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Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces
Veterinary Microbiology 116 (2006) 239–245 www.elsevier.com/locate/vetmic
Short communication
Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces Nyree D. Phillips, Tom La, David J. Hampson * School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia Received 7 December 2005; received in revised form 24 March 2006; accepted 28 March 2006
Abstract Avian intestinal spirochaetosis (AIS) is an infection of the caeca and/or colo-rectum of laying and meat breeder hens caused by anaerobic intestinal spirochaetes of the genus Brachyspira. AIS can result in a variety of symptoms, including delayed and/or reduced egg production, and increased faecal water content. The two most commonly reported Brachyspira species involved in AIS are Brachyspira pilosicoli and Brachyspira intermedia, and their detection and identification can be difficult and time consuming. In the current study a two-step nested duplex PCR (2S-N-D-PCR) was developed for the detection of these two species, using DNA extracted from washed chicken faeces. In the first step, a duplex PCR (D-PCR) amplifying Brachyspira genus-specific portions of the 16S rRNA and NADH oxidase (nox) genes was undertaken on the washed faeces. In the second step, a nested D-PCR was used that amplified species-specific portions of the 16S rRNA gene of B. pilosicoli and the nox gene of B. intermedia from the amplicons produced in the first step. The 2S-N-D-PCR was rapid and specific, and could be used to detect approximately 103 cells of each spirochaete species per gram of washed faeces. When tested on 882 chicken faecal samples from infected flocks, it detected 4–5% more positive faecal samples than did the standard method of selective anaerobic culture followed by individual species-specific PCR assays conducted on the growth on the primary plate. The application of this new technique should improve diagnostic capacity, and facilitate further studies on AIS. Crown Copyright # 2006 Published by Elsevier B.V. All rights reserved. Keywords: Avian intestinal spirochaetes; Chicken; Brachyspira; PCR; Detection; Faeces
1. Introduction Avian intestinal spirochaetosis (AIS) is a condition of laying and meat breeder hens in which there is * Corresponding author. Tel.: +61 8 9360 2287; fax: +61 8 9310 4144. E-mail address: [email protected] (D.J. Hampson).
delayed and/or reduced egg production, and an increased faecal water content associated with wet litter and faecal staining of eggshells (Davelaar et al., 1986; Griffiths et al., 1987; Dwars et al., 1992; Swayne et al., 1992; Trampel et al., 1994; Stephens and Hampson, 2001; Swayne, 2003). AIS results from infection of the caeca and/or colo-rectum by anaerobic intestinal spirochaetes of the genus Brachyspira. The
0378-1135/$ – see front matter. Crown Copyright # 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.03.020
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two most commonly reported species in AIS are Brachyspira pilosicoli and Brachyspira intermedia (McLaren et al., 1997; Stephens and Hampson, 1999, 2001; Swayne, 2003; Stephens et al., 2005), although B. alvinipulli also has been reported as a cause of disease in a chicken flock in USA (Swayne et al., 1992; Stanton et al., 1998), and in geese in Hungary (Nemes et al., 2006). Currently, microbiological diagnosis of AIS requires cultivation of the spirochaetes using specialised media and anaerobic growth conditions, followed by biochemical testing and/or the use of species-specific PCR assays (Stephens and Hampson, 2001; Swayne, 2003). Growth and identification of the spirochaetes often takes at least 7–10 days. Despite the apparent high prevalence rates of the infection(s) that have been revealed in epidemiological studies (Dwars et al., 1989; McLaren et al., 1996; Smit et al., 1998; Stephens and Hampson, 1999), AIS is not commonly diagnosed. In part, this is due to the difficulties associated with the current laboratory diagnostic methods used for these spirochaetes. Although PCR assays for Brachyspira species have been conducted on DNA extracted from human and pig faeces (e.g. Mikosza et al., 2001; Choi et al., 2002; La et al., 2003, 2006), to date it has not been possible to develop a similar PCR system for chicken faeces, despite extracting DNAwith a range of different commercial kits (Phillips et al., unpublished data). This lack of success is likely to be associated with the low pH of chicken faeces, and the presence of uric acid and other PCR inhibitors. The aim of the current study was to improve diagnostic capacity for AIS by developing a rapid and sensitive method for detecting B. pilosicoli and B. intermedia in the faeces of chickens with AIS. This was achieved by washing faeces samples to remove inhibitors prior to DNA extraction, and then subjecting the DNA to a two-step nested duplex PCR (2S-N-DPCR) assay to enhance the sensitivity of detection.
