Erysipelas in laying hens is associated with housing system.

Following the change from conventional cages to non-cage housing systems and furnished cages, which in Sweden was finalised by 2005, problems caused by Erysipelothrix rhusiopathiae increased in laying hen flocks. This study aimed to investigate possible associations between housing systems for laying hens and outbreaks of erysipelas. Also, sera from 129 flocks in different housing systems, collected during 2005-2007, were analysed for the presence of antibodies to E rhusiopathiae using an indirect ELISA test. Antibodies were detected in all housing systems. The mean flock absorbance values from free-range flocks were significantly higher than corresponding values from other housing systems. Data on the Swedish laying hen population were compared with the recorded number of erysipelas outbreaks during 1998-2011. Outbreaks occurred on 15 farms with indoor litter-based systems (n=87 farms in 2011). No outbreak was diagnosed on farms with flocks in conventional or furnished cages. The results indicate that the risk for an outbreak was higher in free-range systems than in indoor litter-based systems, and lowest for flocks housed in cages. Absence of erysipelas in the majority of subsequent flocks on the affected farms suggested that proper measures, including vaccination, were undertaken.

Occurrence of pathogens in wild rodents caught on Swedish pig and chicken farms.

A total of 207 wild rodents were caught on nine pig farms, five chicken farms and five non-farm locations in Sweden and surveyed for a selection of bacteria, parasites and viruses. Lawsonia intracellularia and pathogenic Yersinia enterocolitica were only detected in rodents on pig farms (9% and 8% prevalence, respectively) which indicate that these agents are more likely to be transmitted to rodents from pigs or the environment on infected farms. Brachyspira hyodysenteriae (1%), Brachyspira intermedia (2%), Campylobacter jejuni (4%), Campylobacter upsaliensis (2%), leptospires (7%) and encephalomyocarditis virus (9%) were also detected from rodents not in contact with farm animals. Giardia and Cryptosporidium spp. were common, although no zoonotic types were verified, and Salmonella enterica was isolated from 1/11 mice on one farm but not detected by PCR from any of the rodents. Trichinella spp. and Toxoplasma gondii were not detected.

Typing of Brachyspira spp. from rodents, pigs and chickens on Swedish farms.

The aim of the current study was to look for evidence of possible cross-species transmission of Brachyspira species between rodents and farm animals. To do this, previously collected and characterised Brachyspira isolates from rodents, pigs and chickens on the same farms were analysed by random amplified polymorphic DNA (RAPD). Isolates with similar RAPD banding patterns were further typed by pulsed-field gel electrophoresis (PFGE). Identical isolates of Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira murdochii and Brachyspira innocens from pigs and rodents and of B. murdochii from laying hens and rodents were found, indicating cross-species transmission at farm level. PFGE data from rodent isolates of Brachyspira hyodysenteriae were compared with PFGE data from previously typed field isolates of B. hyodysenteriae from pigs with swine dysentery and isolates from mallards (Anas platyrhynchos). Three of four isolates of B. hyodysenteriae from rodents were similar to porcine field isolates by PFGE. PCR analyses of the plasmid-encoded and potential virulence determinants rfb genes B, A, D and C showed that they were present in isolates of B. hyodysenteriae of porcine, mallard and rodent origin.

Occurrence of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis in small wild rodents.

Rodents are a potential source of pathogenic Yersinia enterocolitica and Y. pseudotuberculosis. In order to study this, 190 rodents were captured and sampled on seven pig farms (n=110), five chicken farms (n=55) and six other locations (n=25) in Sweden. Pigs from three of the pig farms were also sampled (n=60). Pathogenic Y. enterocolitica was detected by TaqMan PCR in about 5% of rodent samples and 18% of pig samples. Only rodents caught on pig farms tested positive for the pathogen. Y. enterocolitica bioserotype 4/O:3 strains isolated from the rodent and pig samples were compared by pulsed-field gel electrophoresis and revealed a high degree of similarity, which was confirmed by random amplified polymorphic DNA. Y. pseudotuberculosis was only detected in one rodent sample. Thus, rodents may be vectors for the transmission of pathogenic Y. enterocolitica to pigs, acting as carriers rather than a reservoir, and should therefore remain an important issue in hygiene control measures on farms.

Characterization and epidemiological relationships of Spanish Brachyspira hyodysenteriae field isolates.

