Adhesion of Streptococcus gallolyticus strains to extracellular matrix proteins

Fourteen pigeon Streptococcus gallolyticus strains of differing virulence, were tested for their ability to adhere to immobilised fibronectin, collagen types I, III and IV. Eight, 2 and 13 strains were able to bind fibronectin, collagen types III and IV, respectively. None of the strains adhered to collagen type I. Heat treatment, proteolytic digestion or periodate treatment reduced the binding of S. gallolyticus to fibronectin and collagen type IV, suggesting that surface receptors contain proteins and carbohydrates. Although binding to these extracellular matrix proteins can play a role in the pathogenesis of streptococcosis in pigeons, binding properties could not be related to virulence, indicating that other factors determine differences in virulence among pigeon S. gallolyticus strains. Adhesion to collagen type IV may account in part for the distribution pattern of the lesions observed in naturally and experimentally infected pigeons.     

Genomic fingerprinting of pigeon Streptococcus gallolyticus strains of different virulence by randomly amplified polymorphic DNA (RAPD) analysis

Randomly amplified polymorphic DNA (RAPD) analysis was performed on 95 pigeon S. gallolyticus strains of different virulence and belonging to different biotypes and different culture supernatant phenotypes as determined by SDS-PAGE. Four distinct RAPD patterns, designated A, B, C and D, were distinguished using primer OPM6 (5'CTGGGCAACT). All 76 strains generating RAPD pattern A or B were designated highly virulent on the basis of their SDS-PAGE pattern. Five of seven strains generating RAPD pattern C and 11 of 12 strains generating RAPD pattern D belonged to the moderately virulent and low virulent culture supernatant phenotype groups, respectively. Only one RAPD group C strain belonged to a highly virulent culture supernatant phenotype group. There was a correlation between biotype and RAPD patterns. These findings indicate that there is a high correlation between RAPD pattern and virulence for pigeons. Therefore, RAPD typing seems a rapid, reliable method to distinguish pigeon S. gallolyticus strains of high, moderate and low virulence.     

Streptococcus gallolyticus infections in racing pigeons, a literature review

S. gallolyticus, formerly known as S. bovis is known since 1988 as a facultative pathogen of racing pigeons. Important clinical signs include acute mortality, inability to fly, lameness, weight loss and slimy green diarrhea. A pathognomonic sign at post mortem examination is the presence of well circumscribed areas of necrosis in the pectoral muscle. Furthermore tenosynovitis of the supracoracoid muscle and arthritis of the knee, shoulder and hock can be observed. In one study S. gallolyticus septicaemia was diagnosed in 10% of necropsied pigeons. Since S. gallolyticus was also isolated from nearly 40% of clinical healthy pigeons it is regarded as a facultative pathogen. Various biotypes, serotypes and culture supernatant phenotypes can be distinguished. Supernatant phenotypes are identified on the basis of the presence of either a T1, T2 or T3 protein triplet and the presence or absence of an extracellular A protein. S. gallolyticus strains with A protein are highly virulent, while strains with only T3 or T2 protein are of moderately or low virulence respectively. Fimbriae are only seen in highly virulent and some of the moderately virulent strains. Possible virulence factors include survival in macrophages, adhesion to cells and toxin production. Infection with serotype 1 and 2 induces some degree of protection against re-infection with serotype 1, which offers perspectives for the development of a vaccine. Experimentally ampicillin, doxycycline and erythromycin have shown therapeutic effects. For the treatment of clinical cases the use of ampicillin is advocated, together with hygienic measures, such as the use of grid floors and avoiding overcrowding.     

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.     

ARDRA and RAPD analyses of human and animal isolates of Streptococcus gallolyticus

A total of 23 Streptococcus gallolyticus strains, consisting of 12 strains from feces of healthy animals and 11 from clinical cases of human or cow mastitis milk, were examined genealogically. Four strains of S. bovis "biotype II/1" and 3 strains of S. equinus, the closely related organisms to S. gallolyticus, were also analyzed for outgroup comparison. Neither the amplified ribosomal DNA restriction analysis (ARDRA) nor the randomly amplified polymorphic DNA (RAPD) analysis that had been designed to recognize S. gallolyticus strains virulent in pigeons could differentiate clinical strains from the others of S. gallolyticus. No correspondence between the DNA profile in either analysis and the host animal species was detected.     