2. Materials and methods 2.1. Control bacterial strains and culture conditions Control Brachyspira spp. strains (n = 108) and strains of other bacterial species (n = 17) were
obtained from the culture collection held at the Reference Centre for Intestinal Spirochaetes, Murdoch University, Western Australia. The strains used originated from North America, Europe and Australia, and included B. hyodysenteriae (n = 20), B. innocens (n = 10), B. intermedia (n = 20), B. murdochii (n = 10), B. pilosicoli (n = 20), ‘‘B. canis’’ (n = 2), ‘‘B. pulli’’ (n = 20), B. alvinipulli (n = 3), and B. aalborgi (n = 3). Non-Brachyspira enteric bacteria used included Escherichia coli (n = 3), Campylobacter species (n = 4), Lawsonia intracellularis (n = 1), Helicobacter species (n = 3), Arcobacter species (n = 3) and Salmonella enterica serovars (n = 3). All strains were used in the evaluation of the specificity of the PCRs. 2.2. Faecal samples, culture and species-specific PCR assays A total of 882 fresh faeces samples from commercial laying hens in flocks with AIS, fed on different commercial diets, were tested. Samples were collected into sterile containers, held at 4 8C and transported to the laboratory, where they were processed within 6 h of collection. Cotton-tipped swabs were used to stir the faecal samples, and this homogenised material was used to inoculate selective bacteriology plates before streaking out. The plates consisted of Trypticase Soy Agar (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD), containing 5% (v/v) defibrinated ovine blood, 400 mg of spectinomycin per milliliter, and 25 mg each of colistin and vancomycin (Sigma– Aldrich Pty. Ltd., Castle Hill, Australia) per milliliter. They were incubated for 7 days at 37 8C in an anaerobic environment of 94% H2 and 6% CO2 generated with anaerobic Gaspak Plus sachets (BBL). Suspected spirochaetal growth was resuspended in phosphate buffered saline (PBS; pH 7.2) and examined under a phase contrast microscope at 400 magnification. Concurrently, a cell-pick method (Atyeo et al., 1998) was used to obtain spirochaetal DNA from the growth on the plates for identification using species-specific PCRs (Phillips et al., 2005). This standard diagnostic procedure was designated ‘‘culture/PCR’’. The species-specific primers used and the PCR conditions have been described (Phillips et al., 2005). The primers amplified an 823 bp region
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Table 1 Primer sequences and their binding sites Species detected
Gene targeted
Primer name
Primer sequence
Brachyspira spp.
NADH oxidase (nox)
Brachy-nox-F
50 -TGGTACATGGGCAGCAAAAAC-30
Brachy-nox-R
50 -CAAATACGCATATAGCGTTAG-30
Predicted size
Equivalent Brachyspira hyodysenteriae base position
991
258–1249a
75–1382b
Brachyspira spp.
16S rRNA
Brachy-16S-F Brachy-16S-R
50 -TGAGTAACACGTAGGTAATC-30 50 -GCTAACGACTTCAGGTAAAAC-30
1307
Brachyspira pilosicoli
16S rRNA
P1
50 -AGAGGAAAGTTTTTTCGCTTC-30
823
165–988b
P2
50 -GCACCTATGTTAAACGTCCTTG-30
Int1
50 -AGAGTTTGATGATAATTATGAC-30
567
516–1073a
Int2
50 -ATAAACATCAGGATCTTTGC-30
Brachyspira intermedia a b
nox
B. hyodysenteriae strain B204 nox gene (GenBank accession number U19610). B. hyodysenteriae strain R1 16S rRNA gene (GenBank accession number U23035).
of the 16S rRNA gene of B. pilosicoli and a 567 bp region of the NADH oxidase (nox) gene of B. intermedia (Table 1), and were designed to give specific PCR products of easily distinguishable sizes and similar optimal annealing temperatures so that the two PCRs also could be run in a single reaction. 2.3. Development of Brachyspira genus-specific PCR primers General Brachyspira 16S rRNA gene primers and nox gene primers were designed with Oligo Explorer (version 1.1.0; University of Kuopio, Kuopio, Finland), using gene sequences available from the GenBank database. Brachy-16S-F and Brachy-16SR were predicted to amplify a 1307 bp region of the 16S rRNA gene (Phillips et al., 2005), whilst Brachynox-F and Brachy-nox-R were designed to amplify a 991 bp region of the nox gene (Table 1). The primers were predicted to give specific PCR products of easily distinguishable sizes, and with similar optimal annealing temperatures so that they could be run in a single reaction. The primers were tested for specificity using Amplify (version 1.2; University of Wisconsin, Madison, WI), then tested in duplicate singly and as a duplex reaction with the control Brachyspira and non-Brachyspira strains. PCR conditions were the same as for the individual speciesspecific PCRs.
2.4. DNA extraction from faeces For each faecal sample, two g of fresh faeces was washed by vortexing for 10 s with 10 ml of sterile PBS, centrifuged at 5000 g for 10 min, and the supernatant discarded. The process was repeated, and the top layer of the final pellet (150 mg) was scraped off and used as the starting material for DNA extraction. DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s instructions. 2.5. PCR assays on DNA extracted from faeces The extracted DNA was amplified in two different PCR systems. The first system involved a speciesspecific duplex-PCR (D-PCR) assay carried out using the same species-specific primers and PCR conditions as described in the culture/PCR procedure, except that both sets of primer pairs were present in the reaction mix. The second system used an initial first step genusspecific D-PCR reaction incorporating both pairs of Brachyspira genus-specific primers, with the same PCR conditions as for the species-specific D-PCR and culture/PCR. Products from this genus-specific DPCR (1 ml of PCR mix) then were used as the template for the second step species-specific nested D-PCR, which was conducted in the same way as described
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when it was used as a one-step D-PCR. To confirm that the sensitivity and specificity of the primer sets had not been compromised, the second step also was run as two separate PCR reactions on the same template DNA generated in the first step PCR.
sequences in GenBank using the BioEdit Sequence Alignment Editor (version 5.09; North Carolina State University, NC, USA).