This research aimed to describe the genetic and phenotypic diversity of 74 Spanish Brachyspira hyodysenteriae field isolates, to establish epidemiological relationships between the isolates and to confirm the presence of tiamulin-resistant isolates in Spain. For these purposes, we performed biochemical tests in combination with diagnostic PCR analysis for the identification of Brachyspira spp. and for detection of the smpA/smpB gene. We also used antimicrobial susceptibility tests, random amplified polymorphic DNA (RAPD) and a new pulsed-field gel electrophoresis (PFGE) protocol. The combination of RAPD and PFGE allowed the study of epidemiological relationships. Both indole-negative and tiamulin-resistant isolates of B. hyodysenteriae are reported in Spain for the first time. The genetic analyses indicated a relationship between these Spanish isolates and indole-negative isolates previously obtained from Germany and Belgium.

Experimental challenge of mallards (Anas platyrhynchos) with Brachyspira hyodysenteriae and "Brachyspira suanatina" isolated from pigs and mallards.

Brachyspira hyodysenteriae, the aetiological agent of swine dysentery, and a recently proposed and closely related enteropathogenic spirochaete "Brachyspira suanatina", originally isolated from pigs or mallards (Anas platyrhynchos), were used to inoculate week-old mallard ducklings orally or cloacally. The colonization rate, clinical outcome, faecal dry matter content, blood leucocyte counts and gross, microscopical and electron microscopical features 14-16 days post-inoculation were investigated at necropsy examination. Strains of "B. suanatina" of pig and mallard origin and B. hyodysenteriae of mallard origin colonized the ducklings by oral inoculation, and colonization was also established by cloacal inoculation with a "B. suanatina" strain of mallard origin. The porcine reference strain of B. hyodysenteriae (B204) failed to colonize the birds. Unchallenged contact birds in one of the challenge groups were readily colonized by a strain of "B. suanatina" of mallard origin. The proportion of colonized birds differed significantly between the challenge groups (P < 0.0001). For each challenge group, the inoculum and a randomly selected subset of recovered isolates had an identical biochemical profile and banding pattern by randomly amplified polymorphic DNA (RAPD) analysis. None of the birds developed clinical signs of gastrointestinal disease during the trial. The faecal dry weight contents, body weights and total leucocyte and heterophil counts did not differ between the various groups of birds. At the microscopical and electron microscopical levels, the caecal mucosa in some of the Brachyspira culture-positive birds had sharply demarcated epithelial cell changes and there were features of irreversible cell damage in crypt necks coinciding with spirochaetal infiltration of the mucosa. The crypts in Brachyspira culture-positive birds were deeper than in culture-negative birds (median: 237 microm and 218 microm, respectively, P = 0.019). This challenge model was well suited for use in mallards and consistent with previous findings that strongly haemolytic Brachyspira spp. may cross the species barrier between pigs and birds.

Phenotypic and molecular characterization of Brachyspira spp. isolated from laying hens in different housing systems.

Several species of intestinal spirochaetes, Brachyspira (B.) alvinipulli, B. intermedia and B. pilosicoli, may cause reduced egg production and faecal staining of eggshells in chickens. The aim of this study was to characterize potentially pathogenic and presumably non-pathogenic Brachyspira spp. from commercial laying hens. Selective culture, phenotyping, PCR and 16S rRNA gene sequencing were used and clinical data were collected. Phenotypic profiles were obtained for 489 isolates and 351 isolates obtained after subculture, and 30 isolates were selected for molecular characterization. Seven isolates were positive by a B. intermedia-specific PCR based on the nox gene, and two were positive in a B. hyodysenteriae-specific 23S rRNA gene based PCR. By comparative phylogenetic analysis in combination with PCR and phenotyping, seven isolates were identified as B. intermedia, eight isolates as B. innocens, five as B. murdochii, and three isolates each as B. alvinipulli and "B. pulli". The remaining four isolates could not be assigned to any presently recognized species. Co-infection with several species or genetic variants of Brachyspira spp. were detected in some flocks and samples, suggesting a high level of diversity. Organic flocks with access to outdoor areas were at higher risk (RR=2.3; 95% CI 1.5-3.6) for being colonized than chickens in other housing systems. No significant differences between colonized and non-colonized flocks were found regarding clinical parameters, i.e. mortality, egg production, faecally contaminated eggshells, and wet litter. Our results show that a combination of traditional laboratory diagnostics, molecular tests and phylogeny is needed for identification of Brachyspira sp. from chickens.

Development of a multilocus sequence typing scheme for intestinal spirochaetes within the genus Brachyspira.

