Acute infection with Brachyspira hyodysenteriae affects mucin expression, glycosylation, and fecal MUC5AC.

Introduction: Infection with strongly ß-hemolytic strains of Brachyspira hyodysenteriae leads to swine dysentery (SD), a production-limiting disease that causes mucohemorrhagic diarrhea and typhlocolitis in pigs. This pathogen has strong chemotactic activity toward mucin, and infected pigs often have a disorganized mucus layer and marked de novo expression of MUC5AC, which is not constitutively expressed in the colon. It has been shown that fucose is chemoattractant for B. hyodysenteriae, and a highly fermentable fiber diet can mitigate and delay the onset of SD. Methods: We used lectins targeting sialic acids in α-2,6 or α-2,3 linkages, N-acetylglucosamine (GlcNAc), α-linked L-fucose, and an immunohistochemical stain targeting N-glycolylneuraminic acid (NeuGc) to investigate the local expression of these mucin glycans in colonic tissues of pigs with acute SD. We used a commercial enzyme-linked immunosorbent assay (ELISA) to quantify fecal MUC5AC in infected pigs and assess its potential as a diagnostic monitoring tool and RNA in situ hybridization to detect IL-17A in the colonic mucosa. Results: Colonic mucin glycosylation during SD has an overall increase in fucose, a spatially different distribution of GlcNAc with more expression within the crypt lumens of the upper colonic mucosa, and decreased expression or a decreased trend of sialic acids in α-2,6 or α-2,3 linkages, and NeuGc compared to the controls. The degree of increased fucosylation was less in the colonic mucosa of pigs with SD and fed the highly fermentable fiber diet. There was a significant increase in MUC5AC in fecal and colonic samples of pigs with SD at the endpoint compared to the controls, but the predictive value for disease progression was limited. Discussion: Fucosylation and the impact of dietary fiber may play important roles in the pathogenesis of SD. The lack of predictive value for fecal MUC5AC quantification by ELISA is possibly due to the presence of other non-colonic sources of MUC5AC in the feces. The moderate correlation between IL-17A, neutrophils and MUC5AC confirms its immunoregulatory and mucin stimulatory role. Our study characterizes local alteration of mucin glycosylation in the colonic mucosa of pigs with SD after B. hyodysenteriae infection and may provide insight into host-pathogen interaction.

A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

Occurrence of blood parasites in seabirds admitted for rehabilitation in the Western Cape, South Africa, 2001-2013.

Blood parasites are generally uncommon in seabirds, and knowledge on their epidemiology is further limited by the fact that they often inhabit remote locations that are logistically difficult or expensive to study. We present a long term data set of blood smear examinations of 1909 seabirds belonging to 27 species that were admitted to a rehabilitation centre in Cape Town (Western Cape, South Africa) between 2001 and 2013. Blood parasites were detected in 59% of species (16/27) and 29% of individuals examined (551/1909). The following blood parasites were recorded: Babesia ugwidiensis, Babesia peircei, Babesia sp., Plasmodium sp., Leucocytozoon ugwidi, Hepatozoon albatrossi, Haemoproteus skuae and Spirochaetales. Several of the records are novel host-parasite associations, demonstrating the potential of rehabilitation centres for parasite and disease surveillance, particularly for species infrequently sampled from which no host-specific parasites have been described.

Alterações bioquímicas, anatômicas e histopatológicas em fígado de Gallus gallus Linnaeus, 1758 experimentalmente infectados por Borrelia anserina Sakharoff, 1891 / Biochemical, anatomical and histopathological changes in the liver of Gallus gallus Linnaeus, 1758 experimentally infected by Borrelia anserine Sakharoff, 1891

