Survival of Clostridium perfringens During Simulated Transport and Stability of Some Plasmid-borne Toxin Genes under Aerobic Conditions

Clostridium perfringens is a pathogen of great concern in veterinary medicine, because it causes enteric diseases and different types of toxaemias in domesticated animals. It is important that bacteria in tissue samples, which have been collected in the field, survive and for the classification of C. perfringens into the correct toxin group, it is crucial that plasmid-borne genes are not lost during transportation or in the diagnostic laboratory. The objectives of this study were to investigate the survival of C. perfringens in a simulated transport of field samples and to determine the stability of the plasmid-borne toxin genes cpb1 and etx after storage at room temperature and at 4°C. Stability of the plasmid-borne genes cpb1 and etx of C. perfringens CCUG 2035, and cpb2 from C. perfringens CIP 106526, JF 2255 and 6 field isolates in aerobic atmosphere was also studied. Survival of C. perfringens was similar in all experiments. The cpb1 and etx genes were detected in all isolates from samples stored either at room temperature or at 4°C for 24–44 h. Repeated aerobic treatment of C. perfringens CCUG 2035 and CIP 106526 did not result in the loss of the plasmid-borne genes cpb1, cpb2 or etx. Plasmid-borne genes in C. perfringens were found to be more stable than generally reported. Therefore, C. perfringens toxinotyping by PCR can be performed reliably, as the risk of plasmid loss seems to be a minor problem

Detection and identification by PCR of Clostridium chauvoei in clinical isolates, bovine faeces and substrates from biogas plant

<p>Abstract</p> <p>Background</p> <p><it>Clostridium chauvoei </it>causes blackleg, an acute disease associated with high mortality in ruminants. The apparent primary port of entry is oral, during grazing on pasture contaminated by spores. Cases of blackleg can occur year after year on contaminated pastures. A method to determine the prevalence of <it>C. chauvoei </it>spores on pasture would be useful.</p> <p>The standard method for <it>C. chauvoei </it>detection is culture and biochemical identification, which requires a pure culture. In most muscle samples from cattle dead from blackleg the amount of <it>C. chauvoei </it>in samples is high and the bacterium can easily be cultured, although some samples may be contaminated. Detection by PCR would be faster and independent of contaminating flora.</p> <p>Digested residues from biogas plants provide an excellent fertiliser, but it is known that spore-forming baeria such as <it>Clostridium </it>spp. are not reduced by pasteurisation. The use of digested residues as fertiliser may contribute to the spread of <it>C. chauvoei</it>. Soil, manure and substrate from biogas plants are contaminated with other anaerobic bacteria which outgrow <it>C. chauvoei</it>. Therefore, detection by PCR is would be useful. This study applied a PCR-based method to detect of <it>C. chauvoei </it>in 25 muscle and blood samples, 114 manure samples, 84 soil samples and 33 samples from the biogas process.</p> <p>Methods</p> <p>Muscle tissues from suspected cases of blackleg were analysed both by the standard culture method followed by biochemical identification and by PCR, with and without preculture. To investigate whether muscle tissue samples are necessary, samples taken by swabs were also investigated. Samples from a biogas plant and manure and soil from farms were analysed by culture followed by PCR. The farms had proven cases of blackleg. For detection of <it>C. chauvoei </it>in the samples, a specific PCR primer pair complementary to the spacer region of the 16S-23S rRNA gene was used.</p> <p>Results</p> <p><it>Clostridium chauvoei </it>was detected in 32% of muscle samples analysed by culture with identification by biochemical methods and in 56% of cases by culture in combination with PCR. <it>Clostridium chauvoei </it>was detected in 3 (out of 11) samples from the biogas plants collected before pasteurisation, but samples taken after pasteurisation and after digestion all tested negative. <it>Clostridium chauvoei </it>was not detected in any soil or silage samples and only one manure samples tested positive.</p> <p>Conclusion</p> <p>The diagnostic method used for <it>C. chauvoei </it>was not applicable in estimating the risk of blackleg on particular pastures from manure or soil samples, but found to be highly useful for clinical samples.</p

