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A cross-sectional study was conducted between May and August 2010 to estimate Brachyspira species prevalence and to determine the risk factors associated with Brachyspira species colonisation in laying and broiler breeder flocks in the UK. In total 1175 pooled fresh chicken faecal samples were collected from 235 sheds on150 farms representing different production and housing types. Questionnaires were used to collect data on birds' breed, age, water treatment with bleach or acidifying agents, egg production, egg collection trays, manure removal system, type of litter, nature of birds' droppings, presence of any wet litter or scouring problems and any vaccination programmes for Brachyspira. For the detection of Brachyspira, a species specific multiplex polymerase chain reaction (M-PCR) was used. Brachyspira species were detected in 43% (95% CI 0.352-0.516) of farms with 40% (95% CI 0.336-0.465) of the tested sheds test positive. Across all the tested sheds, pathogenic and non-pathogenic Brachyspira prevalence were 18.09% (95% CI 1.85-3.61) and 56.38% (95% CI 0.739-0.898) respectively, while the prevalence of mixed infection with both pathogenic and non-pathogenic Brachyspira was 25.53% (95% CI 0.204-0.629). Brachyspira pilosicoli, intermedia, alvinipulli and non-pathogenic Brachyspira prevalences across all the sheds were 17.02%, 31.91%, 9.57% and 81.91% respectively. The prevalence of Brachyspira infection was the highest among commercial layer farms (48 farms, 59.26%). Brachyspira infection was significantly associated with weight losses (P=0.035), yellow frothy droppings (P=0.002), wet litter problems (P=0.008) and reduced egg production (P=0.006). Birds' breed and flock age were found to be significantly associated with Avian Intestinal Spirochaetosis (AIS) (P<0.05). This study indicates the wide spread of different Brachyspira species across the UK, highlighting the potential predisposing factors and emphasis the economic significance of the disease.
Avian Intestinal Spirochaetosis (AIS) is a worldwide endemic disease caused by Brachyspira species, affecting chicken laying flocks (Davelaar et al., 1986; Stephen and Hampson, 2001; Bano et al., 2008; Pattison et al., 2008 and Myers et al., 2009). The disease causes wet litter, yellowish brown chronic diarrhoea, pasty vent and stained eggs. In addition, it leads to a reduction in egg shell quality, egg carotenoid contents, egg weight, egg production, and/or delayed onset of laying (Jordan and Hampson, 2008). Brachyspira are slender, motile, fastidious anaerobic, Gram-negative, helical bacteria which grow optimally at 37-42oC and require 2-10 days to grow depending on the sample type (Swayne, 1997).
To date, seven named species and several proposed species of the genus Brachyspira have been identified (Hampson and Swayne, 2008). However, only reliably Brachyspira pilosicoli, Brachyspira intermedia and Brachyspira alvinipulli have been implicated in AIS and are the most commonly reported pathogenic species in chickens in Europe, the USA and Australia (Stephens and Hampson, 2001; Pattison et al., 2008). The presumed non-pathogenic species in chickens are Brachyspira innocens, Brachyspira murdochii, and 'Brachyspira pulli' (Stanton et al., 1998; Hampson and McLaren, 1999; Stephens and Hampson, 2001 and 2002; Råsbäck, 2007; Bano et al., 2008; Feberwee et al., 2008 and Pattison et al., 2008). Interestingly, Brachyspira hyodysenteriae, which is the aetiological agent of swine dysentery, has also been isolated from chickens, but has not been associated with the clinical disease (Feberwee et al., 2008).
The estimated annual loss to the egg industry in the UK due to AIS has been estimated at £15.5 million a year (Burch, personal communication, 2009) but remains uncertain as the extent of pathogen circulation remains unknown. In addition, AIS in broiler breeder flocks causes economic losses as a result of increased feed intake, decreased egg production and medication costs which were estimated at £10,900 to £11,500 per flock per annum (Smit et al., 1998). Furthermore, AIS leads to poor quality of the chick progeny, where the affected broilers show wet droppings, growth retardation, poor weight gain and feed conversion, impaired bone growth and increased number of weak chicks at hatching (Dwars et al., 1993; Smit et al., 1998). The economic losses in a commercial broiler farm as a result of the infection of the breeders flock were estimated at approximately £10,000 per annum based on average broiler number of 50,000 birds and six turnaround cycles per annum (Dwars et al., 1993; Smit et al., 1998).