The purpose of this study was to evaluate a multilocus sequence typing (MLST) scheme for intestinal spirochaetes of the genus Brachyspira. Eight loci mainly coding for enzymes previously used in multilocus enzyme electrophoresis analysis of Brachyspira species were examined in 66 Brachyspira field isolates and type/reference strains. The isolates and strains were recovered from pigs, birds, dogs and a mouse and originated from seven European countries, the USA and Canada. Forty-six isolates represented recognized Brachyspira species and 20 represented provisionally designated species or isolates that have not been classified. Only two loci gave PCR products for all 66 strains and isolates, but amplicons for seven loci were obtained for 44 of the isolates. Sequences for each locus had a DNA allelic variation of 30-47 and an amino acid allelic variation of 14-47 that gave rise to the same number of sequence and amino acid types (58) for the strains and isolates studied. A population snapshot based on sequence and amino acid types showed a close phylogenetic relationship amongst the porcine isolates from the same geographical regions, and indicated a close evolutionary relationship between isolates recovered from pigs and mallards. A general concordance was obtained between the MLST groupings and classifications based on culture and biochemical tests, 16S rDNA sequence analysis and random amplified polymorphic DNA analysis. This is a first step towards establishing an MLST system for use in identifying Brachyspira species and determining relationships between individual strains and species in the genus.

Survey on the occurrence of Brachyspira species and Lawsonia intracellularis in children living on pig farms.

The occurrence of Brachyspira species and Lawsonia intracellularis was investigated by PCR analyses of faeces from 60 children living on European pig farms. In addition, 60 other children were included as controls. Two samples were positive for B. aalborgi but B. pilosicoli and L. intracellularis were not demonstrated.

Consecutive pathological and immunological alterations during experimentally induced swine dysentery - a study performed by repeated endoscopy and biopsy samplings through an intestinal cannula.

The development of intestinal lesions after inoculation with Brachyspira hyodysenteriae was followed by repeated endoscopy and biopsy sampling through a caecal cannula. Seven eight-week-old pigs were cannulated and inoculated, two were cannulated but not inoculated, and two pigs were inoculated but not cannulated. Endoscopy, biopsy, and blood sampling to determine SAA (serum amyloid A), haptoglobin, cortisol, and WBC counts were performed at scheduled time-points. At the third day of disease, endoscopy showed a hyperaemic, perturbed mucosa and excessive amount of mucus. Histologically, crypt hyperplasia, depletion of goblet cell mucus, and erosions were noted. Simultaneously, elevated acute phase proteins and circulating monocytes, and decreased number of intraepithelial CD3(+) cells were observed. After five days the pigs recovered. Intestinal lesions were demarcated and interspersed among apparently normal mucosa and blood parameters returned to initial values. Endoscopy through an intestinal cannula made it possible to follow the development of intestinal alterations in vivo and describe the sequential events during the course of swine dysentery. The number of animals used in a study could thus be minimised and the precision of the experiment increased.

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli.

Traditional culture and biochemical tests (CBT) were compared with PCR for sensitivity and detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in seeded faeces and clinical samples from diarrhoeic pigs. A duplex PCR system was developed based on primers detecting the tlyA-gene of B. hyodysenteriae and the 16S rRNA-gene of B. pilosicoli. Sensitivities for the PCR system were determined on seeded faeces, using DNA that had been recovered from primary cultures or extracted directly from faeces. Compared to CBT, PCR applied to DNA extracted directly from faeces lowered the sensitivity by a factor of 1000 to 10,000. B. hyodysenteriae and B. pilosicoli detection was compared for CBT and PCR using 200 clinical samples. CBT detected more B. hyodysenteriae isolates in the clinical samples than PCR, but fewer B. pilosicoli positive samples. An atypical strongly haemolytic isolate was detected only by CBT.

The prevalences of Brachyspira spp. and Lawsonia intracellularis in Swedish piglet producing herds and wild boar population.

