Infections with weakly haemolytic Brachyspira species in pigs with miscellaneous chronic diseases

doi:10.1016/j.vetmic.2008.08.017

Veterinary Microbiology

Volume 134, Issues 3–4, 2 March 2009, Pages 311-317

https://doi.org/10.1016/j.vetmic.2008.08.017 Get rights and content

Abstract

The prevalence of infections with different Brachyspira species was assessed in 202 pigs with various chronic herd problems using different methods. Twenty-seven pigs (13.4%) were positive for Brachyspira spp. with at least one of the methods used. The highest number of positives was identified with mucosal scraping-PCR (23), followed by PET-PCR (22) and bacteriological–biochemical analysis (15). With the exception of three cases of B. pilosicoli infections, only weakly pathogenic Brachyspira species were identified. The majority was B. murdochii, followed by B. innocens and B. intermedia. Concurrent infections with two or more Brachyspira species were common and accounted for 37.1% of the total. Presence of weakly haemolytic Brachyspira was associated with wasting and diarrhoea in a number of cases.

This investigation shows that infections with weakly haemolytic Brachyspira spp. may contribute to colonic pathology in pigs with chronic herd problems and that mixed infections seem to occur more frequently than previously noticed.

Section snippets

Introduction and objectives

Diarrhoeic diseases caused by Brachyspira spp. in pigs are widespread and cause considerable economic problems in the swine production (Jacobson et al., 2003).

Of the currently known porcine Brachyspira species B. hyodysenteriae and B. pilosicoli are well known pathogens which cause swine dysentery and spirochaetal colitis/intestinal spirochaetosis, respectively (Moxley and Duhamel, 1999). The pathogenic importance of B. intermedia, B. murdochii and B. innocens was estimated low or non-existent

Animals, necropsy, histology and sampling

Two hundred and two pigs with miscellaneous chronic herd problems, such as diarrhoea, wasting, respiratory and dermatological problems, which had been submitted for diagnostic workup, were investigated. All pigs were subjected to a complete necropsy 1–3 h after euthanasia. After macroscopic examination for the presence of gross lesions, tissue samples from the first and the second colonic loop were fixed in 7% neutral buffered formalin and embedded in paraffin wax for histological examination.

Animal data, necropsy and histological findings

The 202 pigs investigated in this study came from 70 different farms located in the federal states of Upper Austria, Lower Austria, Styria and Burgenland.

Seven males, 107 castrated males and 88 females were investigated. The weight ranged from 4 kg to 113 kg (with the majority between 10 kg and 30 kg). Data on the age of the pigs were fragmentary and are not shown.

In 27 pigs (13.4%) at least one of the applied methods detected Brachyspira. These animals originated from 21 different farms (30% of

Discussion

The results of all four methods for determination of the Brachyspira species present (scraping-PCR, PET-PCR, biochemical classification, PCR of bacteriological isolates) used in the present study were not corresponding in all cases, a fact which may be explained by several reasons. First, there were cases which yielded a positive result by bacteriological–biochemical investigation, but a negative result with PCR. These seem to be cases with low numbers of bacteria, which could be isolated in

Acknowledgements

This study has been partly funded by the Hochschuljubiläumsstiftung of the City of Vienna and been awarded with a stipend from the Farmers Association of Lower Austria. We thank Klaus Bittermann for his help with the digital layout of the figures.

References (23)