4株鸽源沙门氏菌致病性观察

为了解分离自四川地区的鸽源沙门氏菌的致病性,本研究先将分离的4株沙门氏菌,腹腔注射SPF昆明鼠,测定其半数致死量(LD50),再依据小鼠攻毒试验结果,选取致病性较强的纽波特沙门氏菌菌株,通过灌胃方式接种信鸽来观察对信鸽的致病性.结果显示:甲型副伤寒沙门氏菌、纽波特沙门氏菌、伯里沙门氏菌、基桑加尼沙门氏菌的LD50分别为3.37×10^6、2.49×10^7、5.46×10^7、3.58×107 CFU;感染鼠的肝脏、脾脏、肾脏及小肠有明显的炎性病变;纽波特沙门氏菌可造成感染鸽的肝脏、肺脏、肾脏和胰腺纤维素样变性及炎性细胞浸润等病变,回肠、空肠、盲肠的肠绒毛结构不完整、绒毛上皮脱落,绒毛及肌层有不同程度的炎性细胞浸润.以上结果表明,4株分离的沙门氏菌对试验鼠有一定的致病性,纽波特沙门氏菌可造成信鸽气囊等组织器官损伤,继而影响其飞行能力.本研究为鸽源沙门氏菌尤其是纽波特沙门氏菌的致病性研究提供一定的理论参考,具有公共卫生学意义.     

Attaching and effacing bacteria in the intestines of calves and cats with diarrhea

Histopathologic and electron microscopic examination of intestines of three calves and two cats revealed attaching effacing bacteria characteristic of enteropathogenic Escherichia coli (EPEC) in ileum, cecum, and colon. The attaching effacing bacteria in one of the calves contained bacteriophages, and an E. coli isolate from that calf was shown to produce Shiga-like toxin. These findings contribute to emerging evidence that attaching effacing intestinal bacteria are globally distributed pathogens in a variety of host species and that bacteriophage-mediated production of Shiga-like toxin is related to the virulence of such bacteria.     

Secreted antigens as virulence-associated markers in Staphylococcus aureus strains from rabbits

Western blot analysis was performed from the culture supernatant of 59 rabbit Staphylococcus aureus strains, classified as high and low virulence strains according to their epidemiological behaviour in commercial rabbitries, bio-, phage- and RAPD-type. Fourteen extracellular antigen bands (A-N) were recognised using sera of rabbits immunised with washed, viable high virulence S. aureus bacteria. Eleven of these bands were found in high virulence as well as in low virulence strains. The band A, approximately 78 kDa, was not seen in any of the 27 high virulence strains, except for one strain which was also typical in other aspects, was detected in all, but one of the low virulence strains. The M and N bands with molecular masses of approximately 29 and 27 kDa, respectively, were recognised in all high virulence strains except for the atypical strain, but in none of the low virulence strains. This indicates that the latter two antigens may be virulence-associated markers for S. aureus strains from rabbits.     

Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1a to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.     

Expression of type 1 pili by Escherichia coli strains of high and low virulence in the intestinal tract of gnotobiotic turkeys

Highly virulent (strain 1) and weakly virulent (strain 3) Escherichia coli were examined using immunofluorescent and electron microscopic techniques to determine their ability to express type 1 pili in the intestinal tract of 3-week-old gnotobiotic turkeys. Turkeys were necropsied on postinoculation day (PID) 1, 2, 5, 8, and 12. Nonpiliated forms of strains 1 and 3 were more numerous than piliated forms in cecal and colonic contents examined by negative staining electron microscopy. A piliated form of strain 1 was seen in intestinal contents on each PID and was more numerous in cecal contents than in colonic contents. The mucus blanket of the cecum and colon contained large numbers of bacteria, although organisms were rarely intimately associated with the intestinal epithelium. Immunofluorescent staining indicated large numbers of piliated forms of strains 1 and 3 within the mucus blanket of the cecum and colon on PID 2, 5, 8, and 12. Piliated bacteria were infrequently seen in the ileal mucus blanket. Serum antibody titers to type 1 pili increased markedly by PID 5 and persisted in turkeys inoculated with strain 1. In contrast, antibody titers in turkeys exposed to strain 3 increased gradually and varied markedly among birds at each PID. Type 1 pili may not be important for adherence of pathogenic E coli to intestinal epithelium of turkeys.     

