- Email: [email protected]
Faecal excretion of intestinal spirochaetes by urban dogs, and their pathogenicity in a chick model of intestinal spirochaetosis
Research in Veterinary Science 91 (2011) e38–e43
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Faecal excretion of intestinal spirochaetes by urban dogs, and their pathogenicity in a chick model of intestinal spirochaetosis Nuvee Prapasarakul a,⇑, Kittitat Lugsomya a, Sirilak Disatian b, Thawat Lekdumrongsak b, Wijit Banlunara c, Prugsawon Chetanachan d, David J. Hampson e a
Department of Veterinary Microbiology, Chulalongkorn University, Bangkok 10330, Thailand Department of Veterinary Medicine, Chulalongkorn University, Bangkok 10330, Thailand Department of Pathology, Chulalongkorn University, Bangkok 10330, Thailand d Department of Medical Science, National Institute of Medical Science, Bangkok 11000, Thailand e Animal Research Institute, School of Veterinary and Biomedical Sciences, Murdoch University, Perth, Western Australia 6150, Australia b c
a r t i c l e
i n f o
Article history: Received 6 August 2010 Accepted 18 January 2011
Keywords: Brachyspira Spirochaete Dog Chick model Pathogenic potential
a b s t r a c t This study aimed to obtain information about the types of spirochaetes colonising urban dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis. Spirochaetes were isolated from the faeces of six of 47 (12.8%) healthy dogs and 11 of 104 (10.6%) dogs with diarrhoea. Their biochemical properties and 16S ribosomal DNA sequences were analysed. Four isolates were identified as Brachyspira pilosicoli, three resembled ‘‘Brachyspira pulli’’, nine clustered with ‘‘Brachyspira canis’’ and one was similar to Brachyspira intermedia. Canine isolates of B. pilosicoli, ‘‘B. canis’’ and ‘‘B. pulli’’, and control strains of Brachyspira hyodysenteriae, B. pilosicoli and Brachyspira innocens colonised experimentally infected day-old chicks. The chicks did not develop diarrhoea, but were significantly lighter than the non-infected group and those infected with B. innocens after 21 days (P < 0.05). Using immunohistochemistry, spirochaetes were observed covering the surface epithelium and in the crypts of chicks in all three groups challenged with the canine isolates. Variable histopathological changes were seen, with the greatest inflammatory cell infiltration into the lamina propria occurring in the group infected with ‘‘B. pulli’’ . Canine ‘‘B. canis’’, ‘‘B. pulli’’ and B. pilosicoli isolates may have pathogenic potential. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Although various species of intestinal spirochaetes (genus Brachyspira) colonise mammals and birds, to date there has been relatively little information about these bacteria in dogs. These spirochaetes are slow-growing anaerobes, and their speciation is not always straightforward. Weakly b-haemolytic species have been reported in healthy dogs and dogs with diarrhoea (Duhamel et al., 1995; Fellström et al., 2001; Manabe et al., 2004; Trott et al., 1997). Canine intestinal spirochaetes (CIS) have been classified by their biochemical properties, by 16S ribosomal (r) DNA sequencing, multilocus enzyme electrophoresis (MLEE), speciesspecific PCR and pulsed-field gel electrophoresis (PFGE) analysis. By MLEE, two distinct clusters were observed amongst a collection of 30 CIS isolates from Australia and USA, corresponding to Brachyspira pilosicoli and a new group resembling Brachyspira innocens that was designated as ‘‘Brachyspira canis’’ (Duhamel et al., 1998). ⇑ Corresponding author. Tel.: +66 2218 9583; fax: +66 2251 1656. E-mail address: [email protected] (N. Prapasarakul). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.01.015
Using 16S rDNA sequencing, 22 CISs from Scandinavia, the USA, Australia and Germany were identified as B. pilosicoli, ‘‘B. canis’’ and a group that clustered with Brachyspira alvinipulli (Johansson et al., 2004). Most recently, 41 Spanish CIS were identified as B. pilosicoli, or ‘‘B. canis’’, with one isolate resembling Brachyspira intermedia (Hidalgo et al., 2010). The pathogenic potential of canine intestinal spirochaete isolates has not been investigated in detail. Experimental infection studies in dogs have not been conducted, mainly due to welfare considerations. Various other species such as mice, pigs and chickens have been used to test the pathogenic potential of the different Brachyspira species, and of these the day-old chick model has provided particularly useful (Trott et al., 1995; Muniappa and Duhamel, 1997a,b; Trott and Hampson, 1998). Young birds are inexpensive, disease is easy to reproduce, and the pathological changes that occur resemble those seen in the normal host species. The aims of the current study were to obtain information about the types of spirochaetes colonising dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis.
e39
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
2. Materials and methods 2.1. Spirochaete isolation and biochemical testing Single faecal samples were collected from each of 47 healthy dogs and 104 dogs with diarrhoea presented to the Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, in the period October 2004–December 2006. The healthy dogs were attending a vaccination clinic whilst the dogs with diarrhoea had been admitted for treatment of the condition. Diarrhoea was defined as an increase in frequency of defaecation combined with an increase in water content of the stools, with or without the presence of mucus and fresh blood. The consistency of the faeces was subjectively categorised by the attending veterinarian as normal, soft, mucoid or bloody. The age was recorded as <1 year or P1 year. The owners were asked whether their dogs received a commercial diet or a homemade diet. Faecal smears were made and examined for spirochaetes under a phase contrast microscope. The faeces were plated on Trypticase Soy agar (TSA, Oxoid) containing 5% defibrinated sheep blood and 400 lg/mL spectinomycin (Pfizer) (Songer et al., 1976), and were incubated in at 37 °C in an anaerobic atmosphere created using a commercial gas generator (BBL GasPak Plus; Becton–Dickinson). The plates were checked after three days, and then reincubated and checked every 3–4 days up to 14 days. Type or reference strains of Brachyspira hyodysenteriae (B204R; ATCC 31212), B. pilosicoli (P43/6/78T; ATCC 51139), B. innocens (B256T; ATCC 29796), B. alvinipulli (C1T; ATCC51933), Brachyspira murdochii (56-50T; ATCC51284) and B. intermedia (PWS/AT; ATCC51140) were used as controls. Translucent flat growth on the isolation plates was selected and confirmed as representing spirochaetes by phase-contrast microscopy. Following subculture to purity, preliminary identification was based on haemolysis pattern and biochemical properties, as described (Fellström et al., 1997). 2.2. 16S rDNA sequencing and phylogenetic analysis The Thai CIS were subjected to complete 16S rDNA sequencing, as previously described (Ochiai et al., 1997; Pettersson et al., 1996), and the sequences were submitted to GenBank (Supplementary Table 1). The 16S rDNA sequences of the type strains and related isolates also were obtained from GenBank and were used for phylogenetic alignment. The phylogenetic tree was linearised assuming equal evolutionary rates in all lineages (Takezaki et al., 1995). The evolutionary distances were computed using the Maximum Composite Likelihood method and were in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007), using the NeighbourJoining method (Saitou and Nei, 1987). 2.3. Experimental infection of chicks Animal handling protocols were approved by the Ethics Committee on Experimental Animal Usage and Animal Welfare, Chulalongkorn University. Day-old conventional ISA Babcock Brown chicks (n = 96) from a high-health status breeder farm without the major endemic diseases of chickens were randomly divided into six groups of 14 birds that were challenged with spirochaetes and one control group of 12 birds that received sterile broth. Each group was housed in a cage in a separate room, with an average floor area of 40 cm2 per bird. They were offered a starter diet in mash form, not containing antimicrobials, and had free access to water. Movement of personnel between rooms was restricted,
and in each room staff wore clean protective clothing and gloves, and cleaned and disinfected their boots before moving between rooms. The spirochaetes used to infect the chicks where reference strains B. hyodysenteriae ATCC31212, B. pilosicoli ATCC51139, B. innocens ATCC29796 and Thai canine isolates ‘‘B. pulli’’ AT1.8.8, ‘‘B. canis’’ BT7.4.12 and B. pilosicoli PT5.4.12. They were grown on TSA, harvested and adjusted to 108 cfu/mL in Trypticase Soy broth (TSB, Oxoid). The chicks were inoculated by crop tube with 1 mL of the appropriate spirochaetal suspension daily for 3 days. The control group was treated with an equivalent volume of sterile TSB. Chick performance, clinical symptoms and faecal consistency were recorded daily. One chick from each group was submitted for necropsy on days 8, 9 and 10 post-infection (PI), with the remainder killed at 21 days PI. Following intramuscular pre-anaesthesia with 0.1 mL diazepam (Valium, Roche) they were killed by an injection of 95% methanol into the atlanto-occipital joint. The right caecum was examined for lesions and was scraped and cultured for spirochaetes.
