Prevalence,disease associations and risk factors for colonization with intestinal spirochaetes (Brachyspira spp.) in flocks of laying hens in north-eastern Italy

The present study investigated the occurrence of anaerobic intestinal spirochaetes of the genus Brachyspira in laying hen flocks in Treviso province, north-eastern Italy, with respect to prevalence, spirochaete species present, disease associations and risk factors for colonization. A total of 450 faecal samples from 45 sheds on 29 laying hen farms were cultured for intestinal spirochaetes. Nineteen sheds on 12 farms contained chickens with symptoms consistent with avian intestinal spirochaetosis, including reduced egg production, wet litter and/or pasty vents. Spirochaetes were isolated from 157 (34.8%) samples from 21 (72.4%) farms, and from 32 (71.1%) sheds. From these positive samples, 189 spirochaetal isolates were speciated using three polymerase chain reaction assays and a restriction fragment polymorphism analysis of 16S rDNA polymerase chain reaction products. Overall, 52 (27.5%) isolates were identified as pathogenic Brachyspira intermedia, 26 (13.8%) as pathogenic Brachyspira pilosicoli, 93 (49.7%) as non-pathogenic (Brachyspira innocens/Brachyspira murdochii), and 18 (9.6%) were unidentified. Faeces from 14 sheds (31%) on 10 farms (34.5%) contained B. intermedia and/or B. pilosicoli, and disease consistent with avian intestinal spirochaetosis was observed in nine of these sheds on seven farms. There was a significant association (P=0.042) between the presence of spirochaetes and using deep pits rather than conveyor belts for manure disposal. Sheds housing chickens >40 weeks of age were significantly more likely to contain spirochaetes (P=0.048) and pathogenic species (P=007) than sheds housing younger chickens. A significant association (P=0.02) was found between infection with pathogenic spirochaetes and reduced egg production.     

Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia

Faecal samples (n = 1786) from chickens in broiler breeder (n = 28), layer (n = 22) or broiler (n = 19) flocks in the eastern states of Australia were cultured for intestinal spirochaetes. Overall, birds in 42.9% of broiler breeder and 68.2% of layer flocks were colonized with spirochaetes, but no birds in broiler flocks were infected. Colonization rates in infected flocks ranged from 10 to 100% of birds sampled. Faeces from colonized flocks were on average 14% wetter than those from non-colonized flocks. There was a highly significant association between colonization with spirochaetes and the occurrence of wet litter and/or reduced production. A subset of 57 spirochaete isolates from birds in 16 flocks were identified to the species level using a panel of polymerase chain reaction tests. Isolates from nine (56%) of these flocks were spirochaetes that are known to be pathogens of poultry: Serpulina pilosicoli was isolated from birds from five flocks, birds from two flocks were infected with Serpulina intermedia, and in two other flocks both species were identified. Isolates from the other seven flocks belonged to other Serpulina species, which are currently of unknown pathogenicity. This study indicates that infections with intestinal spirochaetes are a common but currently under-diagnosed cause of wet litter and/or reduced egg production in broiler breeder and layer flocks in Australia.     

Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed “B. pulli” also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.     

High prevalence of Brachyspira spp. in layers kept in alternative husbandry systems associated with frequent species variations from end of rearing to slaughter

A longitudinal survey was conducted to investigate the presence of Brachyspira species in layer flocks. A total of 66 layer flocks kept in alternative husbandry systems were sampled at three time points: end of rearing, at peak of lay and at end of lay. Content from caecal samples of freshly killed birds was cultured at each sampling time point and processed for further investigations. Gross pathological lesions in caeca were recorded during post mortem investigation. Spirochaetes were isolated from 50 flocks: three flocks were positive at all three sampling points, 25 flocks at two and 22 flocks at one sampling point, respectively. The presence of Brachyspira spp. could not be related to specific gross pathological caecal lesions or antibiotic treatments. The number of positive flocks increased with the age of birds. Furthermore, organic flocks were more often positive than flocks from barn systems. In total 80 spirochaetal cultures were obtained. B. intermedia (43.8%) was the most common species, followed by B. pulli (13.8%) and B. pilosicoli (12.5%). Brachyspira murdochii and B. innocens were found in 5.0% and 2.5%, respectively, whereas 11.3% of Brachyspira isolates could not be identified to species level. In 11.3% of the samples mixed infections were detected. Finally, the longitudinal survey revealed for the first time a possible shift in the Brachyspira species in a substantial number (32%) of layer flocks during their lifetime.     

