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.     

Aphanomyces invadans and ulcerative mycosis in estuarine and freshwater fish in Florida

In the spring of 1998, the Florida Fish and Wildlife Research Institute received numerous reports of lesioned or ulcerated fish primarily from the St. Lucie Estuary on the southeast coast of Florida, an area known since the late 1970s for lesions of the ulcerative mycosis (UM) type. From these and archived reports, as well as others received from different areas of Florida, we documented that diseased specimens had randomly distributed skin ulcers (usually reddened or hemorrhagic) with raised irregular margins and, in some cases, deeply penetrating hyphae in the surrounding muscle tissue. Since 1998, 256 fish (comprising 18 species) with ulcerative lesions (from 15 different locations) were confirmed with hyphae in fresh squash preparation or by histological evaluation. Squash preparations revealed nonseptate, sparsely branching, thick-walled hyphae; histological sections revealed mycotic granulomas in the dermis that occasionally penetrated into the skeletal muscle. These pathological characteristics were consistent with UM caused by the oomycete Aphanomyces invadans in Southeast Asia, Japan, Australia, and the United States. For specific identification, six isolates from ulcerated fish were cultured and prepared for molecular characterization using established diagnostic methods. Ribosomal RNA gene sequence analysis identified three isolates as Aphanomyces invadans, one as the oomycete Achlya bisexualis, and two as the ascomycete Phialemonium dimorphosporum. A more extensive survey of 67 ulcerated skin samples from fish collected between 1998 and 2003 was performed using a polymerase chain reaction assay specific for Aphanomyces invadans. Of these, 26 (38.8%) samples from seven fish species and nine collection locations were positive. Confirmation of UM associated with Aphanomyces invadans represents new host records in Florida for the sheepshead Archosargus probatocephalus, striped mullet Mugil cephalus, white mullet Mugil curema, silver perch Bairdiella chrysoura, black drum Pogonias cromis, largemouth bass Micropterus salmoides, and American shad Alosa sapidissima.     

Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhea

Forty-six Escherichia coli strains isolated from post-weaning diarrhea of pigs were analysed for their phenotypic and genotypic properties. The isolates were of serogroups O138, O139, and O141 and most of them possessed hemolytic activities. PCR analysis showed that 34 of the isolates harboured the genes for shiga toxin 2e and 32 strains possessed the genes for heat-stable enterotoxins I and II. Ten strains had the fedA gene of F18 fimbriae. The genetic relationships among all isolates were tested by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR analyses. Using the RAPD test with two different primers, six fingerprints were distinguished whereas the ERIC analysis revealed only three DNA patterns. Some strains possessing identical phenotypic and genotypic virulence determinants exhibited distinct RAPD profiles and some isolates with different pathogenic markers showed the same RAPD and ERIC pictures. Thus, RAPD, and to a less extent ERIC techniques, revealed intra- and interserogroup genotypic variations among the E. coli strains analyzed.     

Physiological properties and classification of strains of Treponema sp. isolated from pigs in poland

Fifty-one treponemas were isolated from pigs. Twenty-three isolates with typical morphology and growth characteristic were beta hemolytic, enteropathogenic, produced indole and with exception of three strains did not ferment fructose. These strains were classified as typical T. hyodysenteriae and were usually isolated from pigs with symptoms of mucohemorrhagic diarrhoea. The seventeen other isolates were weakly beta hemolytic after 48 h incubation, enteropathogenic, 12 out of 17 produced indole, 10 out 17 fermented fructose. These strains were usually isolated from pigs with symptoms of gray-green diarrhoea and classified as T. hyodysenteriae 2 biotype or intermediate type. They may be compared with Treponema sp. isolated by Taylor et al. Eleven non enteropathogenic strains showed typical characteristic for T. innocens. Gas chromatography analysis of the fatty acids production from glucose, showed that all isolated treponemas produced acetate and butyrate. Typical T. hyodysenteriae produced additionally propionate. Strains of T. hyodysenteriae biotype 2 produced propionate or isobutyrate as well.     