2.4. Histopathology, immunohistochemistry and bacteriology The proximal, middle and distal sections of the left caecum and rectum from birds were placed in Bouin’s fixative. Sections were processed, paraffin-embedded and stained with haematoxylin– eosin. Immunohistochemistry was carried out using the peroxidase–anti-peroxidase method (Avidin–Biotin Complex; ABC kit, DAKO) (Bratthauer, 1994). Hyperimmune sera were raised in rabbits against the homologous strains to a titer of at least 1:32 to the specific antigen in an immunodiffusion test, as previously described (Abe and Adachi, 2004). The sections were deparaffinated by graded alcohol and xylene, rehydrated in phosphate buffer saline (PBS), digested with a 0.1% trypsin solution and the peroxidase activity was quenched with 3% H2O2 in absolute methanol. After blocking of non-specific binding with 10% skimmed milk (Gupta et al., 1985), the sections were incubated with rabbit hyperimmune serum diluted 1:200 for 1 h at 37 °C, rinsed in PBS, and incubated with biotinylated goat anti-rabbit IgG antibody (DAKO) diluted 1:200 for 1 h at 37 °C (Ofek et al., 1986). Antigens were visualised using an ABC Kit (DAKO) and 0.05% 3,30 -diaminobenzidine tetrahydrochloride (DAB), according to the manufacturer’s recommendations. Microscopic changes were evaluated qualitatively, including crypt proliferation, villus shortening and inflammatory cell infiltration. For each, a scale of 0–3 was used, where 0 = no abnormalities; 1 = mild changes; 2 = moderate changes; 3 = severe changes. The presence and number of spirochaetes also was recorded using a
Table 1 Comparison between faecal consistency, age and food type and whether the dog was positive for spirochaetes by culture. Category
Number of samples
Number and % faecal samples positive for spirochaetes Number positive
%
47 34 36 34
6 3 4 4
12.8 8.8 11.1 11.8
Age <1 year P1 year
91 60
12 5
13.2 8.3
Food type Commercial Homemade
70 81
8 9
11.4 11.1
Faecal consistency Normal Soft Mucoid diarrhoea Bloody diarrhoea
e40
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
Fig. 1. Dendrogram based on 16S rDNA sequences showing evolutionary relationships between Brachyspira spp. including 17 Thai CIS isolates clustered with ‘‘B. pulli’’, B. intermediate, ‘‘B. canis’’ and B. pilosicoli. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replications) is indicated on the tree. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
e41
scale of 0–3, with 0 = no spirochaetes; 1 = a few spirochaetes; 2 = moderate numbers of spirochaetes; 3 = large numbers of spirochaetes. The location of the spirochaetes was also recorded: C = in the crypts; L = in the lamina propria; E = on the epithelium; G = invading goblet cells. Faeces and caecal contents collected at post-mortem were cultured for Brachyspira species as described for the canine samples. The phenotypic profiles of selected isolates were tested to help confirm that cross-contamination of different species of spirochaetes between rooms had not occurred. Faeces were plated to McConkey’s agar (Oxoid) to exclude the possibility of Salmonella enterica serovars being present, and faecal smears were examined for coccidial oocysts.
The relationships of the Thai CIS to those of other Brachyspira species based on 16S rRNA gene sequences are shown in Fig. 1. Four clades of Thai isolates were identified, most closely corresponding to ‘‘B. pulli’’ (n = 3), ‘‘B. canis’’ (n = 9), B. intermedia (n = 1) and B. pilosicoli (n = 4). The three ‘‘B. pulli’’ isolates were from dogs <1 year of age with soft, mucoid and bloody faeces, respectively. The nine ‘‘ B. canis’’ isolates included four from normal dogs (one puppy and three adults) and five from dogs <1 year with soft (n = 2), mucoid (n = 1) or bloody (n = 2) diarrhoea. The B. intermedia isolate was from an adult dog with bloody diarrhoea. The four B. pilosicoli isolates were from two normal dogs (one adult and one puppy), and two young dogs with mucoid diarrhoea.
2.5. Statistical analysis
3.2. Pathogenicity in the chick model
The relationship between altered faecal consistency and spirochaete recovery in dogs was analysed using Fisher’s exact test. Comparison of average daily gain amongst the groups of chicks was by one-way analysis of variance and the Tukey–Kramer multiple comparisons test, using Graphpad Instat software (Graphpad, USA).
One chick in the reference B. hyodysenteriae group developed transient bloody mucoid diarrhoea in the week following infection,
3. Results 3.1. Spirochaete isolation, characterization and statistical associations Spirochaetes were isolated from 17 (11.3%) of the canine faecal samples (Table 1). The culture-positive samples were all positive on the smears. Spirochaetes were isolated from 6 of 47 (12.8%) healthy dogs and from 11 of 104 (10.6%) dogs with diarrhoea. Isolation rates were similar in dogs with the three types of diarrhoea (8.8%, 11.1% and 11.8% for the three types of diarrhoea, respectively). Spirochaetes were more common in dogs less than one year of age (12/91, 13.2%) than in adults (5/60, 8.3%), but this difference was not significant (P > 0.44). There was no significant difference between the type of faecal consistency and the presence of spirochaetes. The diet did not significantly influence spirochaete recovery. The spirochaetes all had a serpentine shape and a spiral movement. All were weakly b-haemolytic and indole negative. Three different biochemical phenotypes were recognized: the ‘‘B. pulli’’ isolates were hippurate negative and positive for a-galactosidase and b-glucosidase activity; the ‘‘B. canis’’ isolates were negative for hippurate and a-galactosidase, but positive for b-glucosidase; the B. pilosicoli isolates were positive for hippurate and a-galactosidase, but negative for b-glucosidase. The profiles of all the Thai CIS subsequently identified by 16S rRNA sequencing as ‘‘B. pulli’’, ‘‘B. canis’’ and B. pilosicoli were consistent with the profiles of the reference strains for the three species. The Thai CIS identified as B. intermedia by 16S rRNA sequencing was indole negative, and had the same biochemical profile as the ‘‘B. canis’’ isolates. Table 2 Comparison of average daily gain in grams amongst the seven groups of experimental chicks at 21 days post-infection. Group
Number
Mean ± SD
B. hyodysenteriae B. pilosicoli B. innocens ‘‘B. pulli’’ ‘‘B. canis’’ Thai B. pilosicoli Control
11 11 11 11 11 11 9
3.78 ± 0.15 3.69 ± 0.22 4.11a ± 0.15 3.19 ± 0.67 3.66 ± 0.44 3.66 ± 0.65 4.26a ± 0.27
a Groups B. innocens and control were significantly heavier than the other groups. No other differences were significant.