3. Results 2.6. Detection limits of the PCR systems 3.1. Specificity of the PCR assays The detection limits of the culture/PCR method, and of the species-specific D-PCR and the 2S-N-DPCR (both conducted on extracted DNA) were established by seeding seven PCR-negative faeces samples with 10-fold serial dilutions of B. pilosicoli strain CSPp-1 (Stephens and Hampson, 2002) or B. intermedia strain HB60-1 (Hampson and McLaren, 1999), or combinations of the two. One hundred microliters of intestinal spirochaetes were added to 150 mg of faeces to give final concentrations of 1– 108 cells/g of faeces, and these were thoroughly mixed into the faeces using a cotton-tipped swab followed by vortexing for 10 s. A faecal sample to which 100 ml of TE (10 mM Tris–HCl–1 mM EDTA [pH 8.0]) buffer was added was included as a negative control. All faecal samples were washed, and then subjected to DNA extraction using the QIAamp DNA Stool Mini Kit. One microliter of purified DNA from each of the dilutions was added into the PCR reactions as template. The procedures were carried out in duplicate. The presence of potential PCR inhibitors in the final eluent from the control sample was assessed by spiking 10 ml aliquots of the eluent with 1 ml of various concentrations of B. intermedia and/or B. pilosicoli DNA prior to PCR. Bacteriology swabs also were taken from the unwashed faecal samples, and inoculated onto selective agar for analysis by the culture/PCR method.
The genus-specific D-PCR, the species-specific DPCR, and the 2S-N-D-PCR all amplified 16S rDNA and nox DNA of the correct predicted size from the appropriate Brachyspira spp. control strains tested, but not from the other bacterial strains. Examples of products generated in the 2S-N-D-PCR from chicken faeces are shown in Fig. 1. When the two genusspecific primer pairs and the two species-specific primer pairs were used individually, the same results were obtained as when the two reactions were run together as a D-PCR. 3.2. Sequencing results Sequence analysis of the 2S-N-D-PCR products generated from field samples showed that the 16 B. intermedia nox amplicons were 99.2% (503 of 507 bp) to 99.6% (505 of 507 bp) identical to the sequence of HB60-1, the avian control strain of B. intermedia (GenBank accession number DQ458796). Based on the pattern of nucleotide differences, the 16 sequences could be divided into three nox genotypes
2.7. Sequencing of PCR products The 2-S-N-PCR amplicons from 26 (16 B. intermedia and 10 B. pilosicoli) of the 882 faecal samples were chosen for direct sequencing. These included B. intermedia and B. pilosicoli amplicons from samples that were negative by culture/PCR. The sequencing methodology has been described previously (Phillips et al., 2005), with the primers used being listed in Table 1. Sequence results were edited, compiled and compared with each other and with
Fig. 1. 2S-N-D-PCR products of Brachyspira pilosicoli (823 bp) and Brachyspira intermedia (567 bp) amplified from chicken faeces. Lanes 1 and 6, molecular weight markers; lane 2, PCR negative control; lane 3, B. pilosicoli and B. intermedia mixed sample; lane 4, B. intermedia; lane 5, B. pilosicoli.
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Table 2 Nucleotide differences between the partial NADH oxidase (nox) gene sequence of B. intermedia strain HB60-1 (GenBank accession number DQ458796) and the 3 nox genotypes of B. intermedia identified amongst the 16 sequenced B. intermedia nox PCR amplicons nox genotype (number of amplicons) Base position and nucleotide changea
1 (4)
2 (5)
3 (7)
3: T–A 495: A–G
3: T–A 405: G–A 449: G–A
3: T–A 363: G–T 405: G–A 449: G–A
a Refers to the position on the 507 bp B. intermedia strain HB60-1 nox sequence, itself equivalent to base positions 448 to 954 of the B. hyodysenteriae strain B204 nox gene (GenBank accession number U19610).
representing amplicons from 4, 5 and 7 faecal samples, respectively. Differences in the nox sequences of these three genotypes from the sequence of HB60-1 are shown in Table 2. The sequenced B. pilosicoli 16S rRNA gene products were 100% (708 of 708 bp) identical to that of CSPp-1 (also known as Q98.0072.38), the avian control strain of B. pilosicoli (GenBank accession no. AY745551). The sequenced product corresponded to nucleotide positions 208 to 914 of the 16S rRNA gene of B. hyodysenteriae strain R1 (GenBank accession number U23035). 3.3. Detection limits The detection limits of the D-PCR on DNA extracted from seeded washed chicken faeces was 106 cells/g of faeces for both spirochaete species, whilst the detection limits of culture/PCR and the 2SN-D-PCR on the same samples both were 103 cells/g of faeces for each species. The same detection limits were obtained with samples seeded with both Brachyspira species. Table 3 Comparison of detection of Brachyspira pilosicoli and B. intermedia in faecal samples from 882 laying hens, using culture/PCR, DNA extraction and species-specific D-PCR, and DNA extraction and 2SN-D-PCR Number of positive samples
Culture/PCR D-PCRa 2S-N-D-PCRa a
Negative
B. pilosicoli
B. intermedia
Both species
145 121 150
153 119 157
0 0 2
584 642 573
PCRs performed on DNA extracted from washed faeces.
3.4. Field investigation of PCR assays The results of culture/PCR, D-PCR on DNA extracted from washed faeces and the 2S-N-D-PCR on the same material for the 882 faecal samples are recorded in Table 3. The culture/PCR protocol on unwashed faeces detected 145 and 153 positives for B. pilosicoli and B. intermedia, respectively. In comparison, the D-PCR on washed faeces detected only 121 and 119 positive samples for the 2 species respectively, whilst with the same starting material the 2S-ND-PCR protocol detected 150 and 157 positive samples, as well as 2 other samples that were positive for both species. All the samples positive by culture/ PCR were positive in the 2S-N-D-PCR.