Effects of Escherichia coli heat-stable enterotoxin b on small intestinal villi in pigs, rabbits, and lambs

Culture supernates from two strains of E. coli were placed into different ligated intestinal sections (loops) of each animal. The two bacterial strains were identical except that one contained a plasmid carrying the heat-stable toxin b (STb) gene, while the other did not. Morphometric techniques were used to assess villous epithelial surface areas and mucosal volumes in both intestinal segments exposed to STb-positive (test) and to STb-negative (control) supernates. In pigs whose intestines were exposed to STb-positive supernatants for 2 hours, both villous epithelial surface area and mucosal volume were significantly smaller in test loops than in control loops (P less than 0.02). In test loops of pigs incubated for 1 hour, and in test loops of lambs incubated for 2 hours, there was a decrease in villous epithelial surface area which approached the test for significance but did not meet it (0.05 less than P less than 0.10). Rabbit test loops did not differ from rabbit control loops in either villous epithelial surface area or mucosal volume. Histological examination of the tissues from all three species revealed epithelial changes in porcine and ovine tissues only. In porcine and ovine tissues, epithelium at villous tips was seen to be cuboidal or squamous, or even to be absent. Villi with similarly altered epithelium were seen in control loops, but were seen much more frequently in test loops. These epithelial changes were seen as early as 30 minutes of incubation in pigs. Intestinal tissues from these pigs were examined by transmission electron microscopy, but no difference between test and control tissues was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops).

The ability of a high virulence strain (AJS 1) and a low virulence strain (AJS 4) of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of black mollies (Poecilia sphenops) was investigated. For that purpose, two groups of 25 black mollies each were immersed in a bath containing 10(6) CFU/ml of F. columnare AJS 1 or AJS 4. At regular intervals from 1 to 12 h after the contact infection, fish were sacrificed and gills, skin, spleen and heart were sampled for bacteriology. Samples of the gills were taken for immunohistochemical and electron microscopic examination. Bacteriological examination proved that the number of gill-associated F. columnare was higher for AJS 1 than for AJS 4. Strain AJS 1 was isolated from the heart and spleen of 6 and 1 of the 16 examined animals, respectively. Strain AJS 4 was not isolated from the internal organs of any fish. When examined immunohistochemically, strain AJS 1 was found closely associated with gill epithelium whereas this was not the case for strain AJS 4. The adherence of bacteria to the gill tissue challenged with the virulent strain AJS 1 was also clearly demonstrated using scanning and transmission electron microscopy. These results indicate that adhesion of F. columnare to the gill tissue constitutes an important step in pathogenesis.     

In vitro adhesion of an avian pathogenic Escherichia coli O78 strain to surfaces of the chicken intestinal tract and to ileal mucus

The role of fimbria in adherence of an avian pathogenic Escherichia coli (APEC) O78 strain 789 to chicken intestine was studied. Bacterial adhesion to tissue sections representing the regions within the chicken intestinal tract was determined by using immunohistochemical methods. E. coli 789 grown to express the type 1 fimbria adhered efficiently to the crop epithelium, to the lamina propria of intestinal villi, and to the apical surfaces of both the mature as well as the crypt-located enterocytes in intestinal villi, whereas no adhesion to mucus-producing goblet cells was detected. The adhesion was inhibited by mannoside and the role of type 1 fimbriae in the observed adhesion was confirmed with a recombinant strain expressing type 1 fimbriae genes cloned from E. coli and Salmonella enterica. E. coli 789 strain grown to favor AC/I fimbriae expression as well as the recombinant E. coli strain expressing the fac genes adhered to goblet cells but only poorly to the other epithelial sites. E. coli strain 789 as well as S. enterica serovar Typhimurium IR715 and S. enterica serovar Enteriditis TN2 strains were able to multiply in ileal mucus medium. The type 1 fimbria expressing bacteria adhered to the ileal mucus, whereas the AC/I fimbriated strains showed poor adherence to the mucus. The adhesion of E. coli 789 onto the crop epithelium and the follicle associated epithelium of the chicken ileum was efficiently inhibited by an adhesive strain ST1 of Lactobacillus crispatus isolated from chicken, whereas poor inhibition of E. coli adherence was observed with the weakly adhesive L. crispatus strain 134mi. The type 1 fimbriae may be important in colonization of the chicken intestine by APEC and Salmonella.     

