 |
Rural Industries Research & Development Corporation |
|| Home || Search || Contact || Publications Eshop || Privacy Statement ||
Rural Industries Research & Development Corporation
Control of Intestinal Spirochaete Infections in Chickens
by David J Hampson & Carol P. Stephens August 2002
RIRDC Publication No 02/087 RIRDC Project No UMU-23J
Background
Over the past 15 years intestinal spirochaetal
bacteria of the genus Brachyspira (formerly
Serpulina) gradually have become
recognised as common potential pathogens of commercial layer and broiler
breeder chickens. The role of species of these bacteria as important pathogens
of pigs has long been accepted. The organisms have fastidious growth requirements
and need anaerobic conditions for their laboratory isolation: this complicates
diagnosis and helps explain why it has taken so long for their significance
to be appreciated in poultry. Several distinct species of these bacteria
infect poultry, but not all of these are pathogens. Despite an increased
awareness of intestinal spirochaete infections in chickens (Avian Intestinal
Spirochaetosis: AIS), relatively few studies have been carried out on the
condition.
Studies in Europe and the United States have found that infection of the caeca with intestinal spirochaetes is associated with the occurrence of wet droppings, delayed onset of egg laying, faecal staining of eggshells, reduced egg weights and reduced carotenoid content of eggs. Broiler chicks hatched from infected hens show reduced weight gain.
In Australia, studies of intestinal spirochaetal infection in commercial poultry flocks have shown that colonisation with these organisms is common. Moreover, spirochaetes are recovered significantly more frequently from layer and broiler breeder flocks with diarrhoea and reduced egg production than from clinically normal flocks. The results of pathogenicity trials in broilers and layers has shown that Australian strains of both Brachyspira intermedia and B. pilosicoli isolated from chickens have the capacity to cause disease and loss of production. Despite these findings, little is currently known about how to control the infections.
The current study was a collaborative effort involving Professor David Hampson and his colleagues at Murdoch University in Western Australia and Carol Stephens and her colleagues at the Toowoomba Veterinary Laboratory in Queensland. The study was undertaken with the aim of providing means to help control the infections. The objectives that were designed to achieve this involved improving the diagnostic infrastructure for AIS that is available in Australia, including evaluating new molecular diagnostic and strain typing techniques for the organisms, by assessing their in vitro antimicrobial sensitivity, and by testing antimicrobials and a commercial exogenous enzymes for their effectiveness in treating the infections in experimentally-infected layers and broiler breeders. The study also had the added benefits of improving collaboration between the laboratories, and providing a project through which Carol Stephens will obtain a PhD degree in the area through the Division of Veterinary and Biomedical Sciences at Murdoch University.
Diagnostic submissions
Over the two years of the study, diagnostic submissions for suspected AIS were received from 36 layer or broiler breeder flocks, most of which were in Queensland. A positive diagnosis of AIS was made in 13 cases (28%), with both B. intermedia and B. pilosicoli identified as being the major agent involved. The isolates were examined further in other parts of the project.
Polymerase chain reaction (PCR) for identification of Brachyspira intermedia
A polymerase chain reaction assay amplifying a portion of the 23S rDNA gene was evaluated for identification of B. intermedia. A total of 34 strains of B. intermedia isolated from chickens and pigs and 195 strains of other related spirochaete species were tested. The optimised assay correctly identified all the B. intermedia strains, but generated 11 false positive reactions, giving a test sensitivity of 100% and a test specificity of 94.3%. The new PCR is a useful addition to the diagnostic armoury for identifying intestinal spirochaete isolates from chickens.
Pulsed field gel electrophoresis (PFGE) for strain typing of Brachyspira intermedia
Pulsed field gel electrophoresis was developed to investigate diversity amongst 34 B. intermedia strains from chickens and pigs. All strains had distinct DNA banding patterns, although three isolates from chickens on the same farm appeared closely related. The strains showed considerable genetic diversity. The most closely related chicken and pig strains shared only 62% similarity. These results suggest that transmission of strains between chickens and pigs is not very likely. The typing technique itself will prove useful for studying transmission of strains within and between flocks.
In vitro antimicrobial sensitivities of avian intestinal spirochaetes
The susceptibilities of 80 intestinal spirochaete isolates from chickens was tested in vitro against seven antimicrobial drugs that are known to inhibit growth of intestinal spirochaetes from pigs. There were some spirochaete species differences in their susceptibility to the drugs, but in general there was a high degree of susceptibility of the isolates to most of the drugs. This is fortunate since it means that there are antimicrobials available that potentially can be used to treat AIS in Australian flocks.