4. Discussion PCR amplification of DNA extracted from chicken faeces is known to be difficult, as this material contains uric acid and other PCR inhibitors. Initial studies in our laboratory showed that Brachyspira spp. PCRs conducted on DNA extracted with a range of commercial extraction kits were inhibited unless the chicken faeces were first washed to remove these inhibitors (Phillips et al., unpublished data). Unfortunately, in washing the faeces, it was inevitable that some spirochaetes were lost from the material used for DNA extraction. This problem was reflected in the fact that the species-specific DPCR conducted on DNA extracted from washed faeces detected only 106 spirochaete cells/g of faeces, compared to 103 cells/g for culture/PCR on unwashed faeces. To overcome this loss of sensitivity using the species-specific D-PCR on washed
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faeces, an initial genus-specific D-PCR was carried out, and then this was followed by the speciesspecific D-PCR conducted as a second step nested reaction on the DNA templates formed in the first step. Nested PCRs previously have been shown to be useful for increasing the sensitivity of detection of Brachyspira species and L. intracellularis in porcine faeces (Choi et al., 2002; Plawinska et al., 2004), although they have not been used for this purpose with chicken faeces. The 2S-N-D-PCR increased the sensitivity of detection to a level similar to culture/ PCR (i.e. 103 cells/g faeces for each species). Indeed, when conducted on chicken faeces from infected flocks, the 2S-N-D-PCR detected slightly more positive samples than culture/PCR (about 4% and 5% more for B. pilosicoli and B. intermedia, respectively). The fact that the increases in the detection rate for the two species were similar, indicates that the 2S-N-D-PCR was not introducing a species-related bias in relation to detection. Furthermore, as the 2S-N-D-PCR detected all of the samples that were positive in culture/PCR, this helped to confirm its specificity. This specificity was further supported by the results obtained by sequencing the PCR products, including samples that were negative by culture/PCR. In addition, the 2S-N-D-PCR gave positive results from birds from different flocks, fed on different diets, helping to confirm that the washing process was effective at removing potential PCR inhibitors from chicken faeces. A major advantage of the 2S-N-D-PCR was that it was far more rapid than culture/PCR, potentially giving results on the same day as sample receipt rather than after 7–10 days. The availability of such rapid tests should greatly facilitate future epidemiological studies on AIS, and improve diagnostic capacity. Although the current 2S-N-D-PCR was only designed to detect B. pilosicoli and B. intermedia, in the future it could be expanded to include detection of B. alvinipulli, the other less commonly described cause of AIS. All that would be required would be to add an addition species-specific amplification to the second step, based on portions of the 16S rRNA gene or the nox gene, following their amplification in the first genus-specific step. Unfortunately, there are few strains of B. alvinipulli currently available, and so it has not been possible to design primers with confirmed specificity for this species at this time.
Acknowledgements This study was conducted with the financial support of the Australian Chicken Meat Council and the former Australian Egg Industry Council, administered through the Rural Industries Research and Development Corporation, and Murdoch University.
References Atyeo, R.F., Oxberry, S.L., Combs, B.G., Hampson, D.J., 1998. Development and evaluation of polymerase chain reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis. Lett. Appl. Microbiol. 26, 126–130. Choi, C., Han, D.U., Kim, J., Cho, W.S., Chung, H.K., Jung, T., Yoon, B.S., Chae, C., 2002. Prevalence of Brachyspira pilosicoli in Korean pigs, determined using a nested PCR. Vet. Rec. 150, 217–218. Davelaar, F.G., Smit, H.F., Hovind-Hougen, K., Dwars, R.M., van der Valk, P.C., 1986. Infectious typhlitis in chickens caused by spirochaetes. Avian Pathol. 15, 247–258. Dwars, R.M., Smit, H.F., Davelaar, F.G., van Veer, T.W., 1989. Incidence of spirochaetal infections in cases of intestinal disorder in chickens. Avian Pathol. 18, 591–595. Dwars, R.M., Davelaar, F.G., Smit, H.F., 1992. Influence of infection with avian intestinal spirochaetes on the faeces of laying hens. Avian Pathol. 21, 427–429. Griffiths, I.B., Hunt, B.W., Lister, S.A., Lamont, M.H., 1987. Retarded growth rate and delayed onset of egg production associated with spirochaetal infection in pullets. Vet. Rec. 121, 35–37. Hampson, D.J., McLaren, A.J., 1999. Experimental infection of layer hens with Serpulina intermedia causes reduced egg production and increased faecal water content. Avian Pathol. 28, 113–117. La, T., Phillips, N.D., Hampson, D.J., 2003. Development of a duplex PCR assay for the detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in pig feces. J. Clin. Microbiol. 41, 3372–3375. La, T., Collins, A.M., Phillips, N.D., Oksa, A., Hampson, D.J., 2006. Development of a multiplex-PCR for rapid detection of the enteric pathogens Lawsonia intracellularis, Brachyspira hyodysenteriae, and Brachyspira pilosicoli in porcine faeces. Lett. Appl. Microbiol. 42, 284–288. McLaren, A.J., Hampson, D.J., Plant, S.J., 1996. The prevalence of intestinal spirochaetes in commercial poultry flocks in Western Australia. Aust. Vet. J. 74, 31–33. McLaren, A.J., Trott, D.J., Swayne, D.E., Oxberry, S.L., Hampson, D.J., 1997. Genetic and phenotypic characterization of intestinal spirochetes colonizing chickens, and allocation of known pathogenic isolates to three distinct genetic groups. J. Clin. Microbiol. 35, 412–417. Mikosza, A.S.J., La, T., Margawani, K.R., Brooke, C.J., Hampson, D.J., 2001. PCR detection of Brachyspira aalborgi and Brachyspira pilosicoli in human faeces. FEMS Microbiol. Lett. 197, 167–170.