A espiroquetose aviária é uma enfermidade septicêmica de curso agudo, cosmopolita, que acomete diversas espécies aviárias, causada por Borrelia anserina e transmitida pelo carrapato Argas miniatus. O experimento teve como objetivos avaliar as alterações bioquímicas e anátomo-histopatológicas no fígado de Gallus gallus, causadas pela infecção experimental por B. anserina. Quarenta aves da espécie G. gallus foram divididas em quatro grupos inteiramente casualizados com 10 animais cada: G1 - inoculado com soro infectado com B. anserina; G2 - inoculado com soro fisiológico a 0,9%; G3 - exposto a ninfas de terceiro ínstar de A. miniatus infectados por B. anserina; G4 - exposto a ninfas de terceiro ínstar de A. miniatus livres de B. anserina. As aves dos Grupos 1 e 3 manifestaram no 3º e 6º dias pós-inoculação (DPI) respectivamente, sintomatologia característica da doença como inapetência, perda de peso, sonolência, diarreia esverdeada, mucosas hipocoradas, penas arrepiadas e hipertermia. Os níveis de ALT do Grupo 1 mostraram-se significativamente mais elevados apenas no 12ºDPI e 24ºDPI em relação ao seu grupo controle (Grupo 2) e no Grupo 3 esses níveis se mantiveram elevados até o 20º DPI em comparação ao seu grupo controle (Grupo 4). Os níveis da enzima AST pouco oscilaram nos grupos experimentais, embora tenham sido encontradas elevações no 12ºDPI nos Grupos 1 e 3. Os fígados das aves dos Grupos 1 e 3 apresentaram à necropsia, moderada hepatomegalia, congestão, superfície irregular e coloração vermelha a cianótica; constataram-se ainda pequenos pontos esbranquiçados na superfície. A histopatologia do fígado revelou congestão, infiltrados inflamatórios mononucleares, focos de necrose fibrinoide, dilatação dos sinusoides e vacuolização de hepatócitos. A coloração de Warthin-Starry revelou, nos fígados das aves dos Grupos 1 e 3, a presença de espiroquetas compatíveis com B. anserina, frequentemente no interior de vasos sanguíneos.(AU)

Spirochetosis avian is a septicemic disease of acute course and cosmopolitan can affect various avian species, caused by Borrelia anserina and transmitted by Argas miniatus. The experiment aimed to evaluate the biochemical, anatomical and histopathological changes in the liver of Gallus gallus caused by experimental infection with B. anserina. A total of 40 fowls of the species G. gallus were divided into four randomized groups of ten fowls each: G1 - inoculated with serum infected with B. anserina; G2 - inoculated with 0.9% saline; G3 - exposed to nymphs of 3rd instar of A. miniatus infected with B. anserina; G4 - exposed to ticks nymphs of 3rd instar of A. miniatus free of B. anserina. The fowls of Groups 1 and 3 expressed at 3 and 6 days post-inoculation (DAI) respectively , symptoms characteristic of the disease as lack of appetite , weight loss , drowsiness, greenish diarrhea, pale mucous membranes , ruffled feathers and hyperthermia. ALT of group 1 levels were significantly higher only at the 12º and 24º day after inoculation (DAI) compared with its control group (group 2), and in group 3 these levels remained high until the 20º DAI as compared with its control group (group 4). AST enzyme fluctuated little in the experimental groups, although elevations at 12ºDAI has been found in group 1 and 3. The liver of fowls in groups 1 and 3, presented at necropsy moderate hepatomegaly, congestion, irregular surface and red color to cyanotic. If found even small whitish spots on the surface. The histopathology revealed congestion, mononuclear inflammatory infiltrates, fibrinoid necrotic foci, dilatation of sinusoids, and vacuolation of hepatocytes. The Warthin-Starry staining revealed in the liver of fowls in groups 1 and 3 the presence of spirochetes compatible with B. anserina, often within blood vessels.(AU)

Brachyspira hyodysenteriae isolated from apparently healthy pig herds following an evaluation of a prototype commercial serological ELISA.

Swine dysentery (SD) is a disease mainly of grower/finisher pigs characterised by severe mucohaemorrhagic colitis. The classical aetiological agent is the anaerobic intestinal spirochaete Brachyspira hyodysenteriae, although "Brachyspira hampsonii" and Brachyspira suanatina also cause SD. This study reports on the unexpected isolation of B. hyodysenteriae from pigs in apparently healthy herds that gave positive reactions when tested with a prototype commercial serological ELISA for detecting herds infected with B. hyodysenteriae (Priocheck(®)Brachyspira porcine Ab ELISA). The ELISA was tested with sera collected at abattoirs from 1770 slaughtered pigs from 30 Australian herds, including 12 with a history of SD and18 that were considered by their consulting veterinarians to be healthy. The latter herds had no history of SD and did not routinely use antimicrobials that may have masked the disease. Based on the recommended ELISA cut-off value, 25 herds were recorded as showing evidence of infection, including 11 of 12 herds that were considered infected by the submitters and 14 of the 18 "healthy" herds. When faecal or colonic wall samples from 11 of the 14 "false positive" herds subsequently were culturing 6-24 months after the original ELISA testing was completed, different strains of B. hyodysenteriae were isolated from six herds, including a high-health status breeding herd. The existence of apparently healthy herds that are colonised by B. hyodysenteriae has major implications for the control of SD. Had the ELISA not been trialled it is unlikely that colonic samples from these herds would have been cultured and the colonisation identified.