Phenotypic and genetic characterisation of bacteriocin-producing strains of Staphylococcus aureus involved in bovine mastitis

Fifty strains of Staphylococcus spp. isolated from bovine mastitis cases in several herds from different Argentinian provinces were screened for antimicrobial substances. Twelve strains exhibited a high antagonistic activity against the indicator strain (Corynebacterium fimi) and were chosen for further characterisation. The antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). These strains were identified as S. aureus by the amplification of the femA gene. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. Eleven strains carried a plasmid with a size similar to that of pRJ6 (8.0kb), which encodes aureocin A70, a bacteriocin produced by the Brazilian S. aureus strain A70 isolated from commercial milk. The other strain harboured a much larger plasmid. PCR experiments, using specific primers for amplification of the bacteriocin operon found in pRJ6, showed that all strains had the expected 525bp amplicon, suggesting that the bacteriocin produced may be related to aureocin A70. The genomic DNA of all Bac(+) strains was then analysed by pulsed-field gel electrophoresis (PFGE) in order to investigate clonal relationships amongst strains. Based on the results of PFGE experiments, 10 out of the 12 Bac(+) strains belonged to the same clone. The remaining two strains are possibly related to the prevalent clone. The aureocin A70 producer-strain belonged to a distinct clone.     

Molecular characterisation of Shiga toxin-producing Escherichia coli O157 strains isolated in Poland

Ten Escherichia coli O157 strains isolated from cattle and children in Poland were investigated by the use of molecular biological methods. All strains possessed the intimin and enterohaemolysin genes and harboured the genetic determinants for Stx2 toxin (five isolates), Stx1 toxin (two strains) or both (three isolates). The genetic relatedness of the strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with Xbal and Notl. Nine closely related RFLP patterns were observed. Comparison of bovine and human E coli O157 isolates based on the analysis of Xbal and Notl digested profiles showed that all strains belonged to one genetic cluster. These results indicate that cattle must be considered as a possible source of human E coli O157 infection in Poland.     

Virulence markers and genetic relationships of Shiga toxin-producing Escherichia coli strains from serogroup O111 isolated from cattle

Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae theta, alpha variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC-hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.     

Detection and genetic characterisation of vanA‐containing Enterococcus strains in healthy Lusitano horses

Lusitano horses were investigated in order to detect the presence of vancomycin‐resistant enterococci. vanA isolates showed high level vancomycin (Minimum inhibitory concentration; MIC ≥128 mg/l) and teicoplanin resistance (MIC 64 mg/l), as well as resistance to ciprofloxacin, erythromycin and tetracycline. The tet(L) and erm(B) genes, associated with tetracycline and erythromycin resistance, respectively, were found in all vanA isolates. The intestinal tract of Lusitano horses can be a potential reservoir for vanA‐containing enterococci.     

Identification of the crayfish plague fungus Aphanomyces astaci by polymerase chain reaction and restriction enzyme analysis

To characterise the DNA of the crayfish plague fungus Aphanomyces astaci, Saprolegniales (Oomycetes), primers were developed to amplify a 1050bp segment of the 28S rDNA region. Restriction enzymes were applied to the amplicon obtained, to distinguish A. astaci from 12 fungal species belonging also to the Saprolegniales and five more distantly related fungi. Most of the fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. A. astaci DNA was distinguishable from the DNA of other fungal species tested by using the primers developed plus restriction enzymes AluI, HindIII and AvaI.Prior to this study, methods for A. astaci-species determination, e.g. spore production and infection experiments, required a protracted period to yield results; the method described in this study is quicker.     

Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish

The specialized crayfish parasite Aphanomyces astaci causes the devastating crayfish plague in European crayfish. Even though A. astaci sporulation has been thoroughly studied under pure culture conditions, little is known about the sporulation dynamic from its live host. Our purpose was to investigate the A. astaci spore dynamic in its native parasite-host relationship by monitoring the sporulation from carrier crayfish into the ambient water using agent specific qPCR. American signal crayfish (Pacifastacus leniusculus) with known positive carrier status were housed individually and communally in two experimental set-ups using multiple replicates and different temperatures. Water samples were collected weekly, and spore numbers were quantified. We demonstrate here that live latent carrier crayfish continuously released a moderate number of A. astaci spores (~2700 spores per crayfish/week) in the absence of death and moulting events. In contrast, a pronounced sporulation increase was seen already one week prior to death in moribund crayfish, suggesting a crayfish plague-like condition developing in weakened or stressed individuals. Significantly more spores were produced at 18°C compared to 4°C, while a negative correlation was detected between spore numbers and temperatures rising from 17 to 23°C. This study is the first attempt to quantify the spore release from carrier crayfish on the basis of qPCR applied on water samples, and demonstrate that the approach successfully unravel A. astaci sporulation patterns. The results emphasize that carrier crayfish pose a constant infection risk to highly susceptible crayfish species regardless of crayfish life cycle state.     