M. Jacobson et al.
Diarrhoea in the growing pig—a comparison of clinical, morphological and microbial findings between animals from good and poor performance herds
Res. Vet. Sci.
(2003)
K. Møller et al.
Detection of Lawsonia intracellularis, Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes, Salmonella enterica, and haemolytic Escherichia coli from swine herds with and without diarrhoea among growing pigs
Vet. Microbiol.
(1998)
T. Råsbäck et al.
Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test
Vet. Microbiol.
(2005)
T. Råsbäck et al.
Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli
J. Microbiol. Methods
(2006)
C. Rosell et al.
Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs
J. Comp. Pathol.
(1999)
H. Weissenböck et al.
Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspira murdochii
Vet. Microbiol.
(2005)
E. Aasen et al.
Amplification of the ZFy and ZFx genes for sex identification in humans, cattle, sheep and goats
Biotechnol.
(1990)
G.E. Duhamel
Comparative pathology and pathogenesis of naturally acquired and experimentally induced colonic spirochetosis
Anim. Health Res. Rev.
(2001)
C. Fellström et al.
Prevalence of Serpulina species in relation to diarrhea and feed medication in pig-rearing herds in Sweden
Am. J. Vet. Res.
(1996)
C. Fellström et al.
The use of culture, pooled samples and PCR for identification of herds infected with Brachyspira hyodysenteriae
Anim. Health Res. Rev.
(2001)

M. Fossi et al.

Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection

Acta Vet. Scand.

(2005)

Cited by (30)