Brachyspira and its role in avian intestinal spirochaetosis.

The fastidious, anaerobic spirochaete Brachyspira is capable of causing enteric disease in avian, porcine and human hosts, amongst others, with a potential for zoonotic transmission. Avian intestinal spirochaetosis (AIS), the resulting disease from colonisation of the caeca and colon of poultry by Brachyspira leads to production losses, with an estimated annual cost of circa £ 18 million to the commercial layer industry in the United Kingdom. Of seven known and several proposed species of Brachyspira, three are currently considered pathogenic to poultry; B. alvinipulli, B. intermedia and B. pilosicoli. Currently, AIS is primarily prevented by strict biosecurity controls and is treated using antimicrobials, including tiamulin. Other treatment strategies have been explored, including vaccination and probiotics, but such developments have been hindered by a limited understanding of the pathobiology of Brachyspira. A lack of knowledge of the metabolic capabilities and little genomic information for Brachyspira has resulted in a limited understanding of the pathobiology. In addition to an emergence of antibiotic resistance amongst Brachyspira, bans on the prophylactic use of antimicrobials in livestock are driving an urgent requirement for alternative treatment strategies for Brachyspira-related diseases, such as AIS. Advances in the molecular biology and genomics of Brachyspira heralds the potential for the development of tools for genetic manipulation to gain an improved understanding of the pathogenesis of Brachyspira.

Swine dysentery: aetiology, pathogenicity, determinants of transmission and the fight against the disease.

Swine Dysentery (SD) is a severe mucohaemorhagic enteric disease of pigs caused by Brachyspira hyodysenteriae, which has a large impact on pig production and causes important losses due to mortality and sub-optimal performance. Although B. hyodysenteriae has been traditionally considered a pathogen mainly transmitted by direct contact, through the introduction of subclinically infected animals into a previously uninfected herd, recent findings position B. hyodysenteriae as a potential threat for indirect transmission between farms. This article summarizes the knowledge available on the etiological agent of SD and its virulence traits, and reviews the determinants of SD transmission. The between-herds and within-herd transmission routes are addressed. The factors affecting disease transmission are thoroughly discussed, i.e., environmental survival of the pathogen, husbandry factors (production system, production stage, farm management), role of vectors, diet influence and interaction of the microorganism with gut microbiota. Finally, prophylactic and therapeutic approaches to fight against the disease are briefly described.

Development of a modified selective medium to enhance the recovery rate of Brachyspira hyodysenteriae and other porcine intestinal spirochaetes from faeces.

AIMS: The aim of this study was to develop a modified selective medium to improve the recovery rate of Brachyspira hyodysenteriae and other clinically significant intestinal spirochaetes from porcine faeces. METHODS AND RESULTS: The susceptibility of five Brachyspira spp. type strains and five Thai field isolates of B. hyodysenteriae to the antimicrobials halquinol and flavomycin was determined by in vitro susceptibility tests in the agar dilution method, and optimal incorporation rates were confirmed by broth dilution. All the spirochaetes were susceptible to halquinol at ≤ 1 µg ml(-1), while 16 µg ml(-1) of flavomycin (F) allowed their growth, and therefore, only the latter was selected for further use. F and different combinations of colistin (C), spectinomycin (S) and rifampacin (R) were incorporated into pre-enrichment broths and/or agar plates, and growth of the spirochaetes from seeded faeces was determined. Two solid media were selected for further testing using faeces from 90 finishing pigs on 10 farms. A previously recommended method of pre-enrichment did not increase the recovery rate. The use of blood agar modified medium (BAM) containing F (16 µg ml(-1)), S (400 µg ml(-1)), R (30 µg ml(-1)) and colistin (C, 100 U ml(-1)) (assigning as BAM-CSRF) reduced the growth of contaminating intestinal microbiota and resulted in a significantly higher rate of spirochaete recovery than the previous recommended medium. CONCLUSION: BAM-CSRF is a useful new selective medium for the isolation of B. hyodysenteriae and other intestinal spirochaetes from pig faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The new selective medium for isolating B. hyodysenteriae and other Brachyspira spp. from pig faeces will improve their recovery and subsequent disease diagnosis.

