The molecular epidemiology of four outbreaks of porcine pasteurellosis

Biochemical profiles, restriction endonuclease analysis (REA) and ribotyping were used to investigate a total of 38 Pasteurella multocida isolates from four separate outbreaks of pasteurellosis in Australian piggeries. Six isolates were obtained from Outbreak 1, 16 from Outbreak 2 and eight each from outbreaks 3 and 4. Outbreaks 1 and 2 were cases of pneumonic pasteurellosis while outbreaks 3 and 4 involved systemic pasteurellosis. Biochemical characterisation established that a number of different types of P. multocida were present in outbreaks 1 and 3 while outbreaks 2 and 4 were associated with a single type of P. multocida. Outbreaks 1 and 3 yielded isolates of P. multocida that belonged to the subspecies multocida and gallicida, with the subspecies multocida isolates being identified as biovar 3 (6 in total) or 12 (1 in total) and the subspecies gallicida isolates (7 in total) being identified as biovar 8. All 24 isolates from outbreaks 2 and 4 belonged to the subspecies multocida and were all biovar 3. REA and ribotyping showed that, in outbreaks 1 and 3, there were three different types of P. multocida in each outbreak with no common strains between the outbreaks. The molecular methods showed that only a single strain of P. multocida was associated with outbreaks 2 and 4, although the outbreaks were associated with strains that differed in REA profiles but shared a ribotype profile. This study has shown that both, systemic and pneumonic pasteurellosis can be associated with either a single strain or multiple strains of P. multocida. The results also indicate that the molecular typing methods of REA and ribotyping are superior to biochemical characterisation for epidemiological investigation of porcine pasteurellosis.     

Characterisation of Pasteurella multocida isolated from fowl cholera outbreaks on turkey farms

SUMMARY: Biochemical profiles, restriction endonuclease analysis (REA) and ribotyping were used to investigate Pasteurella multocida isolates from outbreaks of fowl cholera on 7 turkey farms in New South Wales. While only a single isolate was available from 5 of the farms, multiple isolates, 4 and 12 respectively, were available from the other 2 farms. The available field evidence suggested that 8 outbreaks had occurred with one farm suffering 2 outbreaks. The isolates obtained were all confirmed as Pasteurella multocida . Biochemical profiles allocated the isolates to 4 groups, 3 being variants of P multocida subsp multocida and the fourth being P multocida subsp septica . REA performed with Hpall established 7 groups. Ribotyping using the Hpall digests probed with the 16S rRNA operon of Haemophilus paragallinarum recognised the same 7 groups as REA. Unlike the biochemical profiles, both REA and ribotyping provided a fine subdivision that identified outbreaks as either related or unrelated. The REA and ribotyping patterns as well as biochemical profiles were stable for all isolates from the outbreaks in which multiple isolates were obtained from either the same bird or from different birds. REA and ribotyping were found to be superior to biotyping methods for the investigation of fowl cholera outbreaks.     

Differentiation of Pasteurella multocida isolates from cases of atrophic rhinitis in pigs from Zimbabwe by RAPD and ribotyping

Atrophic rhinitis in pigs is rarely reported in Southern Africa. To determine the relationship between Pasteurella multocida clones from clinical cases of atrophic rhinitis, twenty-one strains were characterised by selected phenotypic and genotypic methods. Biochemical analysis classified 18 strains as P. multocida subspecies multocida, whilst the remainder were grouped into separate unassigned biotypes. Capsular groups A (16/21) and D (l/21) were found among the isolates by PCR. Four ribotype patterns were obtained following HpaII ribotyping, whilst random amplification of polymorphic DNA (RAPD) revealed three main clusters. However, subclusters were also noted for each RAPD cluster. Our results indicate that RAPD offers a better discrimination of strains than ribotyping and that none of the phenotypic characters were directly related to the genotypic clusters.     

Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark

The aim of the present study was to evaluate capsular-typing, plasmid-profiling, phage-typing and ribotyping for epidemiological studies of toxin-producing Pasteurella multocida ssp. multocida in Denmark. The evaluation of methods was based on 68 strains from nasal swabs and 14 strains from pneumonic lungs. Strains from lungs were all of capsular Type A, whereas strains from nasal swabs were of both capsular Types A and D. Only 9% of the strains contained plasmids, which could not be associated with antibiotic resistance. Phage-typing divided 61% of strains into 10 groups, while 39% were non-typable. CfoI ribotyping divided strains into four groups of which one type contained 94% of isolates. HindIII ribotyping divided strains into 18 types. A total of 18 strains from The Netherlands, UK and USA were subjected to HindIII ribotyping, resulting in 13 types of which six were identical to ribotypes of Danish strains. Phage-typing of isolates from an outbreak of atrophic rhinitis involving six herds in 1985 showed the existence of an epidemic strain. This type was recognised in the herd suspected of being the source of the infections and in four of the five infected herds. These findings were supported by HindIII ribotyping, as 85% of isolates from all herds were assigned to one ribotype. In conclusion, HindIII ribotyping seems to represent a useful tool for epidemiological studies of toxigenic P. multocida ssp. multocida.     

Identification of Pasteurella multocida Serogroup F isolates in rabbits

A total of 24 Pasteurella multocida rabbit isolates obtained from 24 rabbit flocks in the Czech Republic during the period of between 2001 and 2004 were analysed by capsular PCR typing. Apart from isolates identified as serogroups A (n = 14, 58.4%) and D (n = 2, 8.3%), eight isolates (33.3%) were identified as members of serogroup F. This serogroup had been predominantly associated with poultry infections so far. The rabbit serogroup F isolates were characterized in detail by ribotyping with restriction to endonuclease MspI revealing two distinct ribotypes. Seven serogroup F isolates were assigned to ribotype 1 and one isolate was assigned to ribotype 2.     

Ribotyping of Indian Isolates of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.     

Genetic diversity and associated pathology of Pasteurella multocida isolated from porcine pneumonia

Pasteurella multocida is a widespread respiratory pathogen in pigs associated with atrophic rhinitis and contributing to aggravation of the pulmonary lesions. The aims of the present study were to characterize isolates of P. multocida from porcine bronchopneumonia by pulsed-field gel electrophoresis (PFGE), PCR based capsular typing and multilocus sequence typing (MLST) and to compare clonal complexes outlined with the type of histological lung lesions to investigate if a correlation between clonal lineages and lesions might exist. Isolates of P. multocida were obtained from cases of cranioventrally located porcine bronchopneumonia. All lung lesions were described and classified according to histological lesions. A total of 139 isolates, from lung (n=111), pericardial sac (n=21) and kidney (n=7) of 111 pigs were described using PFGE with ApaI as the restriction enzyme. Furthermore, 20 and 29 isolates were characterized by capsular serotyping and multilocus sequence typing, respectively. PFGE demonstrated 15 different clusters showing 50% or more similarity. All selected isolates were of capsular serotype A and only three main sequence types (ST) were detected among the isolates. Associations were not found between histopathology and clonal complexes of P. multocida. In conclusion, PFGE demonstrated a high diversity of genotypes of P. multocida associated with porcine bronchopneumonia. However, isolates obtained mainly belonged to few STs, indicating that isolates of P. multocida associated with porcine bronchopneumonia originates from a limited number of clonal lineages and therefore might have adapted to porcine hosts. No correlation was demonstrated between genotypes and types of lesions, and extra-pulmonary spreading was only rarely demonstrated.     

Ribotyping and restriction endonuclease analysis reveal a novel clone of Bordetella bronchiseptica in seals.

The goal of the present study was to characterize, by ribotyping and restriction endonuclease analysis (REA), 35 phocine Bordetella bronchiseptica isolates and to ascertain their relationship to one another and to isolates acquired from other host species. Thirty-four isolates were obtained in Scotland during a 10-year period encompassing the 1988 epizootic; the remaining isolate was obtained independently in Denmark. All phocine isolates had an identical Pvu II ribotype unique from the 18 ribotypes previously detected in strains from heterologous hosts. Alternative restriction enzymes, useful for subgrouping strains within Pvu II ribotypes, also failed to discriminate among isolates from seals. The exclusive occurrence of a single ribotype of B. bronchiseptica in a particular host species has not been previously observed. Similarly, REA based on either HinfI or Dde I profiles did not reveal detectable polymorphisms, although unique patterns were readily distinguished among a limited number of isolates from other host species. This is the first report demonstrating the utility of REA using frequently cutting enzymes for discrimination of B. bronchiseptica strains. These data suggest that B. bronchiseptica-induced respiratory disease in seals along the Scottish shore may be due to the circulation of a single, unique clone.     