Avian Intestinal Spirochaetosis was first observed in the UK by Fantham and Harris in 1910 (Fantham, 1910 and Harris, 1930, as cited by Stephens and Hampson, 2001). In the USA, Mathey and Zander successfully isolated spirochaetes from caseous nodules in chickens' caeca (Mathey and Zander, 1955 as cited by Stephens and Hampson, 2001). In addition, in the USA Brachyspira was isolated from the caeca of commercial layer flocks with diarrhoea and reduced egg production (Swayne et al., 1992 and Trampel et al., 1994) and named Brachyspira alvinipulli (Stanton et al., 1998). In the Netherlands, avian intestinal spirochaetes have been reported and isolated from the caeca of laying hens suffering from diarrhoea (Davelaar et al., 1986). Similarly in Australia, Italy, Sweden and the USA, Brachyspira has been isolated from broiler breeders and laying flocks (McLaren et al., 1996, Bano et al., 2008; Jansson et al., 2008 and Myers et al., 2009). All these reports indicate the worldwide spread of AIS.
To date, a number of AIS surveys in the Netherlands, Australia, Italy and the USA have been published. The authors of these studies reported an overall prevalence ranging between 8.5% to 81%, and a within-flock prevalence ranging between 8% to 100% (Dwars et al., 1989; McLaren et al., 1996; Stephens and Hampson, 1999, Phillips et al., 2005; Bano et al., 2008 and Myers et al., 2009). Past studies found association of reduced egg production, enteritis, and wet litter with AIS, where deep pit manure removal system and birds over 40 weeks of age were identified as risk factors for infection (Dwars et al., 1989; McLaren et al., 1996; Stephens and Hampson, 1999, Phillips et al., 2005; Bano et al., 2008 and Myers et al., 2009). Nevertheless, all these studies were focused on the detection of Brachyspira pilosicoli and Brachyspira intermedia only and did not consider Brachyspira alvinipulli which has recently been reported as a pathogenic species in poultry (Stephens and Hampson, 2001; Phillips et al., 2005 and Pattison et al., 2008).
To date in the UK, there is only one report on Brachyspira presence and its effect on egg production (Burch et al., 2009), but there are not any published epidemiological studies investigating risk factors associated with AIS. This is a significant gap in our understanding of AIS epidemiology, hindering the development of effective, population-based control strategies. Therefore a cross-sectional study was designed to estimate the prevalence of infection with Brachyspira pilosicoli, Brachyspira intermedia, Brachyspira alvinipulli and non-pathogenic Brachyspira and to identify the risk factors associated with the disease in laying and breeder broiler flocks in the UK.
Material and methods
A cross-sectional study was designed to estimate the prevalence and to identify risk factors associated with Brachyspira infection. The sample size of 302 farms was calculated assuming an estimated prevalence of 50% with a 5% desired absolute precision at a 95% level of confidence.
Chicken layer farms were recruited, subject to their willingness to participate, through a number of private poultry veterinarians across the country. The veterinarians selected the participating farms using systematic random sampling from their client register..
Data were collected using a questionnaire developed for the purpose of this study and which had been reviewed by five private poultry veterinarians and nine producers.
The questionnaire collected data on birds' breed, age and number, any current or previous medication, ration type, any changes in management system, feed and water consumption, water treatment with bleach or acidifying agents, birds' weight, egg production (quality and quantity), type of egg collection trays, age at start of production, manure removal system, type of litter, type of disinfectant used, nature of birds' droppings, presence of wet litter or scouring problems and any vaccination programmes for Brachyspira.
After farms recruitment, copies of the introductory information letter, bio-bottles (each containing five universal sampling tubes) and copies of the questionnaire were sent to the farmers through their private veterinarians. Samples were collected following a systematic random sampling protocol, where each shed was divided into 10 equal squares and pooled samples were collected from five squares. Each pooled sample consisted of 10 droppings. All recruited flocks were sampled only once, in addition, any suspected AIS frothy droppings were collected.
