Prevalence of Brachyspira species in pigs with diarrhoea in Spain.
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Animals↗
Biomedical subjects
G E Duhamel
Publications and source records attributed to G E Duhamel.
Cecal spirochetosis caused by Brachyspira pilosicoli in commercial turkeys.
Spirochetes that were identified as Brachyspira pilosicoli were present in the ceca of 7.5- to 18-wk-old turkeys with cecal spirochetosis and typhlitis. The identity of B. pilosicoli was confirmed on the basis of ultrastructural morphology of the cecal epithelium adherent microbes, immunohistochemical staining with a Brachyspira genus-specific monoclonal antibody, and amplification of a B. pilosicoli species-specific 16S ribosomal RNA (rrs gene) sequence by using the polymerase chain reaction and DNA obtained by laser-capture microdissection of the epithelium-adherent microbial fringe. To the author's knowledge, this is the first report of B. pilosicoli in the ceca of turkeys.
Animals↗
Genome diversity among regional populations of Francisella tularensis subspecies tularensis and Francisella tularensis subspecies holarctica isolated from the US.
Francisella tularensis is a highly infectious facultative intracellular pathogen that is considered a potential agent of bioterrorism. Four different F. tularensis subspecies have been identified and they appear to display different ecological and virulence characteristics as well as differences in geographical distribution. One simple explanation for the variation in ecological and virulence characteristics is that they are conferred by differences in genome content. To characterize genome content among stains isolated from United States, we have used a DNA microarray designed from a shotgun library of a reference strain. Polymorphisms distributed among polyphyletic sets of strains was the most common pattern of genome alteration observed, indicating that strain-specific genome variability is significant. Nonetheless, 13 different contiguous segments of the genome were found to be missing exclusively in each of the subsp. holarctica strains tested. All 13 are associated with repeat sequences or transposases that could promote insertion/deletion events. Comparison of the live vaccine strain to other holarctica strains also identified three regions that are absent exclusively in the live vaccine strain derived from holarctica.
Bacterial Vaccines↗
Ileocolitis associated with Anaerobiospirillum in cats.
Ileocolitis associated with spiral bacteria identified as an Anaerobiospirillum sp. was found in six cats. Two cats had acute onset of gastrointestinal signs characterized by vomiting and diarrhea in one cat and vomiting in another cat, one cat had chronic diarrhea that was refractory to medical therapy; one cat had acute onset of anorexia and lethargy, and two cats had clinical signs that were not related to the gastrointestinal tract. The presence of an Anaerobiospirillum sp. was demonstrated on the basis of ultrastructural morphology of spiral bacteria associated with intestinal lesions and PCR amplification of a genus-specific 16S rRNA gene from affected tissues from each cat. The colons of three clinically healthy cats without lesions and one cat with mild colitis not associated with spiral bacteria were negative for Anaerobiospirillum spp. in the same assay. Comparative nucleotide sequence analysis of cloned PCR products from three affected cats further suggested that the spiral bacteria were closely related to Anaerobiospirillum succiniciproducens.
Anaerobiospirillum↗
Colonic spirochetosis of colony-raised rhesus macaques associated with Brachyspira and Helicobacter.
Colonic spirochetosis is an inflammatory bowel disease that affects a broad range of hosts, including human and non-human primates. The disease in humans and non-human primates is characterized by intimate attachment of the anaerobic spirochetes Brachyspira aalborgi and B. pilosicoli, and some unclassified flagellated microbes along the apical membrane of colonic enterocytes. Although the presence of spiral-shaped bacteria with single polar flagella and blunted ends in colonic spirochetosis is well established, the identities of many of these organisms is still unknown. Recently, Helicobacter species with a morphology similar to the flagellated bacteria present in colonic spirochetosis have been cultured from intestinal specimens obtained from rhesus macaques, some with idiopathic colitis. The purpose of the present study was to determine whether or not the flagellated bacteria seen in the colons of rhesus macaques with colonic spirochetosis are Helicobacter. The presence of flagellated bacteria alone (n=2) or together with spirochetes (n=1) in formalin-fixed and paraffin-embedded colons of three rhesus macaques with the naturally occurring disease was demonstrated by immunohistochemical staining and ultrastructural examination. Total DNA extracted from affected and control intestinal specimens was amplified by polymerase chain reaction (PCR) using Helicobacter 16S rRNA gene-specific primers. Comparative nucleotide sequence analysis of PCR products cloned from positive reactions indicated that two distinct Helicobacter genomospecies were present either alone or in combination with Brachyspira in the colons of rhesus macaques with microscopic lesions indicative of colonic spirochetosis.