Fig. 2. Histological findings in the mid-portion of the caecum of chicks at 21 days PI. (A) Cross-section from a chick infected with ‘‘B. canis’’ showing crypt proliferation (40). (B) Immunohistochemical staining showing spirochaetes lining the apical epithelium in a chick infected with B. pilosicoli (1000). (C) ‘‘B. pulli’’ in the luminal crypt and goblet cells (1000).
e42
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
Table 3 Histological lesions and presence and location of spirochaetes in the caecal epithelium of chicks from the seven experimental groups.
a b
Experimental group, degree of change and number affected/total number examinedb
Days post-infection
Histopathology/immunohistochemistry
B. hyodysenteriae
B. pilosicoli
B. innocens
‘‘B. pulli’’
‘‘B. canis’’
Thai B. pilosicoli
Control
8–10
Crypt proliferation Villus shortening Inflammatory cell infiltration Spirochaetesa
+3 (2/3) +2 (3/3) +1 (3/3) +3 (3/3) C, L, E
+2 (3/3) +2 (3/3) +1 (3/3) +3 (2/3) C, E
0 (3/3) 0 (3/3) +1 (1/3) +2 (1/3) C
+2 (1/3) +3 (2/3) +1 (2/3) +3 (2/3) C, E
0 (3/3) +2 (2/3) +1 (1/3) +3 (1/3) C, E
+1 +2 +1 +3 G
0 0 0 0
21
Crypt proliferation Villus shortening
+3 (8/11) +2 (3/11)
+3 (8/11) +2 (3/11)
+1 (11/11) 0 (11/11)
+2 (6/11) +2 (5/11)
+2 (8/11) +3 (3/11)
0 (9/9) 0 (9/9)
Inflammatory cell infiltration Spirochaetesa
+1 (11/11) 0 (11/11)
+1 (11/11) +3 (11/11) C, E, G
+2 (11/11) +3 (11/11) C
+3 (11/11) +3 (2/11) C, L, E, G +2 (9/11) C, L, E, G
+2 (6/11) +3 (3/11) +2 (2/11) +2 (11/11) +3 (8/11) C, E
+1 (11/11) +2 (11/11) C, E, G
0 (9/9) 0 (9/9)
(1/3) (2/3) (3/3) (3/3)
(3/3) (3/3) (3/3) (3/3)
Letters (marked in bold) indicate the location of spirochaetes: C, in crypts; L, in the lamina propria; E, on the epithelium; G, invading goblet cells. The scoring system for the degree of severity of observed changes (0 to + 3) is defined in the text.
but recovered spontaneously. None of the other chicks showed signs of illness during the experiment. At the end of the experiment the chicks in the reference B. innocens and control groups were significantly heavier than the chicks in the other five groups (Table 2). On day 10 PI, spirochaetes of the same type that were inoculated into the chicks were recovered from chicks in their respective groups. The same occurred at 21 days PI, except that B. hyodysenteriae was not recovered from the reference B. hyodysenteriae group. Coccidia and Salmonella enterica were not detected. At necropsy, only the chicks in the control and reference B. innocens groups had normal viscous caecal contents, whilst the caecae of the chicks in the other groups contained green watery/mucoid exudate. The histopathological findings and location of the spirochaetes in relation to the caecal epithelium are summarised in Table 3. The chicks in the control group showed no macroscopic or microscopic changes. The chicks in the reference B. innocens group had spirochaetes in their caecal crypts and showed moderately extensive inflammatory cell infiltration of the lamina propria. Crypt cell proliferation and villus shortening was present in varying numbers of chicks in all the other groups, particularly at 21 days PI (Fig. 2 A). Inflammatory cell infiltration was commonly observed, and was most extensive in the chicks colonised with the Thai ‘‘B. pulli’’ isolate, followed by those challenged with the Thai ‘‘ B. canis’’ isolate and the reference B. innocens strain. On day 21 PI immunochemical staining revealed large numbers of spirochaetes in the crypts of all infected groups except the reference B. hyodysenteriae group. Spirochaetes were present on the epithelial surface in the case of chicks in groups reference B. pilosicoli, Thai B. pilosicoli, ‘‘B. pulli’’ and ‘‘B. canis’’; they were in the goblet cells in the case of reference B. pilosicoli and Thai B. pilosicoli and ‘‘B. pulli’’ isolate groups (Fig. 2 B and C), and in the lamina propria in the case of the Thai ‘‘B. pulli’’ isolate group. Overall, the chicks infected with ‘‘B. pulli’’ had the most extensive changes at 21 days PI. 4. Discussion This study has demonstrated that, as in other countries, urban dogs in Thailand, commonly shed intestinal spirochaetes in their faeces. The 11.2% prevalence rate obtained by culture was similar to the 13.2% recently found in dogs in Spain (Hidalgo et al., 2010), but less than the 18.7% reported in Australia (Lee and Hampson, 1996). As in the recent Spanish study, spirochaetes tended to be isolated more commonly from dogs less than one year of age than in older animals, although this difference was not significant. The diet type did not influence the shedding rates.
Spirochaetes were observed and isolated at a similar prevalence in both healthy dogs and dogs with diarrhoea, and rates were similar whether the faeces were soft, mucoid or haemorrhagic. Interpretation of whether the spirochaetes had clinical significance was complicated by the fact that four different groups of spirochaetes were ultimately identified, based on combined phenotypic and genotypic analysis. Four isolates were identified as B. pilosicoli. The low overall prevalence (2.6%) for this species in urban dogs was generally similar to the findings in dogs living in tea-estates in India where the prevalence was 1% (Munshi et al., 2004), for urban dogs in Spain where it was 4.8% (Hidalgo et al., 2010), and for village dogs in Papua New Guinea where it was 5.3% (Trott et al., 1997); however, it was significantly (P = 0.005) less than the 14.3% detected using PCR on DNA extracted from isolation plates inoculated with faeces from puppies in pet shops in Australia (Oxberry and Hampson, 2003). In the latter case the puppies may have been exposed in the commercial breeding premises from where they originated. B. pilosicoli has been thought to have a pathogenic role in dogs (Duhamel et al., 1998; Johansson et al., 2004; Oxberry and Hampson, 2003), as it does in species such as pigs and chickens. In the recent Spanish study there was a statistically significant (P < 0.001) association between the shedding of B. pilosicoli by dogs and the presence of diarrhoea (Hidalgo et al., 2010); however, in the current study only two of the four isolates were from animals with diarrhoea, and it is not possible to be sure that the spirochaetes had a role in the aetiology of the diarrhoea. The results do reinforce the possibility that dogs could be a source of B. pilosicoli infection for human beings, as well as other species. The largest group of isolates (9/17; 53%) was identified as ‘‘B. canis’’, and this is consistent with other studies where ‘‘B. canis’’ has been found to be the most common Brachyspira species colonising dogs (Duhamel et al., 1998; Hidalgo et al., 2010; Johansson et al., 2004; Oxberry and Hampson, 2003). ‘‘B. canis’’ is generally considered to be a commensal species of dogs, although in the current study five of the nine isolates (56%) came from dogs with forms of diarrhoea. In this study, as in the recent Spanish study (Hidalgo et al., 2010), one isolate was tentatively identified as B. intermedia. As B. intermedia commonly colonises chickens (Stephens and Hampson, 1999), it is possible that these dogs became infected following consumption of contaminated chicken meat or chicken carcases. The Thai dog had bloody diarrhoea but the Spanish dog was healthy (Á. Hidalgo, personal communication), so it is difficult to know whether B. intermedia should be regarded as a potential pathogen in dogs. Three isolates were identified as ‘‘B. pulli’’, a
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