Microbial diversity in bovine papillomatous digital dermatitis in Holstein dairy cows from upstate New York.

Papillomatous digital dermatitis (PDD) is one of the most prevalent diseases of cattle, adversely affecting the dairy industry by its negative effect on milk production and reproductive performance. Our objective was to use culture-independent methods to determine the microbial diversity in different strata of PDD lesions of three Holstein dairy cows, analyzing whether major differences exist compared to foot skin of three non-infected cows. Both group-specific 16S rRNA gene PCR-denaturing gradient gel electrophoresis and clone library sequencing of broad-range 16S rRNA gene showed differences between the microbial composition of healthy dairy cows and the different strata of the lesion. The predominant bacterial community in the lesion, regardless of the stratum, consisted of 166 specific phylotypes belonging to seven bacterial phyla. Spirochetes (particularly, treponemes) was the most prominent group detected in PDD deep biopsies and was only found in samples from the lesion. Additionally, one phylotype phylogenetically affiliated with uncultured Euryarchaeota was detected in two strata of the lesion. Sequences from healthy foot skin samples revealed 86 specific phylotypes that were affiliated with Firmicutes and Proteobacteria. Our study corroborates the theory that treponemes are involved in PDD disease etiology and suggests, for the first time, the presence of archaeal members in this particular bovine infection.

Evidence that the 36 kb plasmid of Brachyspira hyodysenteriae contributes to virulence.

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently the genome of virulent Australian B. hyodysenteriae strain WA1 was sequenced, and a 36 kilobase (kb) circular plasmid was identified. The plasmid contained 31 genes including six rfb genes that were predicted to be involved with rhamnose biosynthesis, and others associated with glycosylation. In the current study a set of PCRs was developed to amplify portions of nine of the plasmid genes. When used with DNA extracted from virulent strain B204, PCR products were generated, but no products were generated with DNA from avirulent strain A1. Analysis of the DNA using pulsed field gel electrophoresis (PFGE) identified a plasmid band in strains WA1 and B204, but not in strain A1. These results demonstrate that strain A1 does not contain the plasmid, and suggests that lack of the plasmid may explain why this strain is avirulent. To determine how commonly strains lacking plasmids occur, DNA was extracted from 264 Australian field isolates of B. hyodysenteriae and subjected to PCRs for three of the plasmid genes. Only one isolate (WA400) that lacked the plasmid was identified, and this absence was confirmed by PFGE analysis of DNA from the isolate and further PCR testing. To assess its virulence, 24 pigs were experimentally challenged with cultures of WA400, and 12 control pigs were challenged with virulent strain WA1 under the same conditions. Significantly fewer (P=0.03) of the pigs challenged with WA400 became colonised and developed SD (13/24; 54%) compared to the pigs infected with WA1 (11/12; 92%). Gross lesions in the pigs colonised with WA400 tended to be less extensive than those in pigs colonised with WA1, although there were no obvious differences at the microscopic level. The results support the likelihood that plasmid-encoded genes of B. hyodysenteriae are involved in colonisation and/or disease expression.

Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle.

Bovine digital dermatitis (BDD) is a severe infectious cause of lameness which has spread through dairy cattle populations worldwide, causing serious welfare and agricultural problems. Spirochetes are the main organisms implicated and have previously proven difficult to isolate. This study aimed to isolate and characterise the range of spirochetes associated with BDD in the UK. Twenty-three spirochete isolates were obtained from 30 BDD lesions, which by 16S rRNA gene and flaB2 gene analysis clustered within the genus Treponema as three phylogroups; groups 1 (Treponema medium/Treponema vincentii-like), 2 (Treponema phagedenis-like) and 3 (Treponema denticola/Treponema putidum-like). The treponemes displayed large genotypic and phenotypic diversity between phylogroups and differed from named treponeme species. A previously isolated contagious ovine digital dermatitis spirochete was located within one of the three phylogroups, group 3, and could also be identified within this group on the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis may share the same aetiological agent. A strain isolated from a bovine interdigital dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine interdigital dermatitis and BDD may have the same causative agent. Two common enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD associated treponemes suggesting common metabolic pathways for sharing this novel niche or even common virulence traits. Further studies are required to determine whether the three groups of novel treponemes are representative of new treponeme taxa and to delineate how they interact with bovine tissues to cause disease.