Characterization of Brachyspira communities from clinical cases of swine mucohaemorrhagic diarrhea through deep sequencing of the NADH oxidase (nox) gene
2018, Veterinary Microbiology
Citation Excerpt :
All clinical cases contained multiple Brachyspira species, though one species typically dominated the community. Traditionally diagnostics attribute one pathogenic species as being responsible for the disease outcome, though there have been reports of multiple Brachyspira species being detected in individual animals (Komarek et al., 2009; La et al., 2016b; Råsbäck et al., 2005; Rohde et al., 2002; Viott et al., 2013). The extent to which these mixed infections contribute to pathogenesis is not known.
Swine dysentery is traditionally associated with Brachyspira hyodysenteriae, but the re-emergence of Brachyspira-associated disease in North America associated with a novel causative species, B. hampsonii, is now a concern for swine producers. The pathogenesis of Brachyspira-associated disease is not completely understood, and it is not known whether mixed infections of Brachyspira spp. are important in disease development. Deep sequencing of partial sequences of the nox gene amplified with genus-specific primers was used to detect Brachyspira spp. in 55 fecal samples from clinical cases of mucohaemorrhagic diarrhea in pigs from Western Canada that had been identified as positive for one or more Brachyspira species using established diagnostic tests. Synthetic mixtures of Brachyspira genomic DNA were included in the study to define detection limits for the technique and identify biases in detection of different species. Multiple species were detected in all clinical cases for which sufficient nox sequence data were generated (n = 47), indicating that mixed species Brachyspira infections are common, although in most cases, one species accounted for at least half of the sequences identified. In all cases, the species detected in the original diagnostic investigation of each case was also detected by nox sequencing. Results from synthetic communities indicated that the method was highly reproducible, but also indicated potential PCR bias against B. hampsonii genomovar I. Deep sequencing of the nox gene target is a suitable method for simultaneous detection of multiple Brachyspira species in clinical case material that may offer advantages over current, more targeted diagnostic approaches for investigating the significance of mixed infections in disease development.
Veterinary Medicine, Eleventh Edition
2016, Veterinary Medicine, Eleventh Edition
Identification of weakly haemolytic Brachyspira isolates recovered from pigs with diarrhoea in Spain and Portugal and comparison with results from other countries
2013, Research in Veterinary Science
Citation Excerpt :
A more recent study from the U.S. confirmed the predominance of B. murdochii amongst Brachyspira isolates from pigs with mild to moderate mucoid diarrhoea (Hammer and Gebhart, 2013). Hence B. murdochii generally is the most commonly identified weakly haemolytic species in pigs with diarrhoea, including in Spain and Portugal, and this is important given that there is histopathological and experimental infection data supporting its likely role as a pathogen (Weissenböck et al., 2005; Komarek et al., 2009; Jensen et al., 2010). The significance of the other weakly haemolytic species is less clear.
Weakly haemolytic anaerobic intestinal spirochaetes of the genus Brachyspira are commonly identified based on species-specific gene sequences. Apart from the pathogenic Brachyspira pilosicoli, the distribution and disease associations of the other weakly haemolytic Brachyspira species in pigs have not been comprehensively investigated. In this study weakly haemolytic Brachyspira isolates (n = 67) from Spanish and Portuguese pigs with diarrhoea, negative in a routine diagnostic PCR for B. pilosicoli, were identified by sequencing their NADH oxidase genes (nox). Nearly half the isolates were identified as Brachyspira murdochii (n = 31; 46.3%). The others were Brachyspira innocens (n = 26; 38.8%), Brachyspira intermedia (n = 7; 10.4%), “Brachyspira pulli” (n = 1; 1.5%) and a potentially novel Brachyspira species (n = 2; 3%). Multilocus sequence typing (MLST) on a subset of 18 isolates confirmed their species designations, including the potential new species, and identified similarities to strains from other countries.
Comparison of atypical Brachyspira spp. clinical isolates and classic strains in a mouse model of swine dysentery
2012, Veterinary Microbiology
Citation Excerpt :
B. pilosicoli is a well-defined, weakly beta-hemolytic spirochete that is the cause of porcine intestinal spirochetosis (Trott et al., 1996) and its virulence has been confirmed experimentally (Thomson et al., 1997). The role of other weakly hemolytic Brachyspira spp. is not as clear as they are commonly present in the colons of pigs with and without diarrhea (Hudson et al., 1976) and often occur as mixed infections coincident with various chronic diseases (Komarek et al., 2009). Weakly beta-hemolytic isolates characteristic of B. intermedia have been recovered from pigs with diarrhea (Binek and Szynkiewicz, 1984; Fellström and Gunnarsson, 1995), but have not been confirmed as pathogens under experimental conditions (Neef et al., 1994).
Multiple Brachyspira spp. can colonize the porcine colon, and the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae is typically associated with clinical swine dysentery. Recently, several Brachyspira spp. have been isolated from the feces of pigs with clinical disease suggestive of swine dysentery, yet these isolates were not identified as B. hyodysenteriae by genotypic or phenotypic methods. This study used a mouse model of swine dysentery to compare the pathogenic potential of seventeen different Brachyspira isolates including eight atypical clinical isolates, six typical clinical isolates, the standard strain of B. hyodysenteriae (B204), and reference strains of Brachyspira intermedia and Brachyspira innocens. Results revealed that strongly beta-hemolytic isolates induced significantly greater cecal inflammation than weakly beta-hemolytic isolates regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as ‘Brachyspira sp. SASK30446’ and B. intermedia by PCR produced lesions indistinguishable from those caused by B. hyodysenteriae in this model.
The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces
2011, Veterinary Microbiology
Citation Excerpt :
In the present investigation, four out of the 12 samples from which B. pilosicoli had been cultivated were found negative by Bpilo-qPCR. Similarly, Komarek et al. (2009) reported that several samples were PCR negative although they were found cultivation positive. Råsbäck et al. (2006) however, reported that compared to cultivation PCR lowered the sensitivity by a factor 103–104 for detection of B. hyodysenteriae and B. pilosicoli in feces.
Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig feces. Standard curves were based on feces spiked with the respective reference strains. The detection limits from the spiking experiments were 10² bacteria/g feces for Bpilo-qPCR and Laws-qPCR, 10³ CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all four qPCR assays was between 0.91 and 1.01 with R² above 0.993. Standard curves, slopes and elevation, varied between assays and between measurements from pure DNA from reference strains and feces spiked with the respective strains. The linear ranges found for spiked fecal samples differed both from the linear ranges from pure culture of the reference strains and between the qPCR tests. The linear ranges were five log units for F4-qPCR, and Laws-qPCR, six log units for F18-qPCR and three log units for Bpilo-qPCR in spiked feces. When measured on pure DNA from the reference strains used in spiking experiments, the respective log ranges were: seven units for Bpilo-qPCR, Laws-qPCR and F18-qPCR and six log units for F4-qPCR. This shows the importance of using specific standard curves, where each pathogen is analysed in the same matrix as sample DNA. The qPCRs were compared to traditional bacteriological diagnostic methods and found to be more sensitive than cultivation for E. coli and B. pilosicoli. The qPCR assay for Lawsonia was also more sensitive than the earlier used method due to improvements in DNA extraction. In addition, as samples were not analysed for all four pathogen agents by traditional diagnostic methods, many samples were found positive for agents that were not expected on the basis of age and case history. The use of quantitative PCR tests for diagnosis of enteric diseases provides new possibilities for veterinary diagnostics. The parallel simultaneous analysis for several bacteria in multi-qPCR and the determination of the quantities of the infectious agents increases the information obtained from the samples and the chance for obtaining a relevant diagnosis.
Investigations on the prevalence and potential pathogenicity of intestinal trichomonads in pigs using in situ hybridization
2011, Veterinary Parasitology
In pigs, three different trichomonad species (Tritrichomonas foetus, Tetratrichomonas buttreyi and Tritrichomonas rotunda) have been described as commensals in the large intestine. The aim of this study was to gain further knowledge on the prevalence and pathogenicity of trichomonads in pigs by using a morphology-based approach. Chromogenic in situ hybridization (ISH) is a technique which allows direct localization of the protozoa in the intestinal tissue and correlation of the infection with pathologic changes. In the present study paraffin-wax embedded colon and ileum samples of 192 pigs were analyzed with this method. Using a probe specific for all known members of the order Trichomonadida (OT) 100 of the 192 pigs were tested positive. Thereof, about 10% showed moderate to high-grade parasitic load with trichomonads invading the lamina propria. Partial 18S ribosomal RNA gene sequencing of six of those animals showed a 100% sequence identity with T. foetus sequences. The majority of these animals were also tested positive for other enteropathogenic agents, such as Brachyspira sp., Lawsonia intracellularis, Escherichia coli, and porcine circovirus type 2. All OT-positive samples were further examined with another probe complementary to all known Tritrichomonas species sequences including T. foetus, T. augusta, T. mobilensis and T. nonconforma resulting in only 48 positives. These results suggest that T. foetus may not only be considered as an intestinal commensal but rather a facultative pathogen of pigs with a tendency for tissue invasion in the presence of other agents. Furthermore, the existence of other – yet to be identified – trichomonad species in the colon of pigs was shown.