species originally identified in laying chickens in Australia (McLaren et al., 1997; Stephens and Hampson, 1999), and subsequently in The Netherlands and Scandinavia (Feberwee et al., 2008; Jansson et al., 2008). The species is generally thought to be non-pathogenic in chickens, but all three canine isolates were recovered from young dogs with diarrhoea. Again it is possible that they may have been exposed to these spirochaetes through contact with chickens, raw chicken meat or avian faeces. The experimental infection model worked well, as the control chicks inoculated with TSB did not develop pathological changes, whilst changes occurred in the chicks inoculated with spirochaetes. The histopathological findings in the chicks in the reference B. hyodysenteriae and B. pilosicoli groups, and the Thai B. pilosicoli group were generally consistent with those in previous studies that used SPF chicks (Trott et al., 1995; Muniappa and Duhamel, 1997a,b; Trott and Hampson, 1998), although even the supposedly non-pathogenic B. innocens did colonise the crypts and induce infiltration of inflammatory cells into the lamina propria. All three Thai CSIs colonised the chicks, being observed on the surface epithelium and in the crypts, and they all caused variable histopathological changes. These findings suggest that all CISs may have some pathogenic potential; nevertheless, they may not necessarily produce obvious pathological lesions or clinical signs in dogs. It was interesting that ‘‘B. pulli’’ seemed to be the most pathogenic of the three Thai CIS isolates tested, although this may simply reflect the fact that this species is adapted to chickens. These findings indicate that further investigation is required into the potential role of ‘‘B. pulli’’ as a pathogen both in chickens and dogs. Acknowledgments This work was supported by the Thailand Research Fund (MRG 4880072). The authors thank Chula Unisearch, Chulalongkorn University for assistance during preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.rvsc.2011.01.015. References Abe, Y., Adachi, Y., 2004. A possibility of application of the 105-kilodaltons protein of Brachyspira alvinipulli cross-reacting with antisera to five species in the genus Brachyspira to diagnosis. The Journal of Veterinary Medical Science 66, 773– 778. Bratthauer, G.L., 1994. The avidin–biotin complex (ABC) method. Methods in Molecular Biology 34, 175–184. Duhamel, G.E., Muniappa, N., Mathiesen, M.R., Johnson, J.L., Toth, J., Elder, R.O., Doster, A.R., 1995. Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis. Journal of Clinical Microbiology 33, 2212–2215. Duhamel, G.E., Trott, D.J., Muniappa, N., Mathiesen, M.R., Tarasiuk, K., Lee, J.I., Hampson, D.J., 1998. Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identified group provisionally designated ‘‘Serpulina canis’’ sp. nov. Journal of Clinical Microbiology 36, 2264–2270. Feberwee, A., Hampson, D.J., Phillips, N.D., La, T., van der Heijden, H.M., Wellenberg, G.J., Dwars, R.M., Landman, W.J., 2008. Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both. Journal of Clinical Microbiology 46, 593–600.
e43
Fellström, C., Pettersson, B., Thomson, J., Gunnarsson, A., Persson, M., Johansson, K.E., 1997. Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR. Journal of Clinical Microbiology 35, 462–467. Fellström, C., Pettersson, B., Zimmerman, U., Gunnarsson, A., Feinstein, R., 2001. Classification of Brachyspira spp. isolated from Swedish dogs. Animal Health Research Reviews 2, 75–82. Gupta, P.K., Myers, J.D., Baylin, S.B., Mulshine, J.L., Cuttitta, F., Gazdar, A.F., 1985. Improved antigen detection in ethanol-fixed cytologic specimens. A modified avidin–biotin-peroxidase complex (ABC) method. Diagnostic Cytopathology 1, 133–136. Hidalgo, Á., Rubio, P., Osorio, J., Carvajal, A., 2010. Prevalence of Brachyspira pilosicoli and ‘‘Brachyspira canis’’ in dogs and their association with diarrhoea. Veterinary Microbiology 146, 356–360. Jansson, D.S., Fellström, C., Råsbäck, T., Vågsholm, I., Gunnarsson, A., Ingermaa, F., Johansson, K.E., 2008. Phenotypic and molecular characterization of Brachyspira spp. isolated from laying hens in different housing systems.. Veterinary Microbiology 130, 348–362. Johansson, K.E., Duhamel, G.E., Bergsjo, B., Engvall, E.O., Persson, M., Pettersson, B., Fellström, C., 2004. Identification of three clusters of canine intestinal spirochaetes by biochemical and 16S rDNA sequence analysis. Journal of Medical Microbiology 53, 345–350. Lee, J.I., Hampson, D.J., 1996. The prevalence of intestinal spirochaetes in dogs. Australian Veterinary Journal 74, 466–467. Manabe, M., Suenaga, I., Ogawa, Y., Adachi, Y., 2004. Brachyspira pilosicoli isolated from two beagles and one mongrel in Japan. The Journal of Veterinary Medical Science 66, 589–592. 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. Journal of Clinical Microbiology 35, 412–417. Muniappa, N., Duhamel, G.E., 1997a. Outer membrane-associated serine protease of intestinal spirochetes. FEMS Microbiology Letters 154, 159–164. Muniappa, N., Duhamel, G.E., 1997b. Phenotypic and genotypic profiles of human, canine, and porcine spirochetes associated with colonic spirochetosis correlates with in vivo brush border attachment. Advances in Experimental Medicine and Biology 412, 159–166. Munshi, M.A., Traub, R.J., Robertson, I.D., Mikosza, A.S., Hampson, D.J., 2004. Colonization and risk factors for Brachyspira aalborgi and Brachyspira pilosicoli in humans and dogs on tea estates in Assam, India. Epidemiology Infection 132, 137–144. Ochiai, S., Adachi, Y., Mori, K., 1997. Unification of the genera Serpulina and Brachyspira, and proposals of Brachyspira hyodysenteriae Comb. Nov., Brachyspira innocens Comb. Nov. and Brachyspira pilosicoli Comb. Nov. Microbiology and Immunology 41, 445–452. Ofek, I., Courtney, H.S., Schifferli, D.M., Beachey, E.H., 1986. Enzyme-linked immunosorbent assay for adherence of bacteria to animal cells. Journal of Clinical Microbiology 24, 512–516. Oxberry, S.L., Hampson, D.J., 2003. Colonisation of pet shop puppies with Brachyspira pilosicoli. Veterinary Microbiology 93, 167–174. Pettersson, B., Fellström, C., Andersson, A., Uhlen, M., Gunnarsson, A., Johansson, K.E., 1996. The phylogeny of intestinal porcine spirochetes (Serpulina species) based on sequence analysis of the 16S rRNA gene. Journal of Bacteriology 178, 4189–4199. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425. Songer, J.G., Kinyon, J.M., Harris, D.L., 1976. Selective medium for isolation of Treponema hyodysenteriae. Journal of Clinical Microbiology 4, 57–60. Stephens, C.P., Hampson, D.J., 1999. Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia. Avian Pathology 28, 447–454. Takezaki, N., Rzhetsky, A., Nei, M., 1995. Phylogenetic test of the molecular clock and linearized trees. Molecular Biology and Evolution 12, 823–833. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596–1599. Trott, D.J., Combs, B.G., Mikosza, A.S., Oxberry, S.L., Robertson, I.D., Passey, M., Taime, J., Sehuko, R., Alpers, M.P., Hampson, D.J., 1997. The prevalence of Serpulina pilosicoli in humans and domestic animals in the Eastern Highlands of Papua New Guinea. Epidemiology and Infection 119, 369–379. Trott, D.J., Hampson, D.J., 1998. Evaluation of day-old specific pathogen-free chicks as an experimental model for pathogenicity testing of intestinal spirochaete species. The Journal of Comparative Pathology 118, 365–381. Trott, D.J., McLaren, A.J., Hampson, D.J., 1995. Pathogenicity of human and porcine intestinal spirochetes in one-day-old specific-pathogen-free chicks: an animal model of intestinal spirochetosis. Infection and Immunity 63, 3705–3710.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Faecal excretion of intestinal spirochaetes by urban dogs, and their pathogenicity in a chick model of intestinal spirochaetosis Nuvee Prapasarakul a,⇑, Kittitat Lugsomya a, Sirilak Disatian b, Thawat Lekdumrongsak b, Wijit Banlunara c, Prugsawon Chetanachan d, David J. Hampson e a
Department of Veterinary Microbiology, Chulalongkorn University, Bangkok 10330, Thailand Department of Veterinary Medicine, Chulalongkorn University, Bangkok 10330, Thailand Department of Pathology, Chulalongkorn University, Bangkok 10330, Thailand d Department of Medical Science, National Institute of Medical Science, Bangkok 11000, Thailand e Animal Research Institute, School of Veterinary and Biomedical Sciences, Murdoch University, Perth, Western Australia 6150, Australia b c
a r t i c l e
i n f o
Article history: Received 6 August 2010 Accepted 18 January 2011
Keywords: Brachyspira Spirochaete Dog Chick model Pathogenic potential