Journal Article↗
First confirmation of porcine colonic spirochaetosis caused by Brachyspira pilosicoli in Iberian pigs in Spain.
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Animals↗
Legionella pneumophila entry gene rtxA is involved in virulence.
Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1, enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to beta(2) integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry gene rtxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.
Animals↗
Comparative pathology and pathogenesis of naturally acquired and experimentally induced colonic spirochetosis.
Research in the past decade has led to the recognition of Brachyspira (formerly Serpulina) pilosicoli as the primary etiologic agent of colonic spirochetosis (CS), an emerging cause of colitis in humans and animals. Attachment of spirochetes to the epithelial surface of the lower intestine is considered to be the hallmark of CS. However, because B. pilosicoli, B. aalborgi and unclassified flagellated bacteria are found singly or together in humans and non-human primates with CS lesions, attachment of spiral-shaped bacteria may not represent the same etiopathogenetic entity in all hosts. Moreover, North American opossums with CS are infected with B. aalborgi-like spirochetes together with flagellated bacteria, whereas B. pilosicoli is found alone in dogs, pigs, chickens and other species of birds with CS. Conversely, guinea-pigs with CS have unidentified spirochetes that may be B. pilosicoli or B. aalborgi. The pig model of CS suggests that attachment of B. pilosicoli to epithelial cells may be transient. By contrast, persistence of B. pilosicoli in the cecal and colonic crypt lumina, chronic inflammation caused by spirochetal invasion into the subepithelial lamina propria and translocation to extraintestinal sites may be more important than previously thought. This review describes the lesions seen in naturally occurring and experimentally induced CS of animals, and it sets the stage for future research into the pathogenic mechanisms of infection and colitis caused by B. pilosicoli.
Animals↗
Minimal prophylactic concentration of dietary zinc compounds in a mouse model of swine dysentery.
Dietary supplementation with 6000 mg of Zn2+/kg of feed has been shown to modify the clinicopathologic expression of Brachyspira hyodysenteriae infection in a laboratory mouse model of swine dysentery. However, this concentration impaired the body weight gain of the mice. The purpose of the present study was to determine a minimal prophylactic concentration of feed-grade zinc compounds that would not affect the growth of mice challenge-exposed with B. hyodysenteriae. A total of 440, 6- to 8-week-old, C3H/HeN mice were allocated randomly to groups and fed either a defined diet or a defined diet containing either 1000, 2000 or 4000 mg/kg ZnO, ZnSO4 or zinc-methionine for 7 days before intragastric inoculation with B. hyodysenteriae. From days 7 to 35 after inoculation, mice in each group were necropsied at weekly intervals for determination of body weight, presence of B. hyodysenteriae in the cecum, and histological assessment of cecal lesions. Only ZnO fed at 2000 mg/kg had a prophylactic effect against B. hyodysenteriae infection without affecting the body weight gain of the mice. The prophylactic effect of Zn2+ against infection with B. hyodysenteriae was also affected by the relative concentration of Fe2+ and Zn2+/Fe2+ ratio of the diet.
Animal Feed↗
Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA.
The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.
Animals↗
Prevalence of Brachyspira species isolated from diarrhoeic pigs in Brazil.
Pathogenic intestinal spirochaetes of pigs include Brachyspira (formerly Serpulina) hyodysenteriae, the cause of swine dysentery, and Brachyspira pilosicoli, the cause of porcine colonic spirochetosis (PCS). The purpose of this study was to assess the relative importance of Brachyspira species in diarrhoeal disease of growing pigs on farms in southern Brazil. The intensity and pattern of haemolysis, the production of indole and the hydrolysis of hippurate by reference and field porcine intestinal spirochaetes were compared with 16S-ribosomal RNA (mRNA)- and 23S-rRNA-based polymerase chain reaction assays for the identification of B hyodysenteriae and B pilosicoli. Between July and October 1998, 206 rectal swabs were taken from pigs on 17 farms with a history of diarrhoea developing within 30 days after they had been moved from nursery to growing facilities. Of 49 beta-haemolytic spirochaetes that were cultured, 29 (59.2 per cent) were grown in pure culture for phenotypic and genotypic characterisation, leaving 20 untyped. Of the 29 typed isolates, eight isolates obtained from six farms were identified as B hyodysenteriae, and 15 isolates obtained from seven other farms were identified as B pilosicoli; the remaining six isolates were identified as weakly beta-haemolytic commensal spirochaetes. There was complete agreement between the results of the phenotypic and genotypic analyses.
Animals↗
Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme-linked oligosorbent assay (PCR-ELOSA).