N.D. Phillips et al. / Veterinary Microbiology 116 (2006) 239–245 Nemes, C.S., Glavits, R., Dobos-Kovacs, M., Ivanics, E., Kaszanyitzky, E., Beregszaszi, A., Szeredi, L., Dencso, L., 2006. Typhlocolitis associated with spirochaetes in goose flocks. Avian Pathol. 35, 4–11. Phillips, N.D., La, T., Hampson, D.J., 2005. A cross-sectional study to investigate the occurrence and distribution of intestinal spirochaetes (Brachyspira spp.) in three flocks of laying hens. Vet. Microbiol. 105, 189–198. Plawinska, J., Jakubowski, T., Rzewuska, M., Binek, M., 2004. Occurrence of Lawsonia intracellularis and Brachyspira spp. infection in swine suffering from diarrhoea. Pol. J. Vet. Sci. 7, 203–206. Smit, H.F., Dwars, R.M., Davelaar, F.G., Wijtten, A.W., 1998. Observations on the influence of intestinal spirochaetosis in broiler breeders on the performance of their progeny and on egg production. Avian Pathol. 27, 133–141. Stanton, T.B., Postic, D., Jensen, N.S., 1998. Serpulina alvinipulli sp. nov., a new Serpulina species that is enteropathogenic for chickens. Int. J. Syst. Bacteriol. 48, 669–676. Stephens, C.P., Hampson, D.J., 1999. Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia. Avian Pathol. 28, 447–454.
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Short communication
Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces Nyree D. Phillips, Tom La, David J. Hampson * School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia Received 7 December 2005; received in revised form 24 March 2006; accepted 28 March 2006
Abstract Avian intestinal spirochaetosis (AIS) is an infection of the caeca and/or colo-rectum of laying and meat breeder hens caused by anaerobic intestinal spirochaetes of the genus Brachyspira. AIS can result in a variety of symptoms, including delayed and/or reduced egg production, and increased faecal water content. The two most commonly reported Brachyspira species involved in AIS are Brachyspira pilosicoli and Brachyspira intermedia, and their detection and identification can be difficult and time consuming. In the current study a two-step nested duplex PCR (2S-N-D-PCR) was developed for the detection of these two species, using DNA extracted from washed chicken faeces. In the first step, a duplex PCR (D-PCR) amplifying Brachyspira genus-specific portions of the 16S rRNA and NADH oxidase (nox) genes was undertaken on the washed faeces. In the second step, a nested D-PCR was used that amplified species-specific portions of the 16S rRNA gene of B. pilosicoli and the nox gene of B. intermedia from the amplicons produced in the first step. The 2S-N-D-PCR was rapid and specific, and could be used to detect approximately 103 cells of each spirochaete species per gram of washed faeces. When tested on 882 chicken faecal samples from infected flocks, it detected 4–5% more positive faecal samples than did the standard method of selective anaerobic culture followed by individual species-specific PCR assays conducted on the growth on the primary plate. The application of this new technique should improve diagnostic capacity, and facilitate further studies on AIS. Crown Copyright # 2006 Published by Elsevier B.V. All rights reserved. Keywords: Avian intestinal spirochaetes; Chicken; Brachyspira; PCR; Detection; Faeces
1. Introduction Avian intestinal spirochaetosis (AIS) is a condition of laying and meat breeder hens in which there is * Corresponding author. Tel.: +61 8 9360 2287; fax: +61 8 9310 4144. E-mail address: [email protected] (D.J. Hampson).