View all citing articles on Scopus

View full text

Article preview

Veterinary Microbiology

Abstract

Section snippets

Introduction and objectives

Animals, necropsy, histology and sampling

Animal data, necropsy and histological findings

Discussion

Acknowledgements

References (23)

Diarrhoea in the growing pig—a comparison of clinical, morphological and microbial findings between animals from good and poor performance herds

Res. Vet. Sci.

Detection of Lawsonia intracellularis, Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes, Salmonella enterica, and haemolytic Escherichia coli from swine herds with and without diarrhoea among growing pigs

Vet. Microbiol.

Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test

Vet. Microbiol.

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli

J. Microbiol. Methods

Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs

J. Comp. Pathol.

Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspira murdochii

Vet. Microbiol.

Amplification of the ZFy and ZFx genes for sex identification in humans, cattle, sheep and goats

Biotechnol.

Comparative pathology and pathogenesis of naturally acquired and experimentally induced colonic spirochetosis

Anim. Health Res. Rev.

Prevalence of Serpulina species in relation to diarrhea and feed medication in pig-rearing herds in Sweden

Am. J. Vet. Res.

The use of culture, pooled samples and PCR for identification of herds infected with Brachyspira hyodysenteriae

Anim. Health Res. Rev.

Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection

Acta Vet. Scand.

Cited by (30)

Characterization of Brachyspira communities from clinical cases of swine mucohaemorrhagic diarrhea through deep sequencing of the NADH oxidase (nox) gene

Veterinary Medicine, Eleventh Edition

Identification of weakly haemolytic Brachyspira isolates recovered from pigs with diarrhoea in Spain and Portugal and comparison with results from other countries

Comparison of atypical Brachyspira spp. clinical isolates and classic strains in a mouse model of swine dysentery

The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces

Investigations on the prevalence and potential pathogenicity of intestinal trichomonads in pigs using in situ hybridization