a b s t r a c t This study aimed to obtain information about the types of spirochaetes colonising urban dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis. Spirochaetes were isolated from the faeces of six of 47 (12.8%) healthy dogs and 11 of 104 (10.6%) dogs with diarrhoea. Their biochemical properties and 16S ribosomal DNA sequences were analysed. Four isolates were identified as Brachyspira pilosicoli, three resembled ‘‘Brachyspira pulli’’, nine clustered with ‘‘Brachyspira canis’’ and one was similar to Brachyspira intermedia. Canine isolates of B. pilosicoli, ‘‘B. canis’’ and ‘‘B. pulli’’, and control strains of Brachyspira hyodysenteriae, B. pilosicoli and Brachyspira innocens colonised experimentally infected day-old chicks. The chicks did not develop diarrhoea, but were significantly lighter than the non-infected group and those infected with B. innocens after 21 days (P < 0.05). Using immunohistochemistry, spirochaetes were observed covering the surface epithelium and in the crypts of chicks in all three groups challenged with the canine isolates. Variable histopathological changes were seen, with the greatest inflammatory cell infiltration into the lamina propria occurring in the group infected with ‘‘B. pulli’’ . Canine ‘‘B. canis’’, ‘‘B. pulli’’ and B. pilosicoli isolates may have pathogenic potential. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Although various species of intestinal spirochaetes (genus Brachyspira) colonise mammals and birds, to date there has been relatively little information about these bacteria in dogs. These spirochaetes are slow-growing anaerobes, and their speciation is not always straightforward. Weakly b-haemolytic species have been reported in healthy dogs and dogs with diarrhoea (Duhamel et al., 1995; Fellström et al., 2001; Manabe et al., 2004; Trott et al., 1997). Canine intestinal spirochaetes (CIS) have been classified by their biochemical properties, by 16S ribosomal (r) DNA sequencing, multilocus enzyme electrophoresis (MLEE), speciesspecific PCR and pulsed-field gel electrophoresis (PFGE) analysis. By MLEE, two distinct clusters were observed amongst a collection of 30 CIS isolates from Australia and USA, corresponding to Brachyspira pilosicoli and a new group resembling Brachyspira innocens that was designated as ‘‘Brachyspira canis’’ (Duhamel et al., 1998). ⇑ Corresponding author. Tel.: +66 2218 9583; fax: +66 2251 1656. E-mail address: [email protected] (N. Prapasarakul). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.01.015
Using 16S rDNA sequencing, 22 CISs from Scandinavia, the USA, Australia and Germany were identified as B. pilosicoli, ‘‘B. canis’’ and a group that clustered with Brachyspira alvinipulli (Johansson et al., 2004). Most recently, 41 Spanish CIS were identified as B. pilosicoli, or ‘‘B. canis’’, with one isolate resembling Brachyspira intermedia (Hidalgo et al., 2010). The pathogenic potential of canine intestinal spirochaete isolates has not been investigated in detail. Experimental infection studies in dogs have not been conducted, mainly due to welfare considerations. Various other species such as mice, pigs and chickens have been used to test the pathogenic potential of the different Brachyspira species, and of these the day-old chick model has provided particularly useful (Trott et al., 1995; Muniappa and Duhamel, 1997a,b; Trott and Hampson, 1998). Young birds are inexpensive, disease is easy to reproduce, and the pathological changes that occur resemble those seen in the normal host species. The aims of the current study were to obtain information about the types of spirochaetes colonising dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis.
e39
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
2. Materials and methods 2.1. Spirochaete isolation and biochemical testing Single faecal samples were collected from each of 47 healthy dogs and 104 dogs with diarrhoea presented to the Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, in the period October 2004–December 2006. The healthy dogs were attending a vaccination clinic whilst the dogs with diarrhoea had been admitted for treatment of the condition. Diarrhoea was defined as an increase in frequency of defaecation combined with an increase in water content of the stools, with or without the presence of mucus and fresh blood. The consistency of the faeces was subjectively categorised by the attending veterinarian as normal, soft, mucoid or bloody. The age was recorded as <1 year or P1 year. The owners were asked whether their dogs received a commercial diet or a homemade diet. Faecal smears were made and examined for spirochaetes under a phase contrast microscope. The faeces were plated on Trypticase Soy agar (TSA, Oxoid) containing 5% defibrinated sheep blood and 400 lg/mL spectinomycin (Pfizer) (Songer et al., 1976), and were incubated in at 37 °C in an anaerobic atmosphere created using a commercial gas generator (BBL GasPak Plus; Becton–Dickinson). The plates were checked after three days, and then reincubated and checked every 3–4 days up to 14 days. Type or reference strains of Brachyspira hyodysenteriae (B204R; ATCC 31212), B. pilosicoli (P43/6/78T; ATCC 51139), B. innocens (B256T; ATCC 29796), B. alvinipulli (C1T; ATCC51933), Brachyspira murdochii (56-50T; ATCC51284) and B. intermedia (PWS/AT; ATCC51140) were used as controls. Translucent flat growth on the isolation plates was selected and confirmed as representing spirochaetes by phase-contrast microscopy. Following subculture to purity, preliminary identification was based on haemolysis pattern and biochemical properties, as described (Fellström et al., 1997). 2.2. 16S rDNA sequencing and phylogenetic analysis The Thai CIS were subjected to complete 16S rDNA sequencing, as previously described (Ochiai et al., 1997; Pettersson et al., 1996), and the sequences were submitted to GenBank (Supplementary Table 1). The 16S rDNA sequences of the type strains and related isolates also were obtained from GenBank and were used for phylogenetic alignment. The phylogenetic tree was linearised assuming equal evolutionary rates in all lineages (Takezaki et al., 1995). The evolutionary distances were computed using the Maximum Composite Likelihood method and were in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007), using the NeighbourJoining method (Saitou and Nei, 1987). 2.3. Experimental infection of chicks Animal handling protocols were approved by the Ethics Committee on Experimental Animal Usage and Animal Welfare, Chulalongkorn University. Day-old conventional ISA Babcock Brown chicks (n = 96) from a high-health status breeder farm without the major endemic diseases of chickens were randomly divided into six groups of 14 birds that were challenged with spirochaetes and one control group of 12 birds that received sterile broth. Each group was housed in a cage in a separate room, with an average floor area of 40 cm2 per bird. They were offered a starter diet in mash form, not containing antimicrobials, and had free access to water. Movement of personnel between rooms was restricted,
and in each room staff wore clean protective clothing and gloves, and cleaned and disinfected their boots before moving between rooms. The spirochaetes used to infect the chicks where reference strains B. hyodysenteriae ATCC31212, B. pilosicoli ATCC51139, B. innocens ATCC29796 and Thai canine isolates ‘‘B. pulli’’ AT1.8.8, ‘‘B. canis’’ BT7.4.12 and B. pilosicoli PT5.4.12. They were grown on TSA, harvested and adjusted to 108 cfu/mL in Trypticase Soy broth (TSB, Oxoid). The chicks were inoculated by crop tube with 1 mL of the appropriate spirochaetal suspension daily for 3 days. The control group was treated with an equivalent volume of sterile TSB. Chick performance, clinical symptoms and faecal consistency were recorded daily. One chick from each group was submitted for necropsy on days 8, 9 and 10 post-infection (PI), with the remainder killed at 21 days PI. Following intramuscular pre-anaesthesia with 0.1 mL diazepam (Valium, Roche) they were killed by an injection of 95% methanol into the atlanto-occipital joint. The right caecum was examined for lesions and was scraped and cultured for spirochaetes.