Multilocus enzyme electrophoresis for establishing genetic relationships between avian intestinal spirochaetes
Sixty two intestinal spirochaete isolates, including 56 from 15 Australian flocks, were analysed using multilocus enzyme electrophoresis (MLEE). The work was undertaken to help support the species identification of chicken isolates, and in particular to help assess the sensitivities and specificities of the PCR techniques being used and developed in the project. The study also provided additional information about the genetic relatedness and diversity of intestinal spirochaete isolates from Australian chickens, and compared these with non-Australian isolates.
For B. pilosicoli isolates there was a perfect correlation between MLEE grouping (m) and PCR results, with 16 isolates from five flocks divided into 6 electrophoretic types (ETs). The 13 B. intermedia isolates were defined by their location in MLEE group g. Of these 13, only four were positive in the nox PCR, but all were positive in the new 23S rRNA PCR. The isolates were from six Australian flocks, and belonged to 11 ETs. As with B. pilosicoli, strains from the same farm differed in some cases. Hence multiple strains of these species may be present in certain flocks, and this may make treatment more difficult if they have different drug sensitivities or vary in some other important properties. The nox PCR for the combined species B. innocens/B. murdochii was positive with nine isolates. Seven of these belonged to MLEE group d, identified as B. murdochii, one was in group b (a new unnamed group), and one in group a (also new and unnamed). Ten other isolates in group d (B. murdochii) were negative in the PCR, as were three in group e (B. innocens). These results indicate that the B. murdochii/B. innocens nox PCR is specific, but has poor sensitivity for detection of these two species. This probably does not present a major diagnostic problem, since neither species is considered pathogenic in poultry. However, these isolates all came from flocks with wet litter or production problems, and it is still necessary to clarify their pathogenic potential by testing them under experimental conditions.
Overall, eight new genetic groups of intestinal spirochaetes were identified by MLEE, although each was only represented by one or two isolates. Further work is required to clarify whether these groups represent new species, and whether they have any significance in relation to disease production. No isolates of B. alvinipulli were identified amongst the Australian isolates. To date B. alvinipulli has only been isolated from a single diseased flock in the USA (Swayne et al., 1995).
Experimental infections of layer hens and response to antimicrobials and enzyme
Three experiments were conducted, with the aim of determining useful means of treating layer hen with AIS.
Experiment 1
Thirty individually caged layer hens were inoculated with Brachyspira intermedia, and 20 control irds remained unchallenged. Birds received a diet containing 100 ppm zinc bacitracin (ZnB), and were monitored for ten weeks. B. intermedia was recovered sporadically from five of the inoculated irds, and there were no significant effects on body weight, faecal water or egg production. ZnB was resumed to be inhibiting spirochaete growth, and when removed from the diet, 18 of the 30 noculated birds rapidly became culture positive. After 4 weeks, 10 of the 30 infected birds were reated with tiamulin at 25mg/kg for 5 days, and 10 were returned to the diet containing ZnB. Birds eceiving tiamulin became spirochaete negative, and maintained their egg production, but reinfection ccurred. The other 20 infected birds had a significant drop in egg production, but those receiving ZnB showed a reduced colonisation by B. intermedia after three weeks. This experiment emonstrates that ZnB at 100ppm in the diet inhibits the growth of B. intermedia. Treatment of nfected birds with tiamulin is effective, but birds can rapidly become reinfected. The study also onfirms that B. intermedia infection in layer hens can reduce egg production.
Experiment 2
Brachyspira intermedia strain HB60 was used to experimentally infect 40 individually caged 22- week-old laying hens. Another 10 control birds were sham inoculated with sterile broth. All chickens eceived an experimental layer diet based on wheat. The infected birds were randomly divided into our groups of 10, with the diet for each group containing either 50 ppm zinc bacitracin (ZnB), 100 pm ZnB, 256g/tonne of dietary enzyme (Avizyme , 1302), or no additive. Birds were kept for six weeks after infection, and faecal excretion of B. intermedia, faecal water content, egg numbers, egg weights and body weights were recorded weekly. Control birds remained uninfected throughout the xperiment. B. intermedia was isolated significantly less frequently from the groups of xperimentally infected birds receiving ZnB at 50 ppm or Avizyme®, than those receiving 100 ppm ZnB, or no treatment. Infected birds had a transient increase in faecal water content in the week ollowing challenge, but no other significant production differences were detected amongst the five roups of birds in subsequent weeks. It was not established how the ZnB at 50 ppm and the dietary nzyme reduced the ability of the spirochaete to colonise, but it may have been by bringing about hanges in the intestinal microflora and/or the intestinal microenvironment.