鸡源致病性奇异变形杆菌的分离鉴定与遗传进化分析

为了解鸡源奇异变形杆菌的耐药情况、致病力和遗传特征,本试验从凉山地区分离得到2株细菌（YLF1、WS）,通过培养特征、镜检特征和16S rRNA序列测定对分离菌进行鉴定及致病性试验;采用（K-B）法检测分离菌对14种常用抗生素（包括β-内酰胺类、头孢烯类、氨基糖苷类、四环素类、氟喹诺酮类、大环内酯类）的敏感性;并与GenBank中公布的21种不同来源的变形杆菌16S rRNA基因序列进行遗传进化分析。结果表明,YLF1、WS菌株均具有迁徙性和β-溶血生长特征,镜检为革兰氏阴性长短不一的杆菌或球菌,经16S rRNA基因鉴定与GenBank中奇异变形杆菌同源性均在99%以上,YLF1、WS菌株确定为奇异变形杆菌,均表现为多重耐药,耐药率分别为78.6%（11/14）和71.4%（10/14）;雏鸡致死率均为80%;YLF1、WS菌株与日本人体分离株、中国新疆黄牛分离株和中国广东土壤分离株同源性最高,达99.9%;与来自中国河南、中国新疆及印度的鸡源奇异变形杆菌同源性较高（98.9%~99.3%）,遗传关系密切。表明凉山地区鸡群中存在奇异变形杆菌感染,分离株有较高的致病力和较强的耐药性,也提示分离菌可能来源于环境、感染鸡或其他动物,同时存在人畜共患的潜在危险。     

Extracellular proteins and virulence in Streptococcus bovis isolates from pigeons

The association between virulence and the occurrence of the extracellular proteins A, T1, T2 and T3 in the culture supernatant of pigeon Streptococcus bovis strains, was examined in experimental infection studies. Fourteen groups of 10–17 pigeons were inoculated intravenously with 1 × 10⁹ CFU of S. bovis strains that belonged to the phenotypes A ⁺T1, A ⁻T1, A ⁺T2, A ⁻T2, A ⁺T3 and A ⁻T3, respectively. The overall postinoculation morbidity in the phenotype groups was 85%, 87%, 70%, 5%, 100% and 37%, respectively. These results indicate that strains producing A or T1 are of high virulence, those producing T3 only are of moderate virulence and those producing T2 are of low virulence. Virulence of S. bovis for pigeons was more clearly correlated with supernatant-phenotype than with serotype.     

The role of hyaluronic acid capsular material of Streptococcus equi subsp. zooepidemicus in mediating adherence to HeLa cells and in resisting phagocytosis.

Hyaluronic acid is thought to be one of the critical virulence factors of Streptococcus equi subsp. zooepidemicus. The present study was designed to study the role of hyaluronic acid capsular material in mediating adherence and to resist the phagocytosis of the host's immune defence. The studies were performed with two encapsulated S. equi subsp. zooepidemicus and two unencapsulated phase variants. The bacteria had been previously isolated from diseased pigs and monkeys in Indonesia. The presence of capsular material was determined using the hyaluronic acid decapsulation test and by electron microscopic studies. Both encapsulated bacteria showed mucoid colonies after cultivation on blood agar, grew with diffuse colonies in soft agar media and reacted negatively in the salt aggregation test. The unencapsulated bacteria grew with small colonies on blood agar, formed compact colonies in soft agar media and reacted positively in the salt aggregation test. Adherence and phagocytosis studies revealed that the encapsulated bacteria adhered significantly more to HeLa cells and were less phagocytosed by murine macrophages compared to unencapsulated bacteria. Pretreatment of the HeLa cells using hyaluronic acid or pretreatment of the bacteria by hyaluronidase decreased the adherence value of encapsulated bacteria. Pretreatment of bacteria with pronase had no effect. The presented results strongly indicate that the hyaluronic acid capsular material contributes to adherence properties of S. equi subsp. zooepidemicus and might help the bacteria to resist phagocytosis by macrophages.     