2.4. Histopathology, immunohistochemistry and bacteriology The proximal, middle and distal sections of the left caecum and rectum from birds were placed in Bouin’s fixative. Sections were processed, paraffin-embedded and stained with haematoxylin– eosin. Immunohistochemistry was carried out using the peroxidase–anti-peroxidase method (Avidin–Biotin Complex; ABC kit, DAKO) (Bratthauer, 1994). Hyperimmune sera were raised in rabbits against the homologous strains to a titer of at least 1:32 to the specific antigen in an immunodiffusion test, as previously described (Abe and Adachi, 2004). The sections were deparaffinated by graded alcohol and xylene, rehydrated in phosphate buffer saline (PBS), digested with a 0.1% trypsin solution and the peroxidase activity was quenched with 3% H2O2 in absolute methanol. After blocking of non-specific binding with 10% skimmed milk (Gupta et al., 1985), the sections were incubated with rabbit hyperimmune serum diluted 1:200 for 1 h at 37 °C, rinsed in PBS, and incubated with biotinylated goat anti-rabbit IgG antibody (DAKO) diluted 1:200 for 1 h at 37 °C (Ofek et al., 1986). Antigens were visualised using an ABC Kit (DAKO) and 0.05% 3,30 -diaminobenzidine tetrahydrochloride (DAB), according to the manufacturer’s recommendations. Microscopic changes were evaluated qualitatively, including crypt proliferation, villus shortening and inflammatory cell infiltration. For each, a scale of 0–3 was used, where 0 = no abnormalities; 1 = mild changes; 2 = moderate changes; 3 = severe changes. The presence and number of spirochaetes also was recorded using a
Table 1 Comparison between faecal consistency, age and food type and whether the dog was positive for spirochaetes by culture. Category
Number of samples
Number and % faecal samples positive for spirochaetes Number positive
%
47 34 36 34
6 3 4 4
12.8 8.8 11.1 11.8
Age <1 year P1 year
91 60
12 5
13.2 8.3
Food type Commercial Homemade
70 81
8 9
11.4 11.1
Faecal consistency Normal Soft Mucoid diarrhoea Bloody diarrhoea
e40
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
Fig. 1. Dendrogram based on 16S rDNA sequences showing evolutionary relationships between Brachyspira spp. including 17 Thai CIS isolates clustered with ‘‘B. pulli’’, B. intermediate, ‘‘B. canis’’ and B. pilosicoli. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replications) is indicated on the tree. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
e41
scale of 0–3, with 0 = no spirochaetes; 1 = a few spirochaetes; 2 = moderate numbers of spirochaetes; 3 = large numbers of spirochaetes. The location of the spirochaetes was also recorded: C = in the crypts; L = in the lamina propria; E = on the epithelium; G = invading goblet cells. Faeces and caecal contents collected at post-mortem were cultured for Brachyspira species as described for the canine samples. The phenotypic profiles of selected isolates were tested to help confirm that cross-contamination of different species of spirochaetes between rooms had not occurred. Faeces were plated to McConkey’s agar (Oxoid) to exclude the possibility of Salmonella enterica serovars being present, and faecal smears were examined for coccidial oocysts.
The relationships of the Thai CIS to those of other Brachyspira species based on 16S rRNA gene sequences are shown in Fig. 1. Four clades of Thai isolates were identified, most closely corresponding to ‘‘B. pulli’’ (n = 3), ‘‘B. canis’’ (n = 9), B. intermedia (n = 1) and B. pilosicoli (n = 4). The three ‘‘B. pulli’’ isolates were from dogs <1 year of age with soft, mucoid and bloody faeces, respectively. The nine ‘‘ B. canis’’ isolates included four from normal dogs (one puppy and three adults) and five from dogs <1 year with soft (n = 2), mucoid (n = 1) or bloody (n = 2) diarrhoea. The B. intermedia isolate was from an adult dog with bloody diarrhoea. The four B. pilosicoli isolates were from two normal dogs (one adult and one puppy), and two young dogs with mucoid diarrhoea.
2.5. Statistical analysis
3.2. Pathogenicity in the chick model
The relationship between altered faecal consistency and spirochaete recovery in dogs was analysed using Fisher’s exact test. Comparison of average daily gain amongst the groups of chicks was by one-way analysis of variance and the Tukey–Kramer multiple comparisons test, using Graphpad Instat software (Graphpad, USA).
One chick in the reference B. hyodysenteriae group developed transient bloody mucoid diarrhoea in the week following infection,
3. Results 3.1. Spirochaete isolation, characterization and statistical associations Spirochaetes were isolated from 17 (11.3%) of the canine faecal samples (Table 1). The culture-positive samples were all positive on the smears. Spirochaetes were isolated from 6 of 47 (12.8%) healthy dogs and from 11 of 104 (10.6%) dogs with diarrhoea. Isolation rates were similar in dogs with the three types of diarrhoea (8.8%, 11.1% and 11.8% for the three types of diarrhoea, respectively). Spirochaetes were more common in dogs less than one year of age (12/91, 13.2%) than in adults (5/60, 8.3%), but this difference was not significant (P > 0.44). There was no significant difference between the type of faecal consistency and the presence of spirochaetes. The diet did not significantly influence spirochaete recovery. The spirochaetes all had a serpentine shape and a spiral movement. All were weakly b-haemolytic and indole negative. Three different biochemical phenotypes were recognized: the ‘‘B. pulli’’ isolates were hippurate negative and positive for a-galactosidase and b-glucosidase activity; the ‘‘B. canis’’ isolates were negative for hippurate and a-galactosidase, but positive for b-glucosidase; the B. pilosicoli isolates were positive for hippurate and a-galactosidase, but negative for b-glucosidase. The profiles of all the Thai CIS subsequently identified by 16S rRNA sequencing as ‘‘B. pulli’’, ‘‘B. canis’’ and B. pilosicoli were consistent with the profiles of the reference strains for the three species. The Thai CIS identified as B. intermedia by 16S rRNA sequencing was indole negative, and had the same biochemical profile as the ‘‘B. canis’’ isolates. Table 2 Comparison of average daily gain in grams amongst the seven groups of experimental chicks at 21 days post-infection. Group
Number
Mean ± SD
B. hyodysenteriae B. pilosicoli B. innocens ‘‘B. pulli’’ ‘‘B. canis’’ Thai B. pilosicoli Control
11 11 11 11 11 11 9
3.78 ± 0.15 3.69 ± 0.22 4.11a ± 0.15 3.19 ± 0.67 3.66 ± 0.44 3.66 ± 0.65 4.26a ± 0.27
a Groups B. innocens and control were significantly heavier than the other groups. No other differences were significant.