Samples were sent directly from the farms to the laboratory and were stored at -20 Co until all samples were received.
DNA was extracted and purified from faecal samples by MaxwellÂ® 16 automated System (Promega, UK) using MaxwellÂ® 16 Tissue DNA Purification Kit (Promega, UK). Briefly, pooled samples from each shed were mixed well together in one pot and three samples, approximately 50 mg each, were added to the purification kits. Then the extracted DNA was filtered by using OneStepâ„¢ PCR inhibitor removal kit (Zymo Research, USA) to remove any PCR inhibitors in the samples such as polyphenolic compounds, humic, fulvic or uric acids.
Brachyspira identification by M-PCR
Sample analysis was performed using a Multiplex Polymerase Chain Reaction (M-PCR) (Abdelrahman et al., 2011). The M-PCR primers used were predicted to amplify specific regions of the corresponding genes, which were easily distinguishable by size when electrophoresed. Brachyspira pilosicoli primers amplified a 127bp region of cpn60 gene whereas, the Brachyspira intermedia, Brachyspira alvinipulli, and non-pathogenic Brachyspira primers amplified 403, 250 and 467bp regions of nox gene, respectively. The M-PCR protocol was as follow; 20 Âµl reaction volume contained 10 Âµl mastermix (containing 2 mM MgCl2), 0.2 ÂµM of each primer of 4 primer pairs, 2 Âµl Q solution (QIAGEN), 0.2 Âµl BSA (QIAGEN), 6 Âµl molecular biology grade water (Promega), and 1 Âµl of Brachyspira strain as a template DNA. The cycling condition of the reaction was 1 cycle at 95o C for 15 minute followed by 30 cycles at 95o C for 30 seconds, 63o C for 30 seconds and 72o C for 60 seconds, and finally 1 cycle at 72o C for 10 minutes. PCR products were electrophoresed on a 2% TAE agarose gel to confirm amplicon size and DNA was visualised using a UV transilluminator (Synegene) emitting at a wavelength of 302 nm. The UV transillumiator was coupled with GeneSnap computer software (Synegene) for image retrieval.
Data management and analysis
All questionnaires data were entered into Microsoft Office Access 2007, and after being checked and cleaned, data were imported to Microsoft Office Excel 2007. For uncompleted questionnaires, the respective farms were contacted to obtain the missing data.
Flock age was categorised into four categories as follows : 12-22 weeks, 23-40 weeks, 41-56 weeks and 57-74 weeks, while flock size was categorised into six categories as follows :2,000-5,000 birds, 5,001-7,500 birds, 7,501-12,500 birds, 12,501-25,000 birds, 25,001-45,000 birds and 45,001-75,000 birds. Egg tray type was categorised into plastic and cardboard trays, the latter were also categorised into new and used trays.
For this study a positive farm was defined as a Brachyspira PCR positive farm regardless of Brachyspira species or the number of sheds infected as farms with more than one shed considered positive for Brachyspira if only one shed came positive as the analysis was performed at the farm level.
Statistical analyses, including descriptive, univariable and multi-variables analysis, was carried out with Stata 9.2 (intercooled Stata 9.2 software, StataCorp LP, Texas, USA).. Risk factors analysis initially involved univariable screening using logistic regression models. Predictor variables with unconditional association with the outcome (P<0.2) were included in the multi-variable logistic regression analysis (Dohoo et al., 2009). Backward stepwise multi-variable logistic regression modelling was performed and the best fitted model was identified using likelihood ratio (P<0.05). The possibility of variables to be confounders were assessed based on the literature and the biological relevance to AIS (Dohoo et al., 2009). Confounding between variables was assessed using Mantel-Haenszel method where adjusted and crude odds ratios were compared to detect the confounder and to estimate the degree of confounding. Variables in the final multi-variables logistic regression model were assessed for interaction and the final logistic regression model was assessed by Hosmer-Lemeshow goodness of fit test.
