus uberis (60), S. dysgalactiae (32), S. agalactiae (15), S. bovis (15), Enterococcus faecium (10) and Ent. faecalis (12) were evaluated. All reference strains were identified correctly by both systems. Vitek GPI card system identified 94.4% of strains, including 95% of S. uberis, 93.8% of S. dygalactiae, 93.3% of S. agalactiae and S. bovis II, 90% of Ent. faecium and 100% of Ent. faecalis. Majority of strains were identified with a 90-99% level of confidence, with an average of 8 h needed for identification. The API Rapid Strep system identified 96.5% of strains correctly, including 95% of S. 96.9% of S. dysgalactiae, 93.3% of S. agalactiae, and 100% of S. bovis II, Ent. faecium, and Ent. faecalis. Majority of strains were identified with excellent level of identification. With the exception of S. uberis, most strains were identified at 4 h of incubation.     

Elucidation of the DNA sequence of Streptococcus uberis adhesion molecule gene (sua) and detection of sua in strains of Streptococcus uberis isolated from geographically diverse locations

Streptococcus uberis is an important environmental pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows throughout the world. S. uberis adhesion molecule (SUAM) was identified recently by our laboratory and we hypothesize that SUAM is a potential virulence factor involved in the pathogenesis of S. uberis mastitis. The first objective of the present study was to clone and sequence the SUAM gene (sua) from S. uberis UT888. The second objective was to determine the prevalence of sua in strains of S. uberis isolated from geographically diverse locations. The 20 amino acid N-terminal sequence of purified SUAM was utilized to identify a single open reading frame (ORF) in the S. uberis O140J (ATCC BAA-854) genome database. Three sets of primers were identified from this sequence for amplification of sub-fragments and the complete gene encoding SUAM. Restriction fragment analysis of the largest polymerase chain reaction (PCR) product confirmed the desired fragment had been amplified. This 2970bp PCR fragment was cloned into plasmid pCR-XL-TOPO and sequenced. The S. uberis UT888 sua sequence (NCBI Accession no. DQ232760) was 99% similar to the S. uberis O140J database sequence. The three pairs of PCR primers were used in a subsequent experiment to identify sua in 12 strains of S. uberis isolated in milk from dairy cows with mastitis in Tennessee (n=6), Colorado (n=1), Washington (n=1), New Zealand (n=1) and from the American Type Culture Collection (n=3). Primer pairs yielded the expected 2970, 2639 and 2362bp PCR fragments in all strains evaluated. In conclusion, we cloned and sequenced sua, which codes for the first described S. uberis adhesin, SUAM. sua was detected in all strains of S. uberis evaluated suggesting that it is conserved.     

Efficacy of extended pirlimycin therapy for treatment of experimentally induced Streptococcus uberis intramammary infections in lactating dairy cattle

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, around the time of calving, and during early lactation. Strategies for controlling S. uberis mastitis have not received adequate research attention and are therefore poorly defined and inadequate. Objectives of the present study were to evaluate the efficacy of extended therapy regimens with pirlimycin for treatment of experimentally induced S. uberis intramammary infections in lactating dairy cows during early lactation and to evaluate the usefulness of the S. uberis experimental infection model for evaluating antimicrobial efficacy in dairy cows. The efficacy of extended pirlimycin intramammary therapy regimens was investigated in 103 mammary glands of 68 dairy cows that became infected following experimental challenge with S. uberis during early lactation. Cows infected with S. uberis in one or both experimentally challenged mammary glands were randomly allocated to three groups, representing three different treatment regimens with pirlimycin, including 2-day (n = 21 cows, 31 mammary quarters), 5-day (n = 21 cows, 32 quarters), and 8-day (n = 26 cows, 40 quarters). For all groups, pirlimycin was administered at a rate of 50 mg of pirlimycin hydrochloride via intramammary infusion. A cure was defined as an experimentally infected mammary gland that was treated with pirlimycin and was bacteriologically negative for the presence of S. uberis at 7, 14, 21, and 28 days after treatment. Experimental S. uberis intramammary infections were eliminated in 58.1% of the infected quarters treated with the pirlimycin 2-day regimen, 68.8% for the 5-day regimen, and 80.0% for the 8-day regimen. Significant differences (P <.05) in efficacy were observed between the 2-day and 8-day treatment regimens. The number of somatic cells in milk decreased significantly following therapy in quarters for which treatment was successful in eliminating S. uberis. However, there was no evidence to suggest that extended therapy with pirlimycin resulted in a greater reduction in somatic cell counts in milk than the 2-day treatment. The S. uberis experimental infection model was a rapid and effective means of evaluating antimicrobial efficacy during early lactation at a time when mammary glands are highly susceptible to S. uberis intramammary infection.     

Epidemiology of Streptococcus uberis intramammary infections in a dairy herd.