Experiment 3
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 50ppm ZnB incorporated in their food. Birds were kept for seven 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 colonised. This difference in colonisation rate was highly significant (P =<0.001). Dietary ZnB at 50 ppm therefore predisposed to colonisation by B. pilosicoli. Despite colonisation, no significant production differences were found between the birds in the three groups.
Experimental infection of broiler breeders and response to antimicrobials
Two experiments were conducted to investigate the pathogenic potential of B. pilosicoli in adult hens (broiler breeders), and to determine whether the infection could be treated with two antimicrobials commonly used to treat intestinal spirochaete infections in pigs (tiamulin and lincomycin).
Experiment 1
The pathogenic potential of Brachyspira pilosicoli and Brachyspira innocens was evaluated in thirty 17-week-old Cobb broiler breeder hens individually caged in three groups of 10 birds. Control birds were sham inoculated with sterile broth medium. Birds in the other two groups 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 brief increase in faecal water content. B. innocens 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, whilst B. innocens is non-pathogenic.
Experiment 2
Brachyspira pilosicoli strain CPSp1 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. The birds failed to become colonised. At 29 weeks of age they were transferred to a diet containing 50 ppm ZnB and were rechallenged with the B. pilosicoli strain at 32 weeks of age, weekly for five weeks. The majority of the birds then became colonised. Ten birds were then treated by crop tube with 25 mg/kg
body weight tiamulin for five days, and 10 with 20mg/kg body weight lincomycin for five days. Both these treatments removed the infection, whilst untreated birds remained infected. The results of this experiment support previous observations that ZnB at 50 ppm in the diet increases the susceptibility of birds to B. pilosicoli infection. The study also demonstratess the usefulness of both tiamulin and lincomycin for treatment of infection with B. pilosicoli in adult birds.
Implications
The fact that regular diagnoses of AIS were made in layer and breeder flocks with production problems throughout this two year project emphasises that infection with intestinal spirochaetes is a real problem to the Industries in Australia. The condition is undoubtedly going undiagnosed in many cases due to insufficient awareness on the part of veterinarians and pathologists and a lack of expertise and facilities in diagnostic laboratories around Australia. Clearly both the Industries and the laboratories that service them need to be more aware of AIS as a possible differential diagnosis where wet litter and production drops are involved, and of what facilities are available for diagnosis. The molecular identification and strain typing techniques developed during this project will improve diagnostic capacity in Australia.
In general the spirochaete species remain susceptible to a range of antimicrobial agents, although resistance against tylosin was evident for a number of strains. The different spirochaete species tended to have a different range of sensitivities to some of the drugs tested. Clearly antimicrobial sensitivities need to be monitored on an ongoing basis.
The pathogenicity studies in adult birds confirmed that both B. intermedia and B. pilosicoli are capable of causing loss of production, although complex interactions which may modulate disease expression appear to occur between the spirochaetes and the intestinal microflora. In particular, in-feed zinc bacitracin increased the susceptibility of birds to B. pilosicoli infection, although it offered protection against B. intermedia. These differences emphasise the need for good diagnostic capacity to identify the spirochaete species involved in individual cases of AIS. Furthermore, research is required to understand the basis of these effects, so that improved control measures can be developed. The study demonstrated that both tiamulin and lincomycin can be used to treat AIS. Both drugs are effective, but a problem arises from the potential for birds to become reinfected after treatment has ceased. Tiamulin has recognised incompatibilities with the use of ionophores (for control of cocidiosis), and this incompatibility would need to be considered before any treatment was undertaken. As most of the antibiotics used in this work require veterinary prescription and many are not registered for use in egg laying poultry, producers should seek veterinary advice before using any antibiotics to control intestinal spirochaetes.
Finally, the observation that addition of xylanase to the diet can reduce colonisation by B. intermedia has important ramifications. The mechanisms behind this require further study, but presumably may be linked to changes in viscosity of the intestinal digesta. If this is the case, there are also likely to be dietary influences on AIS . although the possibility of different responses for B. intermedia and B. pilosicoli does need to be investigated.
Recommendations
The current findings should be disseminated
to chicken producers and their veterinarians in Australia. In particular
the potential for zinc bacitracin to increase susceptibility to B.
pilosicoli should be made known. Similarly
the potential beneficial effects of dietary xylanase preparations in reducing
colonisation by B. intermedia in
adult birds should be made known to the Industries. Research funds should
be made available for future research:
|
