The use of culture, pooled samples and PCR for identification of herds infected with Brachyspira hyodysenteriae.
ABSTRACT The sensitivity of culturing Brachyspira hyodysenteriae was determined after sampling with swabs from porcine fecal specimens inoculated with tenfold dilutions of a field strain of these microbes. After storage of swabs, Brachyspira hyodysenteriae was recovered throughout the first 3 weeks after inoculation from feces with more than 140 cells/g. Viable spirochetes could still be recovered after up to 83 days of storage from feces, with 1.4 x 10(6) cells or more per gram. Culture for Brachyspira spp. was performed on 285 rectal swabs, which were pooled in batches of five. The number of pooled samples positive for B. hyodysenteriae corresponded with the sum results of individual analysis of the corresponding collections of five samples. A PCR system based on the tlyA gene of B. hyodysenteriae was developed and tested on primary cultures of pooled samples. The results of the PCR assay showed a 97% correlation with the culture results. The prevalence of Brachyspira spp. was determined in five swine herds and found to be highest among breeding gilts and boars aged 13-16 weeks and among 6-12-week-old weaned pigs. In contrast, Brachyspira spp. were only rarely found in sows, which may reflect the development of immunity by adult pigs to all species of the genus.
- SourceAvailable from: Stefanie Barth
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- "TlyA is probably linked with the virulence since mutant strains with a disrupted, nonfunctional tlyA gene were of lower virulence in a mouse infection model (ter Huurne et al., 1992b). Among isolates from Sweden the tlyA gene was solely found in B. hyodysenteriae isolates (Fellströ m et al., 2001). Accordingly in Poland and Spain tlyA PCRs were used to detect this species although it is uncertain if the gene is exclusively and invariably present in B. hyodysenteriae (Hidalgo et al., 2010; Pławin´ska et al., 2004). "
ABSTRACT: The distribution of many genes encoding virulence and virulence life-style (VL-S) factors in Brachyspira (B.) hyodysenteriae and other Brachyspira species are largely unknown. Their knowledge is essential e.g. for the improvement of diagnostic methods targeting the detection and differentiation of the species. Thus 121 German Brachyspira field isolates from diarrhoeic pigs were characterized down to the species level by restriction fragment length polymorphism analysis of the nox gene and subsequently subjected to polymerase chain reaction detecting VL-S genes for inner (clpX) and outer membrane proteins (OMPs: bhlp16, bhlp17.6, bhlp29.7, bhmp39f, bhmp39h), hemolysins (hlyA/ACP, tlyA), iron metabolism (ftnA, bitC), and aerotolerance (nox). For comparison, B. hyodysenteriae reference strains from the USA (n=7) and Australia (2) were used. Of all genes tested only nox was detected in all isolates. The simultaneous presence of both the tlyA and hlyA/ACP was restricted to the species B. hyodysenteriae. The hlyA infrequently occurred also in weakly hemolytic Brachyspira. Similarly to tlyA and hlyA all B. hyodysenteriae strains contained the ferritin gene ftnA which was also found in two Brachyspira intermedia isolates. OMP encoding genes were present in B. hyodysenteriae field isolates in rates of 0% (bhlp17.6, bhmp39h), 58.1% (bhlp29.7), and 97.3% (bhmp39f). Since the study revealed a high genetic heterogeneity among German B. hyodysenteriae field isolates differentiating them from USA as well as Australian strains, targets for diagnostic PCR were limited to the nox gene (genus specific PCR) as well as to the species specific nox(hyo) gene and the combination of hlyA and tlyA which allow to specifically detect B. hyodysenteriae.Veterinary Microbiology 10/2011; 155(2-4):438-43. DOI:10.1016/j.vetmic.2011.09.032 · 2.73 Impact Factor
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- "These seem to be cases with low numbers of bacteria, 288 which could be isolated in culture, but their quantity was below the threshold of 289 detection by PCR. It has been shown that culture has a high sensitivity with a 290 detection limit as low as 140 bacterial cells per gram faeces (Fellström et al., 291 2001) which is clearly superior to the detection threshold of the PCRs used in the 292 M a n u s c r i p t 13 present study. Second, an even higher proportion of cases was positive by PCR 293 but negative by culture. "
ABSTRACT: The prevalence of infections with different Brachyspira species was assessed in 202 pigs with various chronic herd problems using different methods. Twenty-seven pigs (13.4%) were positive for Brachyspira spp. with at least one of the methods used. The highest number of positives was identified with mucosal scraping-PCR (23), followed by PET-PCR (22) and bacteriological-biochemical analysis (15). With the exception of three cases of B. pilosicoli infections, only weakly pathogenic Brachyspira species were identified. The majority was B. murdochii, followed by B. innocens and B. intermedia. Concurrent infections with two or more Brachyspira species were common and accounted for 37.1% of the total. Presence of weakly haemolytic Brachyspira was associated with wasting and diarrhoea in a number of cases. This investigation shows that infections with weakly haemolytic Brachyspira spp. may contribute to colonic pathology in pigs with chronic herd problems and that mixed infections seem to occur more frequently than previously noticed.Veterinary Microbiology 09/2008; 134(3-4):311-7. DOI:10.1016/j.vetmic.2008.08.017 · 2.73 Impact Factor
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- "rapid and reliable diagnosis (Atyeo et al., 1998; Fellström et al., 2001; La et al., 2003). Nevertheless, culture and/or PCR-based testing is time consuming, relatively costly, and may not identify pigs that intermittently excrete low numbers of spirochaetes. "
ABSTRACT: Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera (n=1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.Veterinary Microbiology 07/2008; 133(1-2):98-104. DOI:10.1016/j.vetmic.2008.06.003 · 2.73 Impact Factor