From May to August 2010, pooled fresh chicken faecal samples (n=1175) were collected from numerous sheds (n=235) on 150 farms (n=150) located in 45 different counties in the UK. Eighty-one farms were commercial layers, eight farms were layer breeders and 61 farms were broiler breeders, and different housing systems were represented in the sample: in three farms birds were housed in cages, 74 farms were free range and 73 reared their poultry in barns.
Brachyspira species were detected in 65 of 150 farms (95% CI 0.352-0.516) and 94 of 235 sheds (95% CI 0.336-0.465). Across the tested sheds, prevalence of pathogenic Brachyspira (B. pilosicoli, B. intermedia and B. alvinipulli) was 18.09% (95% CI 1.85-3.61), non-pathogenic Brachyspira was 56.38% (95% CI 0.739-0.898) and 25.53% (95% CI 0.204-0.629) was the prevalence of mixed pathogenic and non-pathogenic Brachyspira. Brachyspira pilosicoli, intermedia, alvinipulli and non-pathogenic Brachyspira prevalences across the tested sheds were 17.02%, 31.91%, 9.57% and 81.91% respectively (Table 1). Sixteen broiler breeders farms (26.23%), one layer breeders farm (12.5%) and 48 commercial layer farms (59.26%) were infected with Brachyspira species.
Disease and association with clinical signs and production parameters
Brachyspira infection was significantly associated with weight losses (P=0.035), yellow frothy droppings (P=0.002), wet litter problems (P=0.008) and reduced egg production (P=0.006). At the species level, Brachyspira pilosicoli infection was significantly associated with scouring (P=0.025), while infection with Brachyspira intermedia was significantly associated with production of mixed sized eggs (P=0.005) and scouring (P=0.025). There was no association between infection with non-pathogenic Brachyspira and reduced egg production (P=0.068) (Table 2).
Table 1. Different Brachyspira species occurrence and prevalence in the 94 Brachyspira positive sheds, showing number of sheds with single and mixed infection. Brachyspira species are coded from 1 to 4. The data were collected in a cross-sectional study from 150 farms (235 sheds) located in 45 UK counties.
Number of sheds
* 1=B. pilosicoli, 2=B. intermedia, 3=B. alvinipulli, 4=non-pathogenic Brachyspira
Risk factor analysis
In the univariable analysis, 12 potential risk factors were found to be potentially associated with AIS (P<0.20) and included breed, type of production, housing type, multi-age system farms, flock age, changes in farm management, type and condition of egg collection trays (Table 3). For inclusion in the multi-variable model, the variable breed was recoded and breed A1and A2 were combined as A and compared to all other breeds which were grouped in one group as B.
Multi-variable logistic regression and risk factors
Factors associated with AIS identified in the univariable analysis were included in the multi-variables analysis. In the final model only breed and flock age were found significantly associated with AIS. Flocks with birds of one particular breed (group AB? were less likely to have AIS with OR of 0.275 (95% CI 0.12-0.61, P=0.002) compared to all other breeds and flocks aged â‰¥23 weeks had higher odds to test positive (Table 4). The model was assessed by Hosmer-Lemeshow goodness-of-fit test and indicated good fitness of the final model (P=0.980).
Table 2. Association of AIS with potential clinical signs and impact on egg production. The data were collected in a cross-sectional study from 150 farms (235 sheds) located in 45 UK counties.
Yellow frothy dropping
Wet litter problems
Reduced egg production
Mixed sized eggs
Reduced egg production
Table 3. Univariable analysis of potential risk factors for AIS in a cross-sectional study from 150 farms located in 45 UK counties.
Recent management changes
Bleach and acids water treat.
Manure removal system
Egg trays types
Cardboard trays condition
Table 4. Results of multi-variables logistic regression analysis on data collected in a cross-sectional study conducted between May and August, 2010 on 150 egg producing farms in 45 UK counties showing the protective effect of breed A and birds younger than 23 weeks old against AIS. The final model showed good fitness using Hosmer-Lemeshow goodness-of-fit test (P=0.980).