Identification of nine sequence types of the 16S rRNA genes of Campylobacter jejuni subsp. jejuni isolated from broilers

<p>Abstract</p> <p>Background</p> <p>Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. <it>Campylobacter jejuni </it>is the <it>Campylobacter </it>sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of <it>C. jejuni </it>and <it>C. coli </it>and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of <it>Sma</it>I restricted genomic DNA of the strains.</p> <p>Methods</p> <p>The 16S rRNA genes of 45 strains of <it>C. jejuni </it>and two <it>C. coli </it>strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with <it>Sma</it>I.</p> <p>Results</p> <p>Sequence analyses of the 16S rRNA genes revealed nine sequence types of the <it>Campylobacter </it>strains and the similarities between the different sequence types were in the range 99.6–99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as <it>Campylobacter coli </it>by PCR/REA. The other 10 strains were identified as <it>Campylobacter jejuni</it>. Five of the nine sequence types were also found among the <it>Campylobacter </it>sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains.</p> <p>Conclusion</p> <p><it>C. jejuni </it>and <it>C. coli </it>seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between <it>C. jejuni </it>and <it>C. coli</it>. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only <it>C. jejuni </it>and the other with both <it>C. jejuni </it>and <it>C. coli</it>. Genotyping of the 47 strains by PFGE after digestion with <it>Sma</it>I resulted in 22 subtypes. A potential correlation was found between the <it>Sma</it>I profiles and the 16S rRNA sequences, as a certain <it>Sma</it>I type only appeared in one of the two major phylogenetic groups.</p

Leptospira seroprevalence and associations between seropositivity, clinical disease and host factors in horses

<p>Abstract</p> <p>Background</p> <p>A cross-sectional study was carried out to determine the seroprevalence of different serovars of <it>Leptospira </it>spp. and their association with clinical disease and host factors in Swedish horses.</p> <p>Methods</p> <p>Sera from 2017 horses brought to equine clinics during 1997–98 were investigated. The sera were examined by microscopic agglutination test for the presence of antibodies against the following <it>L. interrogans </it>serovars: Bratislava strain Jez, Icterohaemorrhagiae strain Kantorowicz and Pomona strain Pomona and also <it>L. kirschneri </it>sv Grippotyphosa strain Duyster and <it>L. borgpetersenii </it>sv Sejroe strain M 84. Host factors, disease factors, season, pasture access and outdoor confinement variables were analysed with respect to seropositivity to sv Bratislava and Icterohaemorrhagiae. Multivariable logistic regression was used to model seropositivity to sv Bratislava and Icterohaemorrhagiae (seroprevalence > 8%).</p> <p>Results</p> <p>The seroprevalence, at a cut-off 1:100, were for sv Bratislava (16.6%), Icterohaemorrhagiae (8.3%), Sejroe (1.2%), Pomona (0.5%) and Grippotyphosa (0.4%). In the multivariable analysis, it was demonstrated that seroprevalence increased with age for sv Bratislava and Icterohaemorrhagiae. For sv Bratislava the seasons April – June and October – December and for sv Icterohaemorrhagiae October – December had higher seroprevalences than other seasons. Horses not used for racing had higher levels of seropositivity to sv Bratislava. Furthermore, horses with respiratory problems as well as horses with fatigue had higher levels of seropositivity to sv Bratislava. Ponies and coldbloods, and horses with access to pasture, had lower seroprevalence for sv Icterohaemorrhagiae. Healthy horses had lower seroprevalence for sv Icterohaemorrhagiae, than non-healthy horses.</p> <p>Conclusion</p> <p>There was no significant association between clinical signs and disease and positive titres to sv Bratislava (except for the association between respiratory problems and fatigue and seropositivity to sv Bratislava). The results suggest that horses with increasing age and exposed to factors associated with outdoor life had an increased seroprevalence for sv Bratislava, indicating that horses get infected from outdoor and/or are exposed to shedding from other horses (management dependent). For sv Icterohaemorrhagiae, management possibly plays a role as ponies and coldbloods as well as healthy horses had lower seroprevalence. Overall, the age of the horse should be taken into consideration when evaluating the titre as the average healthy horse has a higher titre than a young horse.</p