Development of a multilocus sequence typing scheme for intestinal spirochaetes within the genus Brachyspira.

The purpose of this study was to evaluate a multilocus sequence typing (MLST) scheme for intestinal spirochaetes of the genus Brachyspira. Eight loci mainly coding for enzymes previously used in multilocus enzyme electrophoresis analysis of Brachyspira species were examined in 66 Brachyspira field isolates and type/reference strains. The isolates and strains were recovered from pigs, birds, dogs and a mouse and originated from seven European countries, the USA and Canada. Forty-six isolates represented recognized Brachyspira species and 20 represented provisionally designated species or isolates that have not been classified. Only two loci gave PCR products for all 66 strains and isolates, but amplicons for seven loci were obtained for 44 of the isolates. Sequences for each locus had a DNA allelic variation of 30-47 and an amino acid allelic variation of 14-47 that gave rise to the same number of sequence and amino acid types (58) for the strains and isolates studied. A population snapshot based on sequence and amino acid types showed a close phylogenetic relationship amongst the porcine isolates from the same geographical regions, and indicated a close evolutionary relationship between isolates recovered from pigs and mallards. A general concordance was obtained between the MLST groupings and classifications based on culture and biochemical tests, 16S rDNA sequence analysis and random amplified polymorphic DNA analysis. This is a first step towards establishing an MLST system for use in identifying Brachyspira species and determining relationships between individual strains and species in the genus.

Experimental study on the role of Brachyspira alvinipulli in intestinal spirochaetosis of geese.

Ten one-day-old goslings were inoculated orally with a Brachyspira alvinipulli strain isolated from the large intestine of geese that had died of intestinal spirochaetosis (Group A), 10 day-old goslings were inoculated orally with a B. hyodysenteriae strain (Group B), and a third group of 10 goslings (Group C) served as uninfected control. The goslings were observed daily for clinical signs. They were sacrificed on days 7, 14, 21 and 35 days postinfection (PI), and necropsied. Segments of the large intestine were subjected to histopathological, immunohistochemical, electron microscopic (TEM, SEM) and microbiological examinations. Mortality did not occur during the experimental period. However, in both groups the caecum of the goslings killed by bleeding was slightly dilated, in its lumen there was a watery, yellowish and frothy content, and the mucous membrane was slightly swollen. By histopathological, immunohistochemical and electron microscopic examination, B. alvinipulli and B. hyodysenteriae could be detected in the caecum or colon, in the lumen of the glands and sometimes among the glandular epithelial cells in goslings of the respective groups, and could be reisolated from these organs by culturing. A mild inflammation of the intestinal mucosa was also noted. In transverse section of the brachyspirae, numerous (16-22) periplasmic flagella could be detected inside the outer sheath, also depending on the plane of section.

The organic acid profiles in the feces of pigs at Brachyspira hyodysenteriae- or B. pilosicoli-positive farms.

We observed a significant difference in the organic acid profile of diarrheal feces between pigs infected with and free from pathogenic spirochetes. Diarrhea and loose feces were collected from growing pigs, held at 15 different commercial farms. A total of 106 samples were measured for organic acid concentration by HPLC and were checked for the presence of B. hyodysenteriae and B. pilosicoli by PCR. B. hyodysenteriae was detected in 3 samples collected from one farm. B. pilosicoli was detected in 5 samples collected from another farm. Lower concentrations of iso-butyrate and iso-valerate were likely associated with development of pathogenic spirochete infection.

A novel enteropathogenic, strongly haemolytic spirochaete isolated from pig and mallard, provisionally designated 'Brachyspira suanatina' sp. nov.

Atypical, strongly haemolytic porcine isolates of intestinal spirochaetes differing genetically from Brachyspira hyodysenteriae were identified and characterized. The isolates were subjected to culture and biochemical tests, antimicrobial susceptibility testing and molecular analyses. None of four species-specific polymerase chain reaction systems targeting genes of B. hyodysenteriae gave a positive reaction. All the atypical porcine isolates were identical in their partial 16S rRNA and nox gene sequences with a previously described isolate from a mallard (Anas platyrhynchos), and differed only slightly from another mallard isolate. All these isolates were distinctly different from all currently recognized Brachyspira species. A challenge study was carried out using recently weaned pigs. Clinical signs and macroscopic changes consistent with swine dysentery were seen both in pigs given the atypical porcine isolate and in control pigs given the reference strain of B. hyodysenteriae (B204(R)). Pigs given the genetically similar isolate from a mallard became colonized and diarrhoea was observed. This is the first study indicating that Brachyspira isolates from mallard can infect pigs and induce diarrhoea. We propose that this atypical spirochaete genotype should be regarded as a new species within the genus Brachyspira, and be provisionally designated 'Brachyspira suanatina' sp. nov.