1株信鸽源鼠伤寒沙门氏菌的分离鉴定及其耐药性和毒力分析

本试验旨在鉴定临床疑似沙门氏菌感染致死信鸽的病原菌并确定致病菌的耐药及毒力状况。通过细菌分离培养、菌落形态观察、染色镜检、生化鉴定、血清分型、16S rRNA基因测序分析和康复信鸽血清平板凝集试验进行鉴定,并通过药敏试验、耐药基因和毒力基因检测进行耐药性和毒力分析。结果显示,分离纯化的细菌在BS、XLD、HE培养基上为黑色菌落,镜检为无荚膜、无芽孢的革兰氏阴性短杆菌;分离菌株生化反应结果符合沙门氏菌生化特性;分离菌株血清型为1,4,12：i：1,2;分离菌株16S rRNA基因序列系统进化树分析显示,该分离菌株与鼠伤寒沙门氏菌聚为一支,同源性>99%,康复信鸽血清与分离菌株发生特异性凝集,结合生化反应和血清分型结果该菌株鉴定为鼠伤寒沙门氏菌;分离菌株对氟苯尼考耐药,经耐药基因检测携带floR、cmlA氯霉素类耐药基因,与耐药表型相符;分离菌株mogA等17种毒力基因检测均为阳性。本试验成功分离到1株信鸽源鼠伤寒沙门氏菌,分离菌株对氟苯尼考耐药且具有较强毒力,为下一步信鸽沙门氏菌的防治和研究提供了参考依据。     

High and low virulence Staphylococcus aureus strains in a rabbit skin infection model

In the present study, an in vivo rabbit skin infection model was developed to reproduce the lesions caused by high and low virulence Staphylococcus aureus strains from rabbits. "O"-shaped dermal skin lesions were induced on the shaved flanks of anaesthetised rabbits using a tattoo pin and pincers. The induced lesions on the flanks of four groups of 10 rabbits were then inoculated by topical application of 0.1 ml of 10(8)cfu S. aureus bacteria. One group was inoculated with a typical high virulence (HV) S. aureus strain from rabbits, one group received an atypical HV strain and two groups were inoculated with low virulence (LV) strains. Five animals were kept as negative controls. The development, appearance and size of abscesses were scored daily for a period of 2 weeks. The infection model showed reproducible results for the different S. aureus inoculation groups. Inoculation of the skin with the typical HV strain resulted in significantly larger abscesses than those caused by the LV strains. The atypical HV strain caused abscesses of a size intermediate to that obtained with the HV and LV strains. In rabbits infected with LV strains, most of the lesions had healed by day 14 post-inoculation. The devised infection model is able to reliably reproduce the virulence properties of HV and LV S. aureus strains.     

Comparative virulence of different bovine rotavirus isolates.

Intestinal loops, ligated in colostrum-deprived calves were used to compare the virulence of four isolates of bovine rotavirus. Histopathological studies were carried out on infected and control loops and measurements of villous length, crypt depth, villus:crypt ratio and crypt mitotic index were recorded. Pathological changes associated with the rotaviruses included villous atrophy, flattening of absorptive epithelium and reduced villus:crypt ratios. The changes were confined to infected intestinal loops in which the presence of virus was demonstrated by specific immunofluorescence. Consistent differences in the measured histopathological changes suggested differences in virulence among the rotavirus isolates tested. The least virulent rotavirus isolate had a polypeptide electrophoretic pattern that differed from the other three more virulent isolates.     

Sequence analysis of a RAPD band differentiating high and low virulence Staphylococcus aureus strains from rabbits.

RAPD typing revealed the presence of a nucleotide band in typical high virulence rabbit Staphylococcus aureus strains which was absent in low virulence strains and in an atypical high virulence strain. The nucleotide sequence of this band was determined. Primers within this sequence were developed and PCR products of eight typical high virulence, one atypical high virulence and nine low virulence rabbit S. aureus strains were sequenced. All low virulence strains and the atypical high virulence strain revealed a constant difference with the typical high virulence strains for nucleotide 377 of the 1055bp sequence. The eight typical high virulence strains possessed a guanine base on this site, while the other strains tested showed an adenine base. These findings support the hypothesis on the clonal origin of typical high virulence rabbit S. aureus strains. After comparison with databases, two open reading frames (ORF) were identified within the sequence, which appeared to encode two structural ribosomal proteins. The single nucleotide mutation does not affect the amino acid sequence of the protein it encodes for.