Fig. 2. Histological findings in the mid-portion of the caecum of chicks at 21 days PI. (A) Cross-section from a chick infected with ‘‘B. canis’’ showing crypt proliferation (40). (B) Immunohistochemical staining showing spirochaetes lining the apical epithelium in a chick infected with B. pilosicoli (1000). (C) ‘‘B. pulli’’ in the luminal crypt and goblet cells (1000).
e42
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
Table 3 Histological lesions and presence and location of spirochaetes in the caecal epithelium of chicks from the seven experimental groups.
a b
Experimental group, degree of change and number affected/total number examinedb
Days post-infection
Histopathology/immunohistochemistry
B. hyodysenteriae
B. pilosicoli
B. innocens
‘‘B. pulli’’
‘‘B. canis’’
Thai B. pilosicoli
Control
8–10
Crypt proliferation Villus shortening Inflammatory cell infiltration Spirochaetesa
+3 (2/3) +2 (3/3) +1 (3/3) +3 (3/3) C, L, E
+2 (3/3) +2 (3/3) +1 (3/3) +3 (2/3) C, E
0 (3/3) 0 (3/3) +1 (1/3) +2 (1/3) C
+2 (1/3) +3 (2/3) +1 (2/3) +3 (2/3) C, E
0 (3/3) +2 (2/3) +1 (1/3) +3 (1/3) C, E
+1 +2 +1 +3 G
0 0 0 0
21
Crypt proliferation Villus shortening
+3 (8/11) +2 (3/11)
+3 (8/11) +2 (3/11)
+1 (11/11) 0 (11/11)
+2 (6/11) +2 (5/11)
+2 (8/11) +3 (3/11)
0 (9/9) 0 (9/9)
Inflammatory cell infiltration Spirochaetesa
+1 (11/11) 0 (11/11)
+1 (11/11) +3 (11/11) C, E, G
+2 (11/11) +3 (11/11) C
+3 (11/11) +3 (2/11) C, L, E, G +2 (9/11) C, L, E, G
+2 (6/11) +3 (3/11) +2 (2/11) +2 (11/11) +3 (8/11) C, E
+1 (11/11) +2 (11/11) C, E, G
0 (9/9) 0 (9/9)
(1/3) (2/3) (3/3) (3/3)
(3/3) (3/3) (3/3) (3/3)
Letters (marked in bold) indicate the location of spirochaetes: C, in crypts; L, in the lamina propria; E, on the epithelium; G, invading goblet cells. The scoring system for the degree of severity of observed changes (0 to + 3) is defined in the text.
but recovered spontaneously. None of the other chicks showed signs of illness during the experiment. At the end of the experiment the chicks in the reference B. innocens and control groups were significantly heavier than the chicks in the other five groups (Table 2). On day 10 PI, spirochaetes of the same type that were inoculated into the chicks were recovered from chicks in their respective groups. The same occurred at 21 days PI, except that B. hyodysenteriae was not recovered from the reference B. hyodysenteriae group. Coccidia and Salmonella enterica were not detected. At necropsy, only the chicks in the control and reference B. innocens groups had normal viscous caecal contents, whilst the caecae of the chicks in the other groups contained green watery/mucoid exudate. The histopathological findings and location of the spirochaetes in relation to the caecal epithelium are summarised in Table 3. The chicks in the control group showed no macroscopic or microscopic changes. The chicks in the reference B. innocens group had spirochaetes in their caecal crypts and showed moderately extensive inflammatory cell infiltration of the lamina propria. Crypt cell proliferation and villus shortening was present in varying numbers of chicks in all the other groups, particularly at 21 days PI (Fig. 2 A). Inflammatory cell infiltration was commonly observed, and was most extensive in the chicks colonised with the Thai ‘‘B. pulli’’ isolate, followed by those challenged with the Thai ‘‘ B. canis’’ isolate and the reference B. innocens strain. On day 21 PI immunochemical staining revealed large numbers of spirochaetes in the crypts of all infected groups except the reference B. hyodysenteriae group. Spirochaetes were present on the epithelial surface in the case of chicks in groups reference B. pilosicoli, Thai B. pilosicoli, ‘‘B. pulli’’ and ‘‘B. canis’’; they were in the goblet cells in the case of reference B. pilosicoli and Thai B. pilosicoli and ‘‘B. pulli’’ isolate groups (Fig. 2 B and C), and in the lamina propria in the case of the Thai ‘‘B. pulli’’ isolate group. Overall, the chicks infected with ‘‘B. pulli’’ had the most extensive changes at 21 days PI. 4. Discussion This study has demonstrated that, as in other countries, urban dogs in Thailand, commonly shed intestinal spirochaetes in their faeces. The 11.2% prevalence rate obtained by culture was similar to the 13.2% recently found in dogs in Spain (Hidalgo et al., 2010), but less than the 18.7% reported in Australia (Lee and Hampson, 1996). As in the recent Spanish study, spirochaetes tended to be isolated more commonly from dogs less than one year of age than in older animals, although this difference was not significant. The diet type did not influence the shedding rates.
Spirochaetes were observed and isolated at a similar prevalence in both healthy dogs and dogs with diarrhoea, and rates were similar whether the faeces were soft, mucoid or haemorrhagic. Interpretation of whether the spirochaetes had clinical significance was complicated by the fact that four different groups of spirochaetes were ultimately identified, based on combined phenotypic and genotypic analysis. Four isolates were identified as B. pilosicoli. The low overall prevalence (2.6%) for this species in urban dogs was generally similar to the findings in dogs living in tea-estates in India where the prevalence was 1% (Munshi et al., 2004), for urban dogs in Spain where it was 4.8% (Hidalgo et al., 2010), and for village dogs in Papua New Guinea where it was 5.3% (Trott et al., 1997); however, it was significantly (P = 0.005) less than the 14.3% detected using PCR on DNA extracted from isolation plates inoculated with faeces from puppies in pet shops in Australia (Oxberry and Hampson, 2003). In the latter case the puppies may have been exposed in the commercial breeding premises from where they originated. B. pilosicoli has been thought to have a pathogenic role in dogs (Duhamel et al., 1998; Johansson et al., 2004; Oxberry and Hampson, 2003), as it does in species such as pigs and chickens. In the recent Spanish study there was a statistically significant (P < 0.001) association between the shedding of B. pilosicoli by dogs and the presence of diarrhoea (Hidalgo et al., 2010); however, in the current study only two of the four isolates were from animals with diarrhoea, and it is not possible to be sure that the spirochaetes had a role in the aetiology of the diarrhoea. The results do reinforce the possibility that dogs could be a source of B. pilosicoli infection for human beings, as well as other species. The largest group of isolates (9/17; 53%) was identified as ‘‘B. canis’’, and this is consistent with other studies where ‘‘B. canis’’ has been found to be the most common Brachyspira species colonising dogs (Duhamel et al., 1998; Hidalgo et al., 2010; Johansson et al., 2004; Oxberry and Hampson, 2003). ‘‘B. canis’’ is generally considered to be a commensal species of dogs, although in the current study five of the nine isolates (56%) came from dogs with forms of diarrhoea. In this study, as in the recent Spanish study (Hidalgo et al., 2010), one isolate was tentatively identified as B. intermedia. As B. intermedia commonly colonises chickens (Stephens and Hampson, 1999), it is possible that these dogs became infected following consumption of contaminated chicken meat or chicken carcases. The Thai dog had bloody diarrhoea but the Spanish dog was healthy (Á. Hidalgo, personal communication), so it is difficult to know whether B. intermedia should be regarded as a potential pathogen in dogs. Three isolates were identified as ‘‘B. pulli’’, a
N. Prapasarakul et al. / Research in Veterinary Science 91 (2011) e38–e43