To our knowledge this study is the first representative study conducted in the UK to estimate AIS prevalence, disease associations and the possible predisposing risk factors. The prevalence of Brachyspira of 43% was not surprising and was comparable to findings of surveys conducted in Western Australia (35.1% in laying flocks and 53.3% in broiler breeder flocks) (McLaren et al., 1996), Eastern Australia (68.2% in laying flocks and 42.9% in broiler breeder flocks) (Stephens and Hampson, 1999) or Northern-Eastern Italy (34.8%) (Bano et al., 2008). Although the prevalence of Brachyspira in broiler breeder farms in the UK was 26.23%, it is still lower than its prevalence in broiler breeder farms in Australia. This could be due to a different management systems followed in these farms which may result in decreased Brachyspira occurrence. Further studies to clarify this may be required.
One of the important findings of this study was that Brachyspira pilosicoli, intermedia and alvinipulli which are known pathogenic species, were identified in the 94 positive sheds in addition to the non-pathogenic species. Similar findings were reported by other authors (Myers et al., 2009), in Pennsylvania, USA, who reported Brachyspira intermedia prevalence of 81% of the tested flocks and Brachyspira pilosicoli prevalence of 23.8% of the tested flocks. Also Stephens and Hampson, 1999 reported 56% prevalence of pathogenic Brachyspira in Eastern Australia, and Bano et al., 2008 in Italy reported 27.5% prevalence of Brachyspira intermedia, 13.8% prevalence of Brachyspira pilosicoli, and 49.7% prevalence of non-pathogenic Brachyspira. Our study however, was the first to include Brachyspira alvinipulli,, which has been confirmed as pathogenic species (Swayne et al., 1992, Trampel et al., 1994 and Stanton et al., 1998). The inclusion of Brachyspira alvinipulli was possible due to the availability of a species specific M-PCR (Abdelrahman et al., 2011). Another important finding of this study was the significant association between AIS and weight losses, yellow frothy droppings, wet litter and reduced egg production which supports previous studies (Trampel et al., 1994; Smith et al., 1998; Swayne et al., 1992; Hampson & McLaren, 1999) and further highlights the economic significance of the disease. Interestingly, there was a significant association between Brachyspira pilosicoli and Brachyspira intermedia, and scouring, however we could not find any significant association between the over-all infection and scouring (95% CI 0.882-4.15, P=0.1). Another interesting finding was the association between Brachyspira intermedia infection and the production of mixed sized eggs which also emphasis the economic significance of AIS, which indicates the need to further investigate the economic impact of the disease.
In this study no significant association between reduced egg production and non-pathogenic Brachyspira infection was found, unlike a recent report by Burch et al., 2009. Either non-pathogenic Brachyspira are truly not associated with decreased egg production or the impact is rather small so that with the sample size used here, we were not able to identify a significant difference. There was however, a significant association between infection with these species and yellow frothy droppings. These findings could explain why the presence of yellow frothy droppings does not necessarily mean the presence of pathogenic Brachyspira.
Even though most factors identified in the univariable analysis were non-significant in the final multi-variable model, their importance for AIS cannot be ruled out. For example the use of used egg trays in multi-age farms results in decreased farms' bio-security and is known to contribute to many other poultry diseases (Pattison et al, 2008). Surprisingly there was no significant association of free range and AIS in the final multi-variables logistic regression model, even though housing type seemed to be a potential risk factor in the univariable analysis.
The two risk factors found to be associated with AIS in this study were flock age and breed. Flocks aged â‰¥ 23 weeks were significantly associated with infection than younger flocks where the odds of infection had positive trend with age increase, which has previously reported in different studies (Stephens and Hampson, 1999; Bano et al., 2008). Meanwhile, compared to other breeds, breed A seemed less susceptible for colonisation, which indicates the potential of breed selection for controlling AIS beside vaccination programme or the use of probiotics. However it needs to be noticed, that most breeds were underrepresented in our study and further investigations on the susceptibility of different breeds to colonisation are needed. This study which was conducted during the warmest months of the year (May to August), showed high prevalence and wide spread of Brachyspira species in the UK laying flocks. Further studies are needed to investigate the potential seasonal effect on Brachyspira prevalence and risk factors.