Preliminary characterisation of paramyxovirus isolated from a parrot.

A virus, designated 0121, which was isolated from a parrot, was shown to be a paramyxovirus, which was serologically related to the paramyxoviruses Bangor/flinch/N. Ireland/73 (Bangor) and Yucaipa/chicken/California/60(PMY). However, the 0121 virus differed in several properties from both the PMY and the Bangor viruses. The virus was not pathogenic for chickens. I was designated Paramyxovirus: Parrot/England/0121/74.     

Limited genetic diversity of Aerococcus viridans strains isolated from clinical and subclinical cases of bovine mastitis in Slovakia

The Aerococcus viridans isolates from bovine mastitis in Slovakia were isolated and characterized by classical microbiological and biochemical, and molecular techniques including IGS-PCR and rep-PCR, ARDRA and 16S rDNA gene sequencing. The substantial variability of antibiotic resistance patterns was observed. The majority of strains were resistant to beta-lactam antibiotics, the resistance to tetracycline was observed in 3 tested strains, resistance to lincomycin was found in 4 strains and practically all tested strains were sensitive to neomycin and ciprofloxacin. While variable at a phenotypic level, no significant genetic variability among A. viridans isolates was detected by molecular DNA based methods. The data obtained suggest that a few A. viridans strains spread among cow's population in Slovak farms.     

Antigenic and genetic characterisation of Newcastle disease viruses isolated from outbreaks in domestic fowl and turkeys in Great Britain during 1997

Antigenic and genetic analyses of viruses from the 11 outbreaks of Newcastle disease in Great Britain, 12 of the outbreaks in Northern Ireland and the single outbreak in the Republic of Ireland which occurred in 1997, indicated that they were all essentially similar. In addition, the viruses from the British Isles were very similar to viruses isolated from three outbreaks in pheasants in Denmark between August and November 1996, from a goosander in Finland in September 1996, from an outbreak in chickens in Norway in February 1997, and from an outbreak in chickens in Sweden in November 1997. Viruses from outbreaks in other countries during 1995 to 1997 could be distinguished antigenically and/or genetically from the 1996 to 1997 Scandinavian/British Isles isolates, as could viruses responsible for two separate outbreaks in caged birds in quarantine premises in Great Britain in March 1997. Minor nucleotide differences in the 413-base region of the fusion gene and the 187-base region of the haemagglutinin-neuraminidase gene sequenced in this study allowed the 1996 to 1997 Scandinavian/British Isles isolates to be divided into groups. These groups broadly corresponded to the clusters of disease outbreaks, but suggested that the discrete outbreak in Scotland was probably the result of virus spread from Northern Ireland. Overall, the antigenic and genetic analyses of these viruses were consistent with the theory that the virus was introduced into the British Isles by migratory birds moving from north-east Europe. However, it was not possible to rule out other sources, such as the movement of pheasants from Denmark.     

Antibiogram of staphylococcal strains isolated from milk and milk-products

A study of the resistance patterns of 248 staphylococcal isolates from milk and milk products to eight antimicrobial agents using the disc method showed that 80 (32.3%) were resistant to sulphafurazole, 75 (30.2%) to penicillin G, 63 (25.4%) to ampicillin, 23 (9.3%) to cloxacillin, 19 (7.7%) to tetracycline, 17 (6.9%) to streptomycin, 14 (5.6%) to erythromycin and 12 (4.8%) to chloramphenicol. One hundred and sixteen (46.8%) of the 248 staphylococcal isolates were sensitive to all the agents tested. A significant percentage (P less than 0.05) of the isolates from raw milk were resistant to erythromycin, sulphafurazole, cloxacillin, penicillin G and streptomycin compared to isolates from fermented milk. The minimum inhibitory concentrations (MIC) as determined by the tube method for isolates resistant by the disc method, were greater than or equal to 16 micrograms for ampicillin in 5 (11.9%) isolates; greater than or equal to 40 micrograms for cloxacillin, 9 (36.0%) and greater than or equal to 12 international units for penicillin G amongst 12 (22.2%) isolates. MIC values of greater than or equal to 40 micrograms were recorded for 9 (90.0%), 9 (69.2%), 8 (73.7%) and 7 (70.0%) isolates to chloramphenicol, streptomycin, erythromycin and tetracycline respectively. The relatively high level of resistance to antimicrobial agents is a reflection of misuse or abuse of these agents in the environment.