Differentiation of field isolates of Pasteurella multocida serotype 3,4 from live vaccine strain by genotypic characterization

Fifty-five serotype 3,4 isolates of Pasteurella multocida, isolated from turkeys dead from fowl cholera, were characterized (fingerprinted) genotypically for comparison with the serotype 3,4 live fowl cholera vaccine principally used in turkeys in California. Twenty-three isolates were obtained from turkeys vaccinated with the M9 live vaccine, and 32 additional isolates were from turkeys not vaccinated for fowl cholera. Methods of characterization included restriction endonuclease analysis of chromosomal DNA and ribotyping, a technique for highlighting restriction site heterogeneity of highly conserved ribosomal RNA genes and associated sequences using a radiolabeled rRNA probe. Eight different genotypes or ribotypes were detected in these isolates by the above methods. Of 23 isolates from M9-vaccinated turkeys flocks, 19 were the same ribotype as M9. Thirty of 32 isolates recovered from unvaccinated turkeys were different ribotypes from M9. The remaining two isolates resembled M9 and were recovered from two different flocks placed in succession on a turkey farm where a flock placed previously had been vaccinated with M9, suggesting interflock transmission. Ribotyping and restriction endonuclease analysis appear to be useful tools to aid in the determination of the role that the live vaccine plays in fowl cholera epidemiology.     

Diversity among isolates of Actinobacillus equuli and related organisms as revealed by ribotyping

Objective The objective of this work was to examine the diversity within Australian isolates of Actinobacillus equuli and related organisms by the genotypic method of ribotyping.
Design Ribotyping, performed using the enzyme Hae III, was used to examine the diversity in 12 field isolates of A equuli (five being capable of fermenting L-arabinose), one field isolate of Pasteurella caballi and two unclassifiable field isolates. Isolates were obtained from Australian horses, except for three isolates of A equuli (one L-arabinose positive and two L-arabinose negative) which were obtained from horses and a pig in Africa. In addition, the type strains for A equuli and P caballi and a reference strain for Bisgaard Taxon 9 were included in the study.
Results The ribotype patterns were analysed by computerised cluster analysis, yielding five clusters (A to E). All five of the L-arabinose positive A equuli were assigned to cluster A, with all the other seven A equuli isolates (all L-arabinose negative) and the type strain being assigned to cluster B. One of the two unclassified isolates formed cluster C along with the reference strain for Bisgaard Taxon 9. The remaining unclassified isolate formed cluster D. Cluster E consisted of the field isolate and reference strain of P caballi .
Conclusion The results of this study indicate that A equuli is a diverse species, with L-arabinose positive isolates of A equuli being quite distinct from typical L-arabinose negative isolates. Ribotyping appears to be a useful tool in confirming the identity of A equuli -like organisms from horses.     

Evaluation and application of ribotyping for epidemiological studies of Actinobacillus pleuropneumoniae in Denmark

The aim of the present study was to evaluate ribotyping as an epidemiological tool for Actinobacillus pleuropneumoniae and apply the method in studies of A. pleuropneumoniae infections in Danish pig herds. The evaluation of ribotyping was based on the 13 international reference strains and 106 epidemiologically unrelated Danish field strains representing the nine serotypes of biotype 1 (1, 2, 5A/B, 6, 7, 8, 10, 12, and K2:O7) and one serotype 14 of biotype 2. Enzymes CfoI and HindIII were chosen for generation of ribotype patterns. Ribotyping of the reference strains resulted in 10 CfoI types and 11 HindIII types. Ribotyping of the Danish strains resulted in 17 different CfoI ribotypes and 24 different HindIII ribotypes. Combining HindIII- and CfoI-ribotyping divided the Danish strains into 26 different types. The stability, reproducibility and typability of ribotype patterns were good, and the discriminatory power was between 0.85–0.89. The relatively low discriminatory power was caused by four predominant types, containing 61% of the isolates. The typing system was applied in studies of routes of infection of specific pathogen-free (SPF) pig herds and included 112 strains of A. pleuropneumoniae. Airborne transmission from neighboring conventional pig farms was investigated in 12 cases of infected SPF herds. Transmission via vehicles transporting pigs between SPF herds was investigated in nine cases while transmission by trading of pigs between SPF herds was investigated in two cases. Serotype 2 was isolated from all SPF herds included in this study, except one, emphasizing the high prevalence of this serotype in Denmark. By ribotyping, airborne transmission was indicated in five of 12 cases, transmission via pig transporting vehicle was indicated in six of nine cases, and transmission via trading was indicated in one of two cases. In many cases findings of predominant ribotypes made interpretations of suspected routes of transmission difficult. The relationship of strains based on ribotypes was calculated using Dices coefficient and clustered by UPGMA. HindIII ribotypes of serotype 2 strains were closely related, though only showing 43% similarity to HindIII ribotypes of remaining serotypes.     

Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin

One hundred and fifty-three bovine Pasteurella multocida strains recovered primarily from cases of pneumonia and mastitis in England and Wales over an 11-year period were characterised by capsular PCR typing, comparison of outer membrane protein (OMP) profiles, and multilocus sequence analysis. All of the strains were of capsular type A with the exception of a single capsular type F isolate. Thirteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. However, 85% of the isolates were represented by just five OMP-types and 39% of the strains were of a single OMP-type. Multilocus sequence analysis revealed a limited degree of genetic diversity among bovine P. multocida isolates; strains of the same OMP-type have identical genetic backgrounds and represent distinct clones. Analysis of OMP variation was more discriminating than multilocus sequence analysis because strains of different OMP-types had the same, or similar, genetic backgrounds. The association of a small number of clones with the majority of cases of bovine pneumonia suggests that these clones have an increased capacity to cause disease compared to less frequently recovered clones. Molecular mass heterogeneity of OmpA and OmpH, in strains of the same or similar genetic background, suggests that these proteins are subject to diversifying selection within the host and might play important roles in host-pathogen interactions. Comparison of the OMP profiles of bovine isolates with those of avian, ovine and porcine strains showed that a high proportion of the respiratory tract infections in each of these species are caused by different strains of P. multocida. However, the presence of small numbers of closely related strains in more than one host species suggests that transmission of bacteria between different host species is also a factor in the population biology of P. multocida.     

Associations between biovar and virulence factor genes in Pasteurella multocida isolates from pigs in Spain

Two hundred and five isolates of Pasteurella multocida from pigs were phenotypically and genetically characterised by determining their biovar, capsular type, virulence-associated genes and pulsed-field gel electrophoresis (PFGE) profiles. All isolates were identified as P multocida subspecies multocida and most were assigned to biovar 3 (58 per cent) and biovar 2 (39.5 per cent). Biovar 1 represented 2.4 per cent of the isolates. According to the capsular type, the great majority of the isolates (79.0 per cent) belonged to capsular type A, 18.5 per cent belonged to capsular type D and 2.4 per cent were of capsular type F. All isolates harboured ompH, psl, oma87, ptfA, nanB, nanH, tonB, hgbA, sodA and sodC genes, while none of them possessed the transferrin-binding protein gene tbpA. The prevalence of toxA, pfhaA and hgbB genes was variable (7.8, 40.5 and 60.5 per cent of the isolates, respectively). After PFGE typing, isolates of biovar 2 and 3 were grouped in two different clusters (A and B) at a level of 45 per cent similarity. In addition, isolates of biovar 2 and 3 exhibited statistically significant differences (P<0.05) in the virulence-associated hgbB and pfhA genes (biovar 3 was hgbB(+) pfhA(-), while biovar 2 was hgbB- pfhA(+)).     

Phenotypic and genotypic characterisation of Pasteurella multocida strains isolated from pigs in Hungary

A total of 146 Pasteurella multocida strains isolated from swine in Hungary in the last 20 years were examined. Biochemical characterisation and PCR-based techniques were used to determine species, subspecies, biovar, capsule type and presence of the toxA gene. Eighty-seven percent of the isolates belonged to P. multocida ssp. multocida, and 98% of these had biovar 3 or were trehalose- or lactose-fermenting or ornithine decarboxylase negative variants of that. Ten percent of the strains were P. multocida ssp. septica, and within this group 80% of the strains showed sorbitol-negative biovars (5, 6 and 7). The rest of the strains (20%) were lactose positive. Only 3% of the porcine isolates were P. multocida ssp. gallicida and 3 out of the 4 strains belonged to the dulcitol-fermenting biovar 8. Using a capsule-specific multiplex PCR, 60% of the strains belonged to capsule type D, 38% to capsule type A, and only 1 isolate had capsule type F. In contrast with data published in the literature, only 3% of capsule type D isolates carried the toxA gene, while this ratio was 41% for the type A strains. A remarkable regional distribution of toxA gene positive strains was observed. All but two isolates were found in swine herds located in the Transdanubian region, separated from other parts of Hungary by the river Danube.     