species originally identified in laying chickens in Australia (McLaren et al., 1997; Stephens and Hampson, 1999), and subsequently in The Netherlands and Scandinavia (Feberwee et al., 2008; Jansson et al., 2008). The species is generally thought to be non-pathogenic in chickens, but all three canine isolates were recovered from young dogs with diarrhoea. Again it is possible that they may have been exposed to these spirochaetes through contact with chickens, raw chicken meat or avian faeces. The experimental infection model worked well, as the control chicks inoculated with TSB did not develop pathological changes, whilst changes occurred in the chicks inoculated with spirochaetes. The histopathological findings in the chicks in the reference B. hyodysenteriae and B. pilosicoli groups, and the Thai B. pilosicoli group were generally consistent with those in previous studies that used SPF chicks (Trott et al., 1995; Muniappa and Duhamel, 1997a,b; Trott and Hampson, 1998), although even the supposedly non-pathogenic B. innocens did colonise the crypts and induce infiltration of inflammatory cells into the lamina propria. All three Thai CSIs colonised the chicks, being observed on the surface epithelium and in the crypts, and they all caused variable histopathological changes. These findings suggest that all CISs may have some pathogenic potential; nevertheless, they may not necessarily produce obvious pathological lesions or clinical signs in dogs. It was interesting that ‘‘B. pulli’’ seemed to be the most pathogenic of the three Thai CIS isolates tested, although this may simply reflect the fact that this species is adapted to chickens. These findings indicate that further investigation is required into the potential role of ‘‘B. pulli’’ as a pathogen both in chickens and dogs. Acknowledgments This work was supported by the Thailand Research Fund (MRG 4880072). The authors thank Chula Unisearch, Chulalongkorn University for assistance during preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.rvsc.2011.01.015. References Abe, Y., Adachi, Y., 2004. A possibility of application of the 105-kilodaltons protein of Brachyspira alvinipulli cross-reacting with antisera to five species in the genus Brachyspira to diagnosis. The Journal of Veterinary Medical Science 66, 773– 778. Bratthauer, G.L., 1994. The avidin–biotin complex (ABC) method. Methods in Molecular Biology 34, 175–184. Duhamel, G.E., Muniappa, N., Mathiesen, M.R., Johnson, J.L., Toth, J., Elder, R.O., Doster, A.R., 1995. Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis. Journal of Clinical Microbiology 33, 2212–2215. Duhamel, G.E., Trott, D.J., Muniappa, N., Mathiesen, M.R., Tarasiuk, K., Lee, J.I., Hampson, D.J., 1998. Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identified group provisionally designated ‘‘Serpulina canis’’ sp. nov. Journal of Clinical Microbiology 36, 2264–2270. Feberwee, A., Hampson, D.J., Phillips, N.D., La, T., van der Heijden, H.M., Wellenberg, G.J., Dwars, R.M., Landman, W.J., 2008. Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both. Journal of Clinical Microbiology 46, 593–600.
e43
Fellström, C., Pettersson, B., Thomson, J., Gunnarsson, A., Persson, M., Johansson, K.E., 1997. Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR. Journal of Clinical Microbiology 35, 462–467. Fellström, C., Pettersson, B., Zimmerman, U., Gunnarsson, A., Feinstein, R., 2001. Classification of Brachyspira spp. isolated from Swedish dogs. Animal Health Research Reviews 2, 75–82. Gupta, P.K., Myers, J.D., Baylin, S.B., Mulshine, J.L., Cuttitta, F., Gazdar, A.F., 1985. Improved antigen detection in ethanol-fixed cytologic specimens. A modified avidin–biotin-peroxidase complex (ABC) method. Diagnostic Cytopathology 1, 133–136. Hidalgo, Á., Rubio, P., Osorio, J., Carvajal, A., 2010. Prevalence of Brachyspira pilosicoli and ‘‘Brachyspira canis’’ in dogs and their association with diarrhoea. Veterinary Microbiology 146, 356–360. Jansson, D.S., Fellström, C., Råsbäck, T., Vågsholm, I., Gunnarsson, A., Ingermaa, F., Johansson, K.E., 2008. Phenotypic and molecular characterization of Brachyspira spp. isolated from laying hens in different housing systems.. Veterinary Microbiology 130, 348–362. Johansson, K.E., Duhamel, G.E., Bergsjo, B., Engvall, E.O., Persson, M., Pettersson, B., Fellström, C., 2004. Identification of three clusters of canine intestinal spirochaetes by biochemical and 16S rDNA sequence analysis. Journal of Medical Microbiology 53, 345–350. Lee, J.I., Hampson, D.J., 1996. The prevalence of intestinal spirochaetes in dogs. Australian Veterinary Journal 74, 466–467. Manabe, M., Suenaga, I., Ogawa, Y., Adachi, Y., 2004. Brachyspira pilosicoli isolated from two beagles and one mongrel in Japan. The Journal of Veterinary Medical Science 66, 589–592. 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. Journal of Clinical Microbiology 35, 412–417. Muniappa, N., Duhamel, G.E., 1997a. Outer membrane-associated serine protease of intestinal spirochetes. FEMS Microbiology Letters 154, 159–164. Muniappa, N., Duhamel, G.E., 1997b. Phenotypic and genotypic profiles of human, canine, and porcine spirochetes associated with colonic spirochetosis correlates with in vivo brush border attachment. Advances in Experimental Medicine and Biology 412, 159–166. Munshi, M.A., Traub, R.J., Robertson, I.D., Mikosza, A.S., Hampson, D.J., 2004. Colonization and risk factors for Brachyspira aalborgi and Brachyspira pilosicoli in humans and dogs on tea estates in Assam, India. Epidemiology Infection 132, 137–144. Ochiai, S., Adachi, Y., Mori, K., 1997. Unification of the genera Serpulina and Brachyspira, and proposals of Brachyspira hyodysenteriae Comb. Nov., Brachyspira innocens Comb. Nov. and Brachyspira pilosicoli Comb. Nov. Microbiology and Immunology 41, 445–452. Ofek, I., Courtney, H.S., Schifferli, D.M., Beachey, E.H., 1986. Enzyme-linked immunosorbent assay for adherence of bacteria to animal cells. Journal of Clinical Microbiology 24, 512–516. Oxberry, S.L., Hampson, D.J., 2003. Colonisation of pet shop puppies with Brachyspira pilosicoli. Veterinary Microbiology 93, 167–174. Pettersson, B., Fellström, C., Andersson, A., Uhlen, M., Gunnarsson, A., Johansson, K.E., 1996. The phylogeny of intestinal porcine spirochetes (Serpulina species) based on sequence analysis of the 16S rRNA gene. Journal of Bacteriology 178, 4189–4199. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425. Songer, J.G., Kinyon, J.M., Harris, D.L., 1976. Selective medium for isolation of Treponema hyodysenteriae. Journal of Clinical Microbiology 4, 57–60. Stephens, C.P., Hampson, D.J., 1999. Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia. Avian Pathology 28, 447–454. Takezaki, N., Rzhetsky, A., Nei, M., 1995. Phylogenetic test of the molecular clock and linearized trees. Molecular Biology and Evolution 12, 823–833. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596–1599. Trott, D.J., Combs, B.G., Mikosza, A.S., Oxberry, S.L., Robertson, I.D., Passey, M., Taime, J., Sehuko, R., Alpers, M.P., Hampson, D.J., 1997. The prevalence of Serpulina pilosicoli in humans and domestic animals in the Eastern Highlands of Papua New Guinea. Epidemiology and Infection 119, 369–379. Trott, D.J., Hampson, D.J., 1998. Evaluation of day-old specific pathogen-free chicks as an experimental model for pathogenicity testing of intestinal spirochaete species. The Journal of Comparative Pathology 118, 365–381. Trott, D.J., McLaren, A.J., Hampson, D.J., 1995. Pathogenicity of human and porcine intestinal spirochetes in one-day-old specific-pathogen-free chicks: an animal model of intestinal spirochetosis. Infection and Immunity 63, 3705–3710.