delayed and/or reduced egg production, and an increased faecal water content associated with wet litter and faecal staining of eggshells (Davelaar et al., 1986; Griffiths et al., 1987; Dwars et al., 1992; Swayne et al., 1992; Trampel et al., 1994; Stephens and Hampson, 2001; Swayne, 2003). AIS results from infection of the caeca and/or colo-rectum by anaerobic intestinal spirochaetes of the genus Brachyspira. The
0378-1135/$ – see front matter. Crown Copyright # 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.03.020
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two most commonly reported species in AIS are Brachyspira pilosicoli and Brachyspira intermedia (McLaren et al., 1997; Stephens and Hampson, 1999, 2001; Swayne, 2003; Stephens et al., 2005), although B. alvinipulli also has been reported as a cause of disease in a chicken flock in USA (Swayne et al., 1992; Stanton et al., 1998), and in geese in Hungary (Nemes et al., 2006). Currently, microbiological diagnosis of AIS requires cultivation of the spirochaetes using specialised media and anaerobic growth conditions, followed by biochemical testing and/or the use of species-specific PCR assays (Stephens and Hampson, 2001; Swayne, 2003). Growth and identification of the spirochaetes often takes at least 7–10 days. Despite the apparent high prevalence rates of the infection(s) that have been revealed in epidemiological studies (Dwars et al., 1989; McLaren et al., 1996; Smit et al., 1998; Stephens and Hampson, 1999), AIS is not commonly diagnosed. In part, this is due to the difficulties associated with the current laboratory diagnostic methods used for these spirochaetes. Although PCR assays for Brachyspira species have been conducted on DNA extracted from human and pig faeces (e.g. Mikosza et al., 2001; Choi et al., 2002; La et al., 2003, 2006), to date it has not been possible to develop a similar PCR system for chicken faeces, despite extracting DNAwith a range of different commercial kits (Phillips et al., unpublished data). This lack of success is likely to be associated with the low pH of chicken faeces, and the presence of uric acid and other PCR inhibitors. The aim of the current study was to improve diagnostic capacity for AIS by developing a rapid and sensitive method for detecting B. pilosicoli and B. intermedia in the faeces of chickens with AIS. This was achieved by washing faeces samples to remove inhibitors prior to DNA extraction, and then subjecting the DNA to a two-step nested duplex PCR (2S-N-DPCR) assay to enhance the sensitivity of detection.
2. Materials and methods 2.1. Control bacterial strains and culture conditions Control Brachyspira spp. strains (n = 108) and strains of other bacterial species (n = 17) were
obtained from the culture collection held at the Reference Centre for Intestinal Spirochaetes, Murdoch University, Western Australia. The strains used originated from North America, Europe and Australia, and included B. hyodysenteriae (n = 20), B. innocens (n = 10), B. intermedia (n = 20), B. murdochii (n = 10), B. pilosicoli (n = 20), ‘‘B. canis’’ (n = 2), ‘‘B. pulli’’ (n = 20), B. alvinipulli (n = 3), and B. aalborgi (n = 3). Non-Brachyspira enteric bacteria used included Escherichia coli (n = 3), Campylobacter species (n = 4), Lawsonia intracellularis (n = 1), Helicobacter species (n = 3), Arcobacter species (n = 3) and Salmonella enterica serovars (n = 3). All strains were used in the evaluation of the specificity of the PCRs. 2.2. Faecal samples, culture and species-specific PCR assays A total of 882 fresh faeces samples from commercial laying hens in flocks with AIS, fed on different commercial diets, were tested. Samples were collected into sterile containers, held at 4 8C and transported to the laboratory, where they were processed within 6 h of collection. Cotton-tipped swabs were used to stir the faecal samples, and this homogenised material was used to inoculate selective bacteriology plates before streaking out. The plates consisted of Trypticase Soy Agar (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD), containing 5% (v/v) defibrinated ovine blood, 400 mg of spectinomycin per milliliter, and 25 mg each of colistin and vancomycin (Sigma– Aldrich Pty. Ltd., Castle Hill, Australia) per milliliter. They were incubated for 7 days at 37 8C in an anaerobic environment of 94% H2 and 6% CO2 generated with anaerobic Gaspak Plus sachets (BBL). Suspected spirochaetal growth was resuspended in phosphate buffered saline (PBS; pH 7.2) and examined under a phase contrast microscope at 400 magnification. Concurrently, a cell-pick method (Atyeo et al., 1998) was used to obtain spirochaetal DNA from the growth on the plates for identification using species-specific PCRs (Phillips et al., 2005). This standard diagnostic procedure was designated ‘‘culture/PCR’’. The species-specific primers used and the PCR conditions have been described (Phillips et al., 2005). The primers amplified an 823 bp region
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Table 1 Primer sequences and their binding sites Species detected
Gene targeted
Primer name
Primer sequence
Brachyspira spp.
NADH oxidase (nox)
Brachy-nox-F
50 -TGGTACATGGGCAGCAAAAAC-30
Brachy-nox-R
50 -CAAATACGCATATAGCGTTAG-30
Predicted size
Equivalent Brachyspira hyodysenteriae base position
991
258–1249a
75–1382b
Brachyspira spp.
16S rRNA
Brachy-16S-F Brachy-16S-R
50 -TGAGTAACACGTAGGTAATC-30 50 -GCTAACGACTTCAGGTAAAAC-30
1307
Brachyspira pilosicoli
16S rRNA
P1
50 -AGAGGAAAGTTTTTTCGCTTC-30
823
165–988b
P2
50 -GCACCTATGTTAAACGTCCTTG-30
Int1
50 -AGAGTTTGATGATAATTATGAC-30
567
516–1073a
Int2
50 -ATAAACATCAGGATCTTTGC-30
Brachyspira intermedia a b
nox
B. hyodysenteriae strain B204 nox gene (GenBank accession number U19610). B. hyodysenteriae strain R1 16S rRNA gene (GenBank accession number U23035).
of the 16S rRNA gene of B. pilosicoli and a 567 bp region of the NADH oxidase (nox) gene of B. intermedia (Table 1), and were designed to give specific PCR products of easily distinguishable sizes and similar optimal annealing temperatures so that the two PCRs also could be run in a single reaction. 2.3. Development of Brachyspira genus-specific PCR primers General Brachyspira 16S rRNA gene primers and nox gene primers were designed with Oligo Explorer (version 1.1.0; University of Kuopio, Kuopio, Finland), using gene sequences available from the GenBank database. Brachy-16S-F and Brachy-16SR were predicted to amplify a 1307 bp region of the 16S rRNA gene (Phillips et al., 2005), whilst Brachynox-F and Brachy-nox-R were designed to amplify a 991 bp region of the nox gene (Table 1). The primers were predicted to give specific PCR products of easily distinguishable sizes, and with similar optimal annealing temperatures so that they could be run in a single reaction. The primers were tested for specificity using Amplify (version 1.2; University of Wisconsin, Madison, WI), then tested in duplicate singly and as a duplex reaction with the control Brachyspira and non-Brachyspira strains. PCR conditions were the same as for the individual speciesspecific PCRs.