Survival of intestinal spirochaete strains from chickens in the presence of disinfectants and in faeces held at different temperatures

This study aimed to evaluate the efficacy of some commonly used disinfectants in inactivating the pathogenic avian intestinal spirochaetes Brachyspira intermedia and Brachyspira pilosicoli, and to examine spirochaete survival in chicken caecal faeces held at 4°C, 25°C or 37°C. Six disinfectants were evaluated at their recommended working concentrations: alkaline salts, quaternary ammonium, iodine as an iodophor, chlorine from a chlorine-release agent, glutaraldehyde and hydrogen peroxide. All but alkaline salts inactivated two different concentrations of both spirochaete species in less than 1 min in the presence of organic matter. Both spirochaete species at three different cell concentrations survived in caecal faeces at 37°C for between 2 and 17 h. B. intermedia tended to survive for longer than B. pilosicoli, but the maximum survival time for both species at 4°C was only 72 to 84 h. Hence, avian intestinal spirochaetes are rapidly inactivated by several common disinfectants, and their survival time in chicken caecal faeces is much less than has been reported for porcine intestinal spirochaetes in porcine faeces. It should be relatively easy to break the cycle of infection between batches of laying birds by resting sheds for a few days, and by using disinfectants on any residual faecal matter.     

Antimicrobial susceptibility of Brachyspira spp. isolated from commercial laying hens and free-living wild mallards (Anas platyrhynchos)

In vitro antimicrobial susceptibility to tylosin, valnemulin, tiamulin, doxycycline, lincomycin and ampicillin was investigated by broth dilution in 48 Brachyspira spp. isolates from commercial laying hens (n=30) and free-living wild mallards (Anas platyrhynchos) (n=18). Presumed pathogens (Brachyspira alvinipulli, Brachyspira intermedia, Brachyspira pilosicoli), commensals (Brachyspira murdochii, Brachyspira innocens, “Brachyspira pulli”), and isolates of undetermined species affiliation were included. The laying hens had not been exposed to therapeutic levels of antimicrobials for at least 50 weeks before sampling, and low levels of environmental antimicrobial exposure were presumed in mallards. No isolates with decreased susceptibility to tylosin, valnemulin, tiamulin or doxycycline were found. Decreased susceptibility to lincomycin (minimum inhibitory concentration 16 µg/ml) was detected in two isolates (Brachyspira sp.) from laying hens. Five isolates showed decreased susceptibility to ampicillin (minimum inhibitory concentration 16 to >32 µg/ml), including two “B. pulli” and one B. alvinipulli from laying hens, and isolates of B. pilosicoli and “B. pulli” from mallards. Decreased susceptibility to ampicillin was associated with β-lactamase activity in four isolates. A new variant of a class D β-lactamase gene designated bla _oxa-192 was identified in a B. pilosicoli isolate of mallard origin. This is the first time the genetic basis for antimicrobial resistance is described in Brachyspira spp. from a free-living wild bird. Isolates displaying decreased susceptibility to ampicillin were accompanied by fully susceptible isolates of the same species or other genotypes within three laying hen flocks. This underlines the need for performing antimicrobial susceptibility tests on single clones/genotypes, and to analyse multiple isolates from the same flock.     

Typhlocolitis associated with spirochaetes in duck flocks

The aetiology of increased mortality observed in two breeder duck flocks (Flock A consisting of 3500 laying ducks and Flock B comprising 4300 laying ducks) during the first egg-laying season was studied. In Flocks A and B, 773 ducks and 715 ducks (18.4% and 16.6%) died within a 24-week and a 20-week period, respectively. Death was preceded by clinical signs including movement difficulties, lack of appetite and depression lasting for 1 to 2 days. Diarrhoea was not observed. On gross pathological examination, the ducks were found to have haemorrhagic to fibrinonecrotic typhlocolitis, renal degeneration accompanied by fibrosis and mineralization, hepatic and splenic amyloidosis, and swelling of some of the metatarsal and phalangeal joints. Histopathological and immunohistochemical examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. On the basis of their cultural and biochemical properties and polymerase chain reaction sequencing analysis, four out of seven spirochaete strains isolated from the ducks (Flock A) by culture on special media under anaerobic conditions were identified as Brachyspira hyodysenteriae, and five out of eight strains (Flock B) were identified as Brachyspira pilosicoli. This is the first report on the isolation of B. hyodysenteriae and B. pilosicoli from laying ducks affected by fibrinonecrotic typhlocolitis.     