The aim of the present study was to survey the prevalences of the enteric pathogens Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in Swedish growing pigs and in the Swedish wild boar population and to relate these findings to clinical signs. The study included 105 randomly selected herds, constituting approximately one third of Swedish herds with a herd size of >100 sows. The herds were located all over the country. In these herds, growth promoters were not used and pigs sampled were not subjected to any medication. From each herd, samples were taken from 10 growing pigs aged 8-12 weeks, corresponding to approximately 2.5% of all growing pigs present in the herd at the sampling occasion. If possible, the samples were taken from pigs with diarrhoea. Forty-eight faecal samples and 71 rectal swabs were also taken from free-living wild boars (31 piglets, 19 growers and 21 adult animals) at shooting. The samples were analysed by culture and biochemical tests for the presence of Brachyspira spp. and by nested PCR for the presence of L. intracellularis. Brachyspira hyodysenteriae was not demonstrated in any sample. Brachyspira intermedia was detected in 22 samples originating from 15 herds, Brachyspira innocens/Brachyspira murdochii was detected in 370 samples from 82 herds and B. pilosicoli was detected in 134 samples originating from 34 herds. In 21 herds and in 534 samples, no Brachyspira spp. were detected. Lawsonia intracellularis was demonstrated in 285 samples from 50 herds. Further, 418 samples from conventional herds were negative with respect to L. intracellularis and in 345 samples the PCR had been inhibited. All samples from the wild boars were negative for Brachyspira spp., 12 of 48 samples were negative for L. intracellularis, and in 36 wild boar samples, the PCR was inhibited.

Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test.

There is no ring test for quality assessment available in Europe for diagnostics and antimicrobial susceptibility testing of the fastidious, anaerobic bacteria of the genus Brachyspira. Therefore, an international ring test for Brachyspira spp. was performed once a year during 2002-2004. Two sets of coded samples were prepared and distributed on each occasion. One set comprised six swabs dipped in pig faeces spiked with Brachyspira spp. intended for diagnostics. The other set comprised two pure strains intended only for susceptibility testing. All methods used were in-house methods. The species used were Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira innocens, Brachyspira murdochii and Brachyspira intermedia. In most cases, the correct Brachyspira spp. were detected. However, the results showed that Brachyspira spp. could be difficult to identify, especially if two Brachyspira spp. were mixed or if the concentration of Brachyspira in faeces was low. Additionally, some laboratories reported Brachyspira growth in control samples that were not seeded with any spirochaetes. The lowest detection level was 10(2) bacteria/ml faeces for both B. hyodysenteriae and B. pilosicoli. The susceptibility tests performed showed that disc diffusion was not recommendable for Brachyspira spp. Extended antimicrobial dilution series gave most congruent results. The diversity of the results highlights the importance of ring tests for a high quality of diagnostics and antimicrobial susceptibility tests for Brachyspira spp. This is the first ring test described for Brachyspira spp.

Antimicrobial resistance in Brachyspira pilosicoli with special reference to point mutations in the 23S rRNA gene associated with macrolide and lincosamide resistance.

A point mutation in the 23S rRNA gene causes macrolide and lincosamide resistance in Brachyspira hyodysenteriae. The possible occurrence of a similar mutation in Brachyspira pilosicoli was studied and the MICs of six antimicrobial agents for Swedish field isolates of B. pilosicoli were determined. Of 10 isolates with high MICs of macrolide and lincosamide antibiotics, six had a mutation in nucleotide position 2058 or 2059 in the 23S rRNA gene as compared to the wild type of Escherichia coli, whereas none of 10 tylosin-susceptible isolates were mutated in this region. The mutations found in position 2058 were A --> T transversions, and in position 2059 either A --> G transitions or A --> C transversions. The MICs at which 90% of the B. pilosicoli field isolates were inhibited by tylosin, erythromycin, clindamycin, virginiamycin, tiamulin, and carbadox, were >256, >256, >4, 4, 2, and 0.125 microg/ml, respectively. In conclusion, point mutations in positions 2058 and 2059 of the 23S rRNA gene can cause macrolide and lincosamide resistance in B. pilosicoli. Macrolide resistance is widespread among Swedish field isolates of B. pilosicoli. Notably also a few isolates with elevated MICs of tiamulin were found.

Routine diagnostics of Lawsonia intracellularis performed by PCR, serological and post mortem examination, with special emphasis on sample preparation methods for PCR.