Molecular epidemiologic features and antimicrobial susceptibility profiles of various ribotypes of Pseudomonas aeruginosa isolated from humans and ruminants

OBJECTIVES: To assess automated ribotyping for characterization of Pseudomonas aeruginosa isolates and to identify their type prevalence and geographic distribution. SAMPLE POPULATION: 39 human and 56 ruminant P aeruginosa isolates. PROCEDURES: Isolates were identified by use of bacteriologic techniques and automated Pvull-based ribotyping. Susceptibility to antimicrobials was tested in vitro. Data were analyzed for index of discrimination; prevalence ratio; geographic distribution of ribotypes found only in humans, only in cows, or only in goats (single-host ribotypes); and geographic distribution of ribotypes found in humans and ruminants (multihost ribotypes). RESULTS: All isolates were typeable (45 ribotypes, 35 single-host ribotypes). Ribotyping index of discrimination was 0.976. More isolates (45.3%) than expected yielded multihost ribotypes (22% of all ribotypes). Although 8.6% of single-host ribotypes were found in 4 or more isolates, 60% of multihost ribotypes were found in 4 or more isolates. Ninety percent of multihost ribotypes were isolated from different geographic areas, whereas 3.0% of single-host ribotypes were isolated from different geographic areas. All ruminant isolates were susceptible to gentamicin and polymyxin B. In contrast, antibiogram profiles differed for human isolates from different geographic areas. Susceptibility to antimicrobials differentiated 6 isolates not distinguished by ribotyping. CONCLUSIONS AND CLINICAL RELEVANCE: Automated ribotyping with Pvull discriminated more isolates than in vitro antimicrobial susceptibility. In combination, both tests provided more information than either test alone. Given the greater prevalence and geographic distribution of multihost ribotypes, immunocompromised humans and lactating ruminants may have a greater risk for disease if exposed to multihost P aeruginosa ribotypes, compared with single-host ribotypes.     

Antimicrobial Susceptibility and rRNA Gene Restriction Patterns among Staphylococcus intermedins from Healthy Dogs and from Dogs Suffering from Pyoderma or Otitis Externa

A total of 60 Staphylococcus intermedins strains from dogs were investigated by their sensitivity to various antibiotics (50 strains) and by their rRNA gene restriction patterns (ribotyping) (60 strains). Fifteen isolates were from healthy dogs, 9 with otitis externa, and 36 with pyoderma, including 10 strains from a previous study. Sixty per cent of the 50 strains tested for antibiotic susceptibility demonstrated resistance to penicillin, 24% to spiramycin, 20% to tetracycline, 16% to chloramphenicol, and 2% to fucidic acid. All isolates were susceptible to amoxycillin with clavulanic acid, enrofloxacin, and sulphonamides with trimethoprim. There were no significant differences in antimicrobial susceptibility patterns observed among isolates from pyoderma, otitis externa or healthy dogs. Among the 60 strains studied by ribotyping, 10 different ribotypes were identified: 6 different ribotypes among isolates from otitis externa, 8 among isolates from pyoderma, and 5 among isolates from healthy dogs. One ribotype (profile C) was dominant among the isolates from healthy dogs while another ribotype (profile A) was dominant among strains from dogs suffering from pyoderma. This profile was not demonstrated in any of the strains from healthy dogs. From 5 different dogs suffering from pyoderma, 2 different clones were demonstrated based on their plasmid profile and antibiogram. In these dogs 1 of the clones always belonged to ribotype A. The results concerning strains of S. intermedins isolated from furunculosis suggest the existence of distinct subpopulations with different pathogenicity to dogs.     

Characterization of Streptococcus suis serotype 7 isolates from diseased pigs in Denmark

Isolates of Streptococcus suis serotype 7 from diseased pigs in Denmark were characterized by ribotyping, pulsed field gel electrophoresis (PFGE), MIC-determinations and detection of resistance genes. Forty-one different ribotype profiles were found among the 103 isolates and could be divided into two main clusters. No obvious relationship between ribotypes and the clinical origin of the isolates could be observed. Fifty-four isolates, including all 24 isolates belonging to the main ribotype profile were examined by PFGE and 50 different profiles were found. A high frequency of resistance to erythromycin (41%), tetracycline (24%) and streptomycin (28%) was observed. Furthermore, almost all isolates (101) were resistant to sulphamethoxazol. Most isolates were susceptible to ceftiofur, chloramphenicol, florfenicol, penicillin, ciprofloxacin, trimethoprim and trimethoprim + sulphonamides. The tet(M) gene was found among 11 (44%) and the tet(O) gene in six (24%) of 25 tetracycline resistant isolates. The tet(L) and tet(S) genes were not detected in any isolates. The erm(B) gene was detected in 39 (93%) of 42 erythromycin resistant isolates.     