Zinc bacitracin enhances colonization by the intestinal spirochaete Brachyspira pilosicoli in experimentally infected layer hens

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 40 individually caged 22-week-old laying hens. Another 10 birds were sham-inoculated with sterile broth. All chickens received a commercial layer diet, but 10 infected birds had 50 parts/10 6 zinc bacitracin (ZnB) incorporated in their food. Birds were kept for 7 weeks, and faecal moisture, egg numbers, egg weights and body weights were recorded weekly. B. pilosicoli was isolated from the faeces of only three of the 30 inoculated birds receiving the diet without ZnB, whereas seven of the 10 inoculated birds receiving ZnB in their diet were colonized. This difference in colonization rate was highly significant ( P = < 0.001). Dietary ZnB at 50 parts/10 6 therefore predisposed to colonization by B. pilosicoli. Despite colonization, no significant production differences were found between the birds in the three groups.     

Vaccination of chickens with the 34 kDa carboxy-terminus of Bpmp72 reduces colonization with Brachyspira pilosicoli following experimental infection

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of a variety of species of mammals and birds, and may result in colitis, diarrhoea and reductions in growth rate. Naturally occurring infections in chickens are largely confined to adult laying and breeding birds. In this study, the 34 kD carboxy-terminus of the prominent outer membrane protein Bmp72 of B. pilosicoli was expressed as a histidine-tagged recombinant protein and used to immunize two groups (B and C) of 15 individually housed layer chickens. Vaccination was with either 100?μg (B) or 1?mg (C) protein emulsified with Freund’s incomplete adjuvant delivered into the pectoral muscles, followed three weeks later by 1?mg of protein in phosphate buffered saline delivered via crop tube. Two weeks later these and 15 non-vaccinated positive control birds (group A) housed in the same room were challenged via crop tube with B. pilosicoli avian strain CPS1. B. pilosicoli was detected in the faeces of all control birds and in 14 of the vaccinated birds in each vaccinated group at some point over the 30-day period following challenge. Colonization was delayed and the duration of excretion was significantly reduced (P?=?0.0001) in both groups of vaccinated birds compared to the non-vaccinated control birds. Fewer immunized birds had abnormal caecal contents at post mortem examination compared to non-vaccinated birds, but the difference was not statistically significant. This study indicates that recombinant Bmp72 C-terminus has potential to be developed for use as a vaccine component to provide protection against B. pilosicoli infections.

RESEARCH HIGHLIGHTS

• Laying chickens were immunized with recombinant Brachyspira pilosicoli membrane protein Bpmp72.

• Immunized birds had a highly significant reduction in the duration of colonization.

• Fewer immunized than control birds had abnormal caecal contents after infection.

• Bpmp72 showed potential for use as a novel vaccine component for B. pilosicoli.

     

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^R) 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 μm and 218 μm, 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.     

Experimental infection of laying hens with Serpulina intermedia causes reduced egg production and increased faecal water content

Serpulina intermedia strain HB60, isolated from an Australian hen with diarrhoea, was used to infect 10 individually caged 14-week-old laying hens. Another 10 birds were sham inoculated with sterile broth. Birds were kept for 16 weeks, and faecal water content, egg production and body weights recorded. Strain HB60 was isolated from the faeces of nine of the infected birds at irregular intervals throughout the experiment, and from their caeca at slaughter. Infected birds tended to be lighter and their faeces, on average, were significantly wetter (by 2.85%; P < 0.002) than those of the controls. Significant reductions in mean number of eggs laid (1.4/week; P < 0.002) and mean egg weights (1.16 g; P < 0.05) were recorded in infected birds. Colonization did not induce any characteristic pathological changes. S. intermedia is potentially an economically significant cause of reduced egg production, and wet faeces in layer and broiler breeder flocks.     