From 1987 to 1991, almost 36,000 quarter samples of mammary secretion representing 1790 lactations of 510 dairy cows from a research herd were collected for bacteriological examination. The percentage of cows infected with Streptococcus uberis ranged from 12 to 16% of cows/year. S. uberis was isolated from 14.2% of lactations over the 5-year period. The prevalence of S. uberis intramammary infection (IMI) was significantly higher in cows with > or = 4 lactations than in cows with 3 or fewer lactations. Regardless of lactation number, the prevalence of S. uberis was highest before parturition, during early lactation and near drying off. The prevalence of S. uberis infected quarters ranged from 1.3 to 2.3% of quarters/year; the prevalence rate for the 5-year period was 2% of quarters. The quarter prevalence of S. uberis was lowest in cows with < or = 3 lactations, increased significantly with lactation number and was highest in cows with > or = 6 lactations. The percentage of quarters infected with S. uberis varied significantly by year. The majority (95%) of S. uberis IMI were subclinical. The ratio of subclinical IMI to clinical IMI was lowest during early lactation, and increased with days in milk, and with lactation age except for cows in their 5th and 6th lactations. Results of this epidemiological investigation suggest that opportunities exist where suitable control measures could be applied to reduce the impact of S. uberis infections in the dairy herd.     

Presence of ISS1-like insertion sequence in wild type Streptococcus uberis strains isolated from cases of bovine mastitis

Streptococcus uberis is a major cause of environmental mastitis worldwide. In spite of significant economic losses caused by S. uberis in many well-managed dairy herds, virulence factors and mechanisms associated with the pathogenesis of S. uberis mastitis are not well known. The ability of S. uberis to attach to and internalize into mammary epithelial cells and subsequent intracellular survival enables it to avoid host defense mechanisms. Research to determine virulence factors responsible for these pathogenic strategies involved creating a random chromosomal mutant library of S. uberis strain UT888 using the thermosensitive plasmid pGh9:ISS1 mutagenesis system. During Southern blot analysis of the mutant library, an endogenous element similar to ISS1 insertion sequence of Lactococcus lactis was found. ISS1 is a transposable bacterial insertion sequence isolated originally from L. lactis and are small phenotypically cryptic sequences of DNA with a simple genetic organization and capable of inserting at multiple sites in a target molecule. They are flanked by inverted repeats; generally encode their own transposition functions. A total of 29 of 34 wild type strains of S. uberis evaluated were positive for ISS1 by Southern blot. Insertion of ISS1 might have a significant phenotypic and genotypic role in the S. uberis genome because of its ability to transpose within the genome.     

Identification of Streptococcus spp. causing bovine mastitis by PCR-RFLP of 16S-23S ribosomal DNA

Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens.     

Investigations on the behavior of C-reactive protein, cell count, lactose content as well as electric conductivity in quarter milk samples of subclinically diseased quarters of the udders in relation to bacteriologic results]

Inflammations of the udder influence the amount of somatic cells and lactose as well as electrical conductivity in milk. They are often caused by bacterial agents. Similar reactions are to be seen in relation to C-reactive protein (CRP). The amount of CRP in milk depends on concentration and characteristics of the agents and the phase of inflammation. We found a significant increase of CRP in milk samples with Streptococcus (Sc.) uberis. There was no significant difference in CRP concentrations in samples with coagulase positive Staphylococcus spp. (CPS) and bacteriological negative samples and such with Corynebacterium (C.) bovis, Micrococcus spp. and Staphylococcus (S.) epidermidis. We only found high correlations to the amount of somatic sells and lactose as well as electrical conductivity in samples with Sc. uberis. The results of our investigations show that CRP may be a parameter for better understanding subclinical inflammations of udder.     

乳铁素参与乳房链球菌对乳腺上皮细胞的粘附

免疫印迹试验表明，试验用的3株乳房链球菌（Streptococcus uberis)菌株均可与乳铁素（Lactoferrin)结合。培养基中加入乳铁素或乳清可以显著促进细菌与乳腺上皮细胞之间的粘附，抗乳铁素抗体可以特异性地抑制乳铁素或乳清预处理细菌与乳腺上皮细胞的粘附。乳铁素在细菌和细胞之间起着桥梁分子作用。有助于细菌与乳腺上皮细胞之间的粘附和乳腺感染的建立。     

Binding of host glycosaminoglycans and milk proteins: possible role in the pathogenesis of Streptococcus uberis mastitis

The role of indirect binding of host proteins through glycosaminoglycans (GAGs) on adherence and internalization of Streptococcus uberis to bovine mammary epithelial cells was evaluated. Preincubation of S. uberis with GAGs followed by incubation with fetal bovine serum (FBS), bovine milk or milk proteins resulted in greater adherence to and internalization of S. uberis into mammary epithelial cells than observed in untreated controls. Highest values were detected, when final incubation was done with milk. Greater adherence to and internalization into mammary epithelial cells were observed when heparin sulfate (HEP) and milk were used compared with any other GAG and FBS. When individual milk proteins were used, greatest adherence and internalization were observed when S. uberis strains were pretreated with HEP followed by treatment with beta-casein. The findings of this study illustrate a pathogenic strategy of S. uberis that may occur during the very early stages of infection.