Relationships among Pasteurellaceae isolated from free ranging chickens and their animal contacts as determined by quantitative phenotyping, ribotyping and REA-typing

One hundred and forty-three Pasteurella spp. strains and 10 unclassified strains obtained from free ranging poultry, dogs and cats were investigated by extended phenotypic characterization. One hundred and forty-nine of these strains were selected for further studies using ribotyping and REA-typing to evaluate the role of dogs and cats in Pasteurella multocida transmission. Seven and six type strains were included for comparison in phenotyping and genotyping, respectively. Eleven clusters and six unclustered strains were revealed by phenotyping. Ribotyping outlined 12 clusters and six unclustered strains. A correlation between clusters obtained by phenotyping and ribotyping was demonstrated which indicated that a genetic basis exists for clusters outlined by quantitative evaluation of phenotypic data. Similarities and differences in hosts, phenotype, ribotype, and zone of isolation were demonstrated among Pasteurella strains investigated. Isolates of P. multocida from ducks were shown to be clonal by both phenotyping and ribotyping. These strains were identical to one of the chickens strains. REA-typing, however, showed that the chicken strain was different underlining that exchange of clones of P. multocida between avian species rarely happens under village conditions. Management practise in the villages suggest the potential for exchange of P. multocida between poultry and animals kept in contact. The present findings, however, did not indicate that clones of P. multocida are widely exchanged between poultry and other animal species, even though close contact exists. In the present investigation exchange of clones of P. multocida was only demonstrated among animals belonging to the same species. Caution is drawn to the use of ribotyping as the sole method for epidemiological typing and tracing of P. multocida. The present results also underline the importance of proper phenotyping in the identification of P. multocida and related species.     

The necrotizing enteritis by Clostridium perfringens type C in piglets: II. Molecular epidemiology study

Investigations were performed on shedding of C. perfringens in sows from four different pig farms. In two farms where no outbreaks of necrotizing enteritis had been observed, no strains of C. perfringens producing beta-toxin were detected in the faeces of sows. In contrast, C. perfringens strains producing beta-toxin were detected in sows on both farms suffering outbreaks of acute necrotizing enteritis. Strains of C. perfringens producing beta-toxin were invariably positive for the beta 2-toxin gene. However, strains carrying the beta 2-toxin gene only (i.e. negative for beta-toxin) were present in animals on all farms with roughly similar frequencies (mean 28.2% carriers). Some sows carried C. perfringens strains of both toxin genotypes simultaneously. Whereas these data further support the role of betatoxin as a cause of necrotizing enteritis, the role of beta 2-toxin in intestinal disease of piglets remains unclear. To establish the role of faecal shedding vs. environmental contamination as reservoirs of C. perfringens type C, strains were isolated from teats and feedlot trough swabs (toxin genotype beta/beta 2), as well as from fodder (genotype beta 2). However, sows carried this pathogen intermittently and in small numbers. This renders an individual, reliable diagnosis of carrier sows very difficult. Ribotyping of 34 C. perfringens isolates of different toxin genotypes showed five distinct profiles. Different toxin genotypes can belong to the same ribotype, and the same toxin genotype can be present in different ribotypes. Thus, even if a majority (79.4%) of strains investigated in a limited geographic region belonged to ribotype 1, ribotyping offered discrimination of strains beyond toxin typing.     

Characterization of Staphylococcus simulans strains isolated from cases of bovine mastitis

This study was conducted to characterize Staphylococcus simulans isolated from cases of bovine mastitis. A total of 134 isolates of S. simulans selected from 80 quarters from 61 cows or heifers in 37 different herds were characterized by EcoRI ribotyping. From 22 quarters two to seven consecutive isolates taken at weekly intervals were selected. Furthermore, three isolates from clinical infections in humans and two reference strains were included. A total of 16 different ribotypes were found, however, two types predominated. In most herds more than one type was found. From the 22 different quarters, where 76 paired or multiple isolates were at disposal, the same ribotype was constantly found in the same quarter. This study showed that S. simulans causing bovine mastitis could be divided into relatively large number of different types, but that two types predominated. More than one type could be found in the same herd and within different quarters of the same cow, but ribotyping confirmed that S. simulans could be the cause of persistent and stable infections.