Experimental infection of broiler breeder hens with the intestinal spirochaete Brachyspira ( Serpulina ) pilosicoli causes reduced egg production

The pathogenic potential of the anaerobic intestinal spirochaetes Brachyspira ( Serpulina ) pilosicoli and Brachyspira innocens was evaluated in adult chickens. Thirty 17-week-old Cobb broiler breeder hens were individually caged in three groups of 10 birds. Control birds (group A) were sham inoculated with sterile broth medium. Birds in the other two groups (groups B and C) were inoculated, respectively, with an isolate of B. innocens or of B. pilosicoli . Birds were monitored daily, and killed at 41 weeks of age. Infection had no consistent effect on body weight gain, but inoculation with B. pilosicoli resulted in a transient increase in faecal water content. B. innocens infection had no effect on egg production, but B. pilosicoli infection caused a delayed onset of laying, and a highly significant reduction in egg production over the first 11 weeks of lay. This study confirms that B. pilosicoli can cause serious egg production losses in adult chickens, while B. innocens is not obviously pathogenic.     

Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.Human intestinal spirochetosis (HIS) is a histologically defined condition of the human distal intestinal tract characterized by helical microorganisms attached at one end to the surface epithelium of the colonic mucosa. This association forms a so-called “false brush border” (13). While certain Brachyspira spp. are recognized as the causative agents of swine dysentery and porcine intestinal spirochetosis (3, 11), their clinical significance and pathogenic potential for humans remain unclear. Various studies have reported on the association of these bacteria with intestinal disorders such as chronic watery diarrhea (8, 12) and on clinical improvement following antimicrobial therapy (29). In contrast, others have suggested that intestinal spirochetes are harmless commensals in humans (7). The prevalence of HIS ranges from 1.2% (23) to >40% (17, 34), depending on presumable patient risk factors, such as origin from developing countries, immunodeficiency, or homosexuality. Recently, Peruzzi et al. (30) discovered a prevalence of 12% in a selected population, indicating that HIS is an important differential diagnosis for patients with chronic gastrointestinal disorders and risk factors.Two intestinal spirochetes have been identified in humans so far: Brachyspira aalborgi (15) and Brachyspira pilosicoli (36). Both species require selective media, and B. aalborgi is an extremely slow growing, fastidious microorganism that requires anaerobic incubation for as long as 4 weeks (4, 34). For this reason, HIS is primarily diagnosed histopathologically. The fuzzy basophilic fringe, 4 to 7 μm thick, on the epithelial layer of the colonic mucosa is visible in hematoxylin-and-eosin (HE)-stained histological sections and is considered pathognomonic for HIS. Tissue morphology usually remains unaltered, and no inflammatory reaction is observed (20).However, diagnosis of HIS on the basis of HE staining requires experienced laboratory personnel and accurate interpretation, and silver staining is often needed to confirm the diagnosis (10). Therefore, a significant portion of cases may be missed, especially since B. pilosicoli might also colonize the epithelium without the characteristic end-on attachment, impeding identification by light microscopy at low magnification (24). Furthermore, histopathology does not provide information about the identity of the microorganisms, thereby precluding epidemiological studies. More importantly, the inability to identify the organism also hampers accurate therapy, since the intestinal spirochetes are suspected to differ in virulence, and therefore some cases of HIS may require antibiotic therapy more urgently than others (5, 29).The genus Brachyspira currently comprises seven established species and several proposed species. Among some Brachyspira species, the high level of 16S rRNA gene conservation precludes interspecies differentiation by 16S rRNA gene methods and necessitates further molecular analyses. However, all known species isolated from humans can be identified and differentiated via their 16S rRNA genes. In line with the genetic variation discovered in Brachyspira species, such as Brachyspira hyodysenteriae (2) and Brachyspira innocens (9), recent molecular studies have also identified human Brachyspira strains genetically distinct from B. aalborgi and B. pilosicoli. This heterogeneity was confirmed by sequencing of 16S rRNA (14, 21) or NADH oxidase (25) genes, fluorescence in situ hybridization (FISH) (16, 17), or multilocus enzyme electrophoresis (33). Pettersson et al. (31) analyzed biopsy samples from two adults by 16S rRNA gene sequencing and consequently proposed to divide the B. aalborgi lineage into three phylogenetic clusters, including the type strain, B. aalborgi 513A, in the first cluster.The extent of intraspecies genetic variation in human intestinal spirochetes is unclear and difficult to estimate, because few complete 16S rRNA gene sequences are available. Further epidemiologic and phylogenetic investigations are needed to elucidate spirochete genetic diversity and to facilitate the evaluation of the molecular diagnostic tools that are presently available.FISH is a microscopic method that allows simultaneous visualization and identification of microorganisms. Jensen and colleagues (3, 16, 17) designed several genus- or species-specific oligonucleotide probes targeting the 16S or 23S rRNA of Brachyspira spp. and applied them successfully to porcine and human intestinal biopsy specimens. However, no genus-specific 16S rRNA-directed probe for diagnostic use targeting all Brachyspira spp. known so far has been developed.In the present study, intestinal biopsy specimens from five patients with possible HIS were analyzed histopathologically and by culture, FISH, PCR amplification, and 16S rRNA gene sequencing. Biopsy specimens from a healthy control group were analyzed retrospectively by histopathology and FISH. The purpose was (i) to acquire further information about the phylogenetic structure of the Brachyspira spp. associated with HIS, (ii) to design a FISH probe covering all Brachyspira spp. based on the currently available sequence data, and (iii) to evaluate FISH as a fast and robust diagnostic screening tool for HIS.     