2.4. DNA extraction from faeces For each faecal sample, two g of fresh faeces was washed by vortexing for 10 s with 10 ml of sterile PBS, centrifuged at 5000 g for 10 min, and the supernatant discarded. The process was repeated, and the top layer of the final pellet (150 mg) was scraped off and used as the starting material for DNA extraction. DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s instructions. 2.5. PCR assays on DNA extracted from faeces The extracted DNA was amplified in two different PCR systems. The first system involved a speciesspecific duplex-PCR (D-PCR) assay carried out using the same species-specific primers and PCR conditions as described in the culture/PCR procedure, except that both sets of primer pairs were present in the reaction mix. The second system used an initial first step genusspecific D-PCR reaction incorporating both pairs of Brachyspira genus-specific primers, with the same PCR conditions as for the species-specific D-PCR and culture/PCR. Products from this genus-specific DPCR (1 ml of PCR mix) then were used as the template for the second step species-specific nested D-PCR, which was conducted in the same way as described
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when it was used as a one-step D-PCR. To confirm that the sensitivity and specificity of the primer sets had not been compromised, the second step also was run as two separate PCR reactions on the same template DNA generated in the first step PCR.
sequences in GenBank using the BioEdit Sequence Alignment Editor (version 5.09; North Carolina State University, NC, USA).
3. Results 2.6. Detection limits of the PCR systems 3.1. Specificity of the PCR assays The detection limits of the culture/PCR method, and of the species-specific D-PCR and the 2S-N-DPCR (both conducted on extracted DNA) were established by seeding seven PCR-negative faeces samples with 10-fold serial dilutions of B. pilosicoli strain CSPp-1 (Stephens and Hampson, 2002) or B. intermedia strain HB60-1 (Hampson and McLaren, 1999), or combinations of the two. One hundred microliters of intestinal spirochaetes were added to 150 mg of faeces to give final concentrations of 1– 108 cells/g of faeces, and these were thoroughly mixed into the faeces using a cotton-tipped swab followed by vortexing for 10 s. A faecal sample to which 100 ml of TE (10 mM Tris–HCl–1 mM EDTA [pH 8.0]) buffer was added was included as a negative control. All faecal samples were washed, and then subjected to DNA extraction using the QIAamp DNA Stool Mini Kit. One microliter of purified DNA from each of the dilutions was added into the PCR reactions as template. The procedures were carried out in duplicate. The presence of potential PCR inhibitors in the final eluent from the control sample was assessed by spiking 10 ml aliquots of the eluent with 1 ml of various concentrations of B. intermedia and/or B. pilosicoli DNA prior to PCR. Bacteriology swabs also were taken from the unwashed faecal samples, and inoculated onto selective agar for analysis by the culture/PCR method.
The genus-specific D-PCR, the species-specific DPCR, and the 2S-N-D-PCR all amplified 16S rDNA and nox DNA of the correct predicted size from the appropriate Brachyspira spp. control strains tested, but not from the other bacterial strains. Examples of products generated in the 2S-N-D-PCR from chicken faeces are shown in Fig. 1. When the two genusspecific primer pairs and the two species-specific primer pairs were used individually, the same results were obtained as when the two reactions were run together as a D-PCR. 3.2. Sequencing results Sequence analysis of the 2S-N-D-PCR products generated from field samples showed that the 16 B. intermedia nox amplicons were 99.2% (503 of 507 bp) to 99.6% (505 of 507 bp) identical to the sequence of HB60-1, the avian control strain of B. intermedia (GenBank accession number DQ458796). Based on the pattern of nucleotide differences, the 16 sequences could be divided into three nox genotypes
2.7. Sequencing of PCR products The 2-S-N-PCR amplicons from 26 (16 B. intermedia and 10 B. pilosicoli) of the 882 faecal samples were chosen for direct sequencing. These included B. intermedia and B. pilosicoli amplicons from samples that were negative by culture/PCR. The sequencing methodology has been described previously (Phillips et al., 2005), with the primers used being listed in Table 1. Sequence results were edited, compiled and compared with each other and with
Fig. 1. 2S-N-D-PCR products of Brachyspira pilosicoli (823 bp) and Brachyspira intermedia (567 bp) amplified from chicken faeces. Lanes 1 and 6, molecular weight markers; lane 2, PCR negative control; lane 3, B. pilosicoli and B. intermedia mixed sample; lane 4, B. intermedia; lane 5, B. pilosicoli.