The effect of fermentable carbohydrates on experimental swine dysentery and whip worm infections in pigs.

An experiment was conducted to study the effect of diets with contrasting fermentability in the large intestine on experimental infections with Brachyspira hyodysenteriae, the causative agent of swine dysentery, and the whip worm, Trichuris suis, in pigs. Two diets with organically grown ingredients were composed. Both diets were based on triticale and barley and supplemented with either rape seed cake (Diet 1) or dried chicory root and sweet lupins (Diet 2). The study had a three-factorial design, with eight groups of pigs receiving Diet 1 or Diet 2, +/-B. hyodysenteriae, and +/-T. suis. Pigs fed Diet 2 and challenged with B. hyodysenteriae did not develop swine dysentery and B. hyodysenteriae was not demonstrated in any of the pigs during the study. In contrast, 94% of the B. hyodysenteriae challenged pigs fed Diet 1 showed clinical symptoms of swine dysentery and all the pigs were shedding B. hyodysenteriae in faeces at some points in time during the experiment. The number of T. suis was lower in pigs fed Diet 2 compared to pigs fed Diet 1, but the differences were not significant. Pigs on Diet 1 and challenged with both pathogens showed clinical symptoms of SD for a longer period than pigs inoculated with B. hyodysenteriae only. The study showed that diets supplemented with highly fermentable carbohydrates from dried chicory roots and sweet lupins can protect pigs against developing swine dysentery, but do not have any significant influence on T. suis.

Blood concentrations of amino acids, glucose and lactate during experimental swine dysentery.

The aim of this study was to examine blood concentrations of amino acids, glucose and lactate in association with experimental swine dysentery. Ten pigs (approximately 23kg) were orally inoculated with Brachyspira hyodysenteriae. Eight animals developed muco-haemorrhagic diarrhoea with impaired general appearance, changes in white blood cell counts and increased levels of the acute phase protein Serum Amyolid A. Blood samples were taken before inoculation, during the incubation period, during clinical signs of dysentery and during recovery. Neither plasma glucose nor lactate concentrations changed during the course of swine dysentery, but the serum concentrations of gluconeogenic non-essential amino acids decreased during dysentery. This was mainly due to decreases in alanine, glutamine, serine and tyrosine. Lysine increased during dysentery and at the beginning of the recovery period, and leucine increased during recovery. Glutamine, alanine and tyrosine levels show negative correlations with the numbers of neutrophils and monocytes. In conclusion, swine dysentery altered the blood concentrations of amino acids, but not of glucose or lactate.

Consecutive pathological and immunological alterations during experimentally induced swine dysentery - a study performed by repeated endoscopy and biopsy samplings through an intestinal cannula.

The development of intestinal lesions after inoculation with Brachyspira hyodysenteriae was followed by repeated endoscopy and biopsy sampling through a caecal cannula. Seven eight-week-old pigs were cannulated and inoculated, two were cannulated but not inoculated, and two pigs were inoculated but not cannulated. Endoscopy, biopsy, and blood sampling to determine SAA (serum amyloid A), haptoglobin, cortisol, and WBC counts were performed at scheduled time-points. At the third day of disease, endoscopy showed a hyperaemic, perturbed mucosa and excessive amount of mucus. Histologically, crypt hyperplasia, depletion of goblet cell mucus, and erosions were noted. Simultaneously, elevated acute phase proteins and circulating monocytes, and decreased number of intraepithelial CD3(+) cells were observed. After five days the pigs recovered. Intestinal lesions were demarcated and interspersed among apparently normal mucosa and blood parameters returned to initial values. Endoscopy through an intestinal cannula made it possible to follow the development of intestinal alterations in vivo and describe the sequential events during the course of swine dysentery. The number of animals used in a study could thus be minimised and the precision of the experiment increased.

Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs.

Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10(2) -10(3) cells/g of faeces for 16S rRNA gene PCR and 10(4) cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.