Infection of broiler parent hens with avian intestinal spirochaetes: Effects on egg production and chick quality

Broiler parent hens were inoculated with avian intestinal spirochaetes several weeks before the onset of egg production. The infection persisted, wet droppings developed, and egg production, mean egg weight and carotenoid contents of the eggs were decreased. Hatching eggs were collected and incubated. In broilers which hatched from these eggs, reduced gain in body weight at 2 and 3 weeks of age, wet droppings, low plasma carotenoid concentration and elevated alkaline phosphatase activity in the blood plasma were observed. Spirochaetes were not detected in these broilers. These findings demonstrated the deleterious effects on chick quality of parental infection with avian intestinal spirochaetes. Avian intestinal spirochaetosis was diagnosed in about 2.5% of all submissions from reproductive flocks in 1991.     

Evaluation of tiamulin and lincomycin for the treatment of broiler breeders experimentally infected with the intestinal spirochaete Brachyspira pilosicoli

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 30 individually caged 22-week-old Cobb 500 broiler breeder hens. Another 10 birds were sham-inoculated with sterile broth. All birds failed to become colonized. At 29 weeks of age, all birds were transferred to a diet containing 50 parts/10 6 zinc bacitracin (ZnB) and were re-challenged with the same B. pilosicoli strain at 32 weeks of age, weekly for 5 weeks. The majority of the inoculated birds then became colonized, confirming previous findings that ZnB can increase susceptibility to colonization with B. pilosicoli. The control group remained uninfected. Infected groups tended to have an increased faecal water content and faecal staining of eggshells. Ten birds were then treated by crop tube with 25 mg/kg body weight tiamulin for 5 days, and 10 birds with 20 mg/kg body weight lincomycin for 5 days. Both treatments removed the infection, while untreated birds remained infected. The results support previous observations that ZnB at 50 parts/10 6 in the diet increases the susceptibility of birds to B. pilosicoli infection, and demonstrated the usefulness of both tiamulin and lincomycin for treatment of infection with B. pilosicoli in adult birds.     

Specific Antibody and Immunoglobulin Responses after Intestinal Colonization of Germ-Free Piglets with Non-Pathogenic Escherichia coli O86

Colonization of the gut with components of commensal microflora profoundly affects the development of the immune system. The aim of the present study was to investigate mucosal and systemic B cell responses during the first few days after intestinal association of colostrum-deprived piglets reared in germ-free (GF) conditions with non-pathogenic Escherichia coli O86. Specific intestinal anti-E. coli antibodies (Ab), among which IgA Ab prevailed, were found 4 days after colonization (72 % of standard) and their amount decreased 11 days later reaching 22 % of standard. In contrast to mucosal Ab, specific serum Ab remained at the level of GF animals at day 4 (less than 10 % of standard) and markedly increased 15 days after colonization (156 % of standard). In addition to the occurrence of specific Ab, increased amounts of total immunoglobulins (Ig) of all isotypes were detected in sera and intestinal washings. Using the ELISPOT method an increased number of IgM, IgG and IgA-secreting lymphocytes were found in spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP) in colonized animals as compared to GF piglets. Contrary to cells from these lymphatic organs, B cells from thymus were not affected by E. coli stimulation. Our results show that at the onset of intestinal colonization, non-pathogenic E. coli specifically and polyclonally stimulate the mucosal and systemic humoral immunity, but relatively soon after stimulation, mucosal-specific responses in gut decreases, indicating the possible beginning of inhibition mechanisms (oral tolerance).     