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Table 2 Nucleotide differences between the partial NADH oxidase (nox) gene sequence of B. intermedia strain HB60-1 (GenBank accession number DQ458796) and the 3 nox genotypes of B. intermedia identified amongst the 16 sequenced B. intermedia nox PCR amplicons nox genotype (number of amplicons) Base position and nucleotide changea
1 (4)
2 (5)
3 (7)
3: T–A 495: A–G
3: T–A 405: G–A 449: G–A
3: T–A 363: G–T 405: G–A 449: G–A
a Refers to the position on the 507 bp B. intermedia strain HB60-1 nox sequence, itself equivalent to base positions 448 to 954 of the B. hyodysenteriae strain B204 nox gene (GenBank accession number U19610).
representing amplicons from 4, 5 and 7 faecal samples, respectively. Differences in the nox sequences of these three genotypes from the sequence of HB60-1 are shown in Table 2. The sequenced B. pilosicoli 16S rRNA gene products were 100% (708 of 708 bp) identical to that of CSPp-1 (also known as Q98.0072.38), the avian control strain of B. pilosicoli (GenBank accession no. AY745551). The sequenced product corresponded to nucleotide positions 208 to 914 of the 16S rRNA gene of B. hyodysenteriae strain R1 (GenBank accession number U23035). 3.3. Detection limits The detection limits of the D-PCR on DNA extracted from seeded washed chicken faeces was 106 cells/g of faeces for both spirochaete species, whilst the detection limits of culture/PCR and the 2SN-D-PCR on the same samples both were 103 cells/g of faeces for each species. The same detection limits were obtained with samples seeded with both Brachyspira species. Table 3 Comparison of detection of Brachyspira pilosicoli and B. intermedia in faecal samples from 882 laying hens, using culture/PCR, DNA extraction and species-specific D-PCR, and DNA extraction and 2SN-D-PCR Number of positive samples
Culture/PCR D-PCRa 2S-N-D-PCRa a
Negative
B. pilosicoli
B. intermedia
Both species
145 121 150
153 119 157
0 0 2
584 642 573
PCRs performed on DNA extracted from washed faeces.
3.4. Field investigation of PCR assays The results of culture/PCR, D-PCR on DNA extracted from washed faeces and the 2S-N-D-PCR on the same material for the 882 faecal samples are recorded in Table 3. The culture/PCR protocol on unwashed faeces detected 145 and 153 positives for B. pilosicoli and B. intermedia, respectively. In comparison, the D-PCR on washed faeces detected only 121 and 119 positive samples for the 2 species respectively, whilst with the same starting material the 2S-ND-PCR protocol detected 150 and 157 positive samples, as well as 2 other samples that were positive for both species. All the samples positive by culture/ PCR were positive in the 2S-N-D-PCR.
4. Discussion PCR amplification of DNA extracted from chicken faeces is known to be difficult, as this material contains uric acid and other PCR inhibitors. Initial studies in our laboratory showed that Brachyspira spp. PCRs conducted on DNA extracted with a range of commercial extraction kits were inhibited unless the chicken faeces were first washed to remove these inhibitors (Phillips et al., unpublished data). Unfortunately, in washing the faeces, it was inevitable that some spirochaetes were lost from the material used for DNA extraction. This problem was reflected in the fact that the species-specific DPCR conducted on DNA extracted from washed faeces detected only 106 spirochaete cells/g of faeces, compared to 103 cells/g for culture/PCR on unwashed faeces. To overcome this loss of sensitivity using the species-specific D-PCR on washed
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faeces, an initial genus-specific D-PCR was carried out, and then this was followed by the speciesspecific D-PCR conducted as a second step nested reaction on the DNA templates formed in the first step. Nested PCRs previously have been shown to be useful for increasing the sensitivity of detection of Brachyspira species and L. intracellularis in porcine faeces (Choi et al., 2002; Plawinska et al., 2004), although they have not been used for this purpose with chicken faeces. The 2S-N-D-PCR increased the sensitivity of detection to a level similar to culture/ PCR (i.e. 103 cells/g faeces for each species). Indeed, when conducted on chicken faeces from infected flocks, the 2S-N-D-PCR detected slightly more positive samples than culture/PCR (about 4% and 5% more for B. pilosicoli and B. intermedia, respectively). The fact that the increases in the detection rate for the two species were similar, indicates that the 2S-N-D-PCR was not introducing a species-related bias in relation to detection. Furthermore, as the 2S-N-D-PCR detected all of the samples that were positive in culture/PCR, this helped to confirm its specificity. This specificity was further supported by the results obtained by sequencing the PCR products, including samples that were negative by culture/PCR. In addition, the 2S-N-D-PCR gave positive results from birds from different flocks, fed on different diets, helping to confirm that the washing process was effective at removing potential PCR inhibitors from chicken faeces. A major advantage of the 2S-N-D-PCR was that it was far more rapid than culture/PCR, potentially giving results on the same day as sample receipt rather than after 7–10 days. The availability of such rapid tests should greatly facilitate future epidemiological studies on AIS, and improve diagnostic capacity. Although the current 2S-N-D-PCR was only designed to detect B. pilosicoli and B. intermedia, in the future it could be expanded to include detection of B. alvinipulli, the other less commonly described cause of AIS. All that would be required would be to add an addition species-specific amplification to the second step, based on portions of the 16S rRNA gene or the nox gene, following their amplification in the first genus-specific step. Unfortunately, there are few strains of B. alvinipulli currently available, and so it has not been possible to design primers with confirmed specificity for this species at this time.
Acknowledgements This study was conducted with the financial support of the Australian Chicken Meat Council and the former Australian Egg Industry Council, administered through the Rural Industries Research and Development Corporation, and Murdoch University.
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