The aim of this study was to find suitable and reliable tools for demonstrating Lawsonia intracellularis in routine clinical diagnosis. Firstly, a method to prepare tissue samples before a polymerase chain reaction (PCR) was evaluated in pigs submitted for necropsy. Secondly, seven different faecal preparation methods and four different DNA polymerases were tested in single or nested PCR, with co-amplification of a mimic molecule. Thirdly, in selected pigs submitted for necropsy, tissue and faecal samples were examined histopathologically and by PCR, and blood samples were analysed serologically. Detection of L. intracellularis in tissue preparations by PCR showed good specificity and correlated to lesions found at necropsy. The sensitivity in spiked tissue samples was 10(1)-10(2) mimic molecules per tube. In faecal samples, nested PCR on boiled lysate gave the best result with a sensitivity of 10(2)-10(3) mimic molecules per reaction tube. However, because of the time-consuming procedure and the increased risk for contamination, a commercially available kit was preferred for routine diagnoses, despite a somewhat lower detection rate in subclinically infected pigs. In a few cases, the serological results differed from those obtained by PCR and by necropsy but the reason for this is not clear. This study indicates that the best method for diagnosis of acute enteritis in growers is PCR on faecal or tissue samples. To determine the presence of the bacteria in a herd, serology or repeated faecal sampling for PCR from target animals, or both, should be used.

Antimicrobial susceptibility testing of porcine Brachyspira (Serpulina) species isolates.

No standardized method for susceptibility testing of Brachyspira spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine Brachyspira spp. representing biochemical groups I, II, and III. Group I corresponds to Brachyspira hyodysenteriae, group II corresponds to B. intermedia, and group III corresponds to B. murdochii and B. innocens. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the B. hyodysenteriae isolates (n = 72) were inhibited in the broth dilution test by tiamulin (0.25 micro g/ml), tylosin (>256 micro g/ml), erythromycin (>256 micro g/ml), clindamycin (>4 micro g/ml), virginiamycin (4 micro g/ml), and carbadox (0.06 micro g/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.

Diarrhoea in the growing pig - a comparison of clinical, morphological and microbial findings between animals from good and poor performance herds.

Diarrhoea among growing pigs (8-13 weeks old) is a significant problem in many herds. Nine herds with poor performance and diarrhoea among growing pigs were selected on the basis of their piglet mean age at a body weight of 25 kg, compared to the overall mean age in Swedish herds. In addition, four herds with good average performance and no problems with diarrhoea were selected. Pigs were necropsied and samples for histology and microbiology were collected. Based on the necropsy findings, the pigs from the good performing herds were all judged to be healthy. The presence of Brachyspira pilosicoli and Lawsonia intracellularis was significantly correlated to poor performing herds and the results indicate that these microbes are main pathogens involved in enteric diseases among Swedish grower pigs. In addition, concomitant infections with other presumptive pathogens were commonly found.

Human intestinal spirochetosis diagnosed with colonoscopy and analysis of partial 16S rDNA sequences of involved spirochetes.

DNA was extracted from colonic biopsies of 33 patients with and three without evidence of intestinal spirochetosis (IS) in the large bowel. The biopsies were subjected to PCR. A pair of primers, generating a 207 bp fragment, were designed to detect specifically the 16S rDNA gene of Brachyspira. PCR products of the expected size were obtained from 33 samples with histologic evidence of IS. The PCR amplicons were used for sequencing. The sequences obtained were aligned to the corresponding 16S rRNA sequences of five type strains of Brachyspira. The sequences of 23 PCR products were 99-100% identical with the corresponding B. aalborgi type strain sequence. Two cases showed 99-100% sequence similarity with the type strain of B. pilosicoli P43/6/78. Six cases could not be referred to any of the known species of Brachyspira. Two PCR products gave incomplete sequences.

The use of culture, pooled samples and PCR for identification of herds infected with Brachyspira hyodysenteriae.

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.

Classification of Brachyspira spp. isolated from Swedish dogs.

Brachyspira spp. were isolated from 21 of 32 sampled dogs (66%) in a colony of Swedish beagle dogs with a history of diarrhea and from 3 of 17 Swedish pet dogs (17%) with diarrhea. All Swedish isolates were weakly beta-hemolytic and gave a negative indole reaction. Eighty-eight percent showed negative alpha-galactosidase and hippurate reactions, but a positive beta-glucosidase reaction. Two isolates were hippurate positive with a negative beta-glucosidase reaction. One additional German isolate diverged by showing a positive indole reaction in combination with a positive hippurate reaction. Sequencing of 16S rDNA indicated that the hippurate-positive isolates belonged to the species Brachyspira pilosicoli. Four representative isolates were examined using pulsed-field gel electrophoresis (PFGE) and compared with six reference strains and five porcine isolates of Brachyspira spp. The canine isolates clustered together in the PFGE analysis. Necropsy examination of a culture-positive B. pilosicoli colony-raised beagle dog revealed macro- and microscopical lesions of colitis with numerous spiral-shaped bacteria in the lumens of the crypts, in goblet cells and within the colonic epithelium.