Development and validation of a drag swab method using tampons and different diluents for the detection of members of Salmonella in broiler houses

Members of the genus Salmonella represent a significant public health concern and also a colonizer of commercial poultry. Therefore, the early detection and management of colonized broiler breeders and their progeny is essential. There have been numerous methods for farm-based detection, with gauze-based drag swabs being the most commonly used. In the present study, the wet (boiled water, buffered peptone water and double-strength skin milk) tampon was compared with the gauze to determine the recovery rate (10² colony-forming units/swab) of five common poultry serovars of Salmonella and after cold (4°C/48 h) storage. The recovery was found to be equivalent when tested using the ISO6572:2002 method, for all diluents (Cohen's κ =1.0; sensitivity = 1.0; specificity = 1.0). The subsequent field trial (n = 15 farms) compared the tampon drag swab (TDS) with a statistically appropriate (90% confidence, detect 10% prevalence) number of faecal swabs (n = 22), which also showed high agreement between the TDS and faecal sampling (κ = 0.86; McNemar's χ² = 1.0; sensitivity = 0.9; specificity = 1.0). However, direct faecal sampling showed a wider diversity of serovars of Salmonella than the corresponding TDS. The TDS is a very sensitive, readily available and cost-effective screening method for salmonellas in broiler breeder houses. This TDS technique may be used for routinely screening of broiler houses, and faecal sampling would only be used to confirm colonization or contamination, and to measure flock serovar variance.     

Drinking water as the source of Campylobacter coli infection in grandparent heavy breeders

The aim of the present study was the molecular identification of a common source of infection of Campylobacter coli in two grandparent breeder farms. Campylobacter jejuni and C. coli were isolated from well water and cloacal swabs from grandparent chickens. Colonies were genotyped using restriction fragment length polymorphism-flaA gene, pulsed field gel electrophoresis and multi-locus sequence typing. The same genotype of C. coli was found in both farms and in the well from which drinking water was supplied to the farms. The well water was epidemiologically linked as the source of C. coli infection. The molecular identification for epidemiological source-tracking of C. coli in breeder farms could aid in combating the colonization of this pathogen and therefore to reduce their incidence in human campylobacteriosis.     

A survey of Eimeria species in commercially-reared chickens in France during 1994

In a survey of chicken coccidia in France during 1994, samples of litter were collected from 41 farms. On 31 of these farms, eimerian oocysts were abundant enough to allow monitoring of their numbers in the litter. Peak total oocyst counts on these farms ranged from 16,200 to 1,254,000/g of litter, but no coccidiosis was observed. The chickens reared without anticoccidial agents in their food (poulets biologiques) produced higher and earlier peak oocyst counts in litter than the chickens given medicated food (poulets labels). The oocysts in litter samples from 22 farms (13 poulet biologique, five poulet label, two standard broiler, one breeder and one layer) of the original 41 were identified. Six of the seven eimerian species known to parasitize chickens were found, using combinations of five methods (oocyst morphology, intestinal lesions, enzyme electrophoresis, growth in embryonating eggs and prepatent time). Multispecific infections predominated (95% of 22 farms), up to six species occurring together. Of farms where oocysts were detected, the percentages with each species were: Eimeria acervulina (100%), E. mitis (82%), E. tenella (77%), E. maxima (73%), E. praecox (45%) and E. brunetti (27%). These appear to be the first definite records of E. mitis and E. praecox for France. Although E. necatrix was not found in this survey, it had recently been detected by other workers in France, so that all seven chicken Eimeria species were known to be contemporaneous.     

The incidence and economic impact of the Escherichia coli peritonitis syndrome in Dutch poultry farming

The incidence and economic impact of the Escherichia coli peritonitis syndrome (EPS), characterized by acute mortality, were estimated in chicken egg-producing farms in the Netherlands in 2013. The incidence was significantly higher (P?相似文献