Proliferative enteropathy (PE) caused by Lawsonia intracellularis is a major diarrheal disease affecting swine worldwide. Routine laboratory diagnosis of PE is done by amplification of L. intracellularis -specific DNA sequences by PCR followed by agarose gel electrophoresis and staining of PCR products with ethidium bromide. We report the development of an enzyme-linked oligosorbent assay (ELOSA) for specific identification of chromosomal L. intracellularis 328-bp PCR amplified products. The ELOSA involved determination of optical density value at 450 nm (OD(450)) after hybridization of biotin-labelled PCR products with an amine-modified internal oligonucleotide capture probe immobilized in microwell plates, and avidin-biotin-peroxidase complex. A positive ELOSA cut-off value of > or =0.375 was established using the mean OD(450)of negative control specimens plus three times the standard deviation. Using this value, the detection limit of PCR amplified L. intracellularis -specific products by ethidium bromide-stained agarose gel electrophoresis, Southern blot, and ELOSA were estimated to be 6.1 ng, between 0.8 and 3.0 ng, and 0.8 ng of DNA, respectively. Comparison of ethidium bromide-stained agarose gel analysis with ELOSA for detection of L. intracellularis -specific PCR products from 315 clinical specimens revealed 78% sensitivity, 100% specificity and 94% accuracy. The ELOSA produced a spectrophotometric signal that confirmed the authenticity of PCR products without subjective interpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis.
Animals↗
Cloning and DNA sequence analysis of an immunogenic glucose-galactose MglB lipoprotein homologue from Brachyspira pilosicoli, the agent of colonic spirochetosis.
Colonic spirochetosis (CS) is a newly emerging infectious disease of humans and animals caused by the pathogenic spirochete Brachyspira (formerly Serpulina) pilosicoli. The purpose of this study was to characterize an antigen that was recognized by antibodies present in sera of challenge-exposed pigs. The gene encoding the antigen was identified by screening a plasmid library of human B. pilosicoli strain SP16 (ATCC 49776) genomic DNA with hyperimmune and convalescent swine sera. The predicted amino acid sequence encoded by the cloned B. pilosicoli gene had a high degree of similarity and identity to glucose-galactose MglB lipoprotein. Located 106 bp downstream of the putative mglB gene was a 3'-truncated open reading frame with 73.8% similarity and 66.3% identity to mglA of Escherichia coli, suggesting a gene arrangement within an operon which is similar to those of other bacteria. A single copy of the gene was present in B. pilosicoli, and homologous sequences were widely conserved among porcine intestinal spirochetes Serpulina intermedia, Brachyspira innocens, Brachyspira murdochii, and the avian Brachyspira alvinipulli, but not in porcine Brachyspira hyodysenteriae, human Brachyspira aalborgi, and porcine Treponema succinifaciens. The deduced molecular weight of the mature MglB lipoprotein was consistent with expression by the cloned gene of a polypeptide with an apparent molecular weight of 36,000, as determined by Western blot analysis and [(3)H]palmitate labeling. Because mucin is the principal constituent of the colonic mucus gel and consists of glycoproteins that can serve as the substrate for growth and chemotaxis of B. pilosicoli in vitro, a role for MglB in mucosal localization of the spirochete appears consistent with the pathogenesis of CS. However, the presence of homologous sequences in closely related but nonpathogenic commensal spirochetes suggests that other virulence determinants may be required for pathogenesis.
Amino Acid Sequence↗
Recovery from colonic infection elicits serum IgG antibodies to specific Serpulina pilosicoli outer membrane antigens (SPOMA).
Colonic spirochetosis caused by S. pilosicoli is a disease of human and animals characterized by intimate attachment of the spirochete to colonic epithelial cells and colitis. To identify antigens that are potentially involved in recovery from the disease, whole-cell lysate (WC) and various detergent extracts including Sarkosyl-soluble (SS) and insoluble (SI), and Triton X-114 detergent phase (TXD) and aqueous phase (TXA) of the human isolate SP16 were examined by Western blotting with Serpulina spp. periplasmic flagellar protein FlaB-specific monoclonal antibody 7G2 as well as pooled pre-immune serum (PS), hyperimmune serum (HS), and convalescent serum (CS) from swine. The HS reacted with several antigens that were not identified by the CS, including the periplasmic flagellar proteins and some lower molecular weight bands. The CS identified three major immunoreactive double (D) or single (S) bands of approximately: (i) 64-kDa in the WC(S), SS(D), and TXD/A(S), (ii) 54-kDa in the WC(S), SS/I(S), and TXD(S), and (iii) 47-kDa in the SS(S) fraction. The data indicate recovery from colonic infection elicits serum IgG antibodies to specific S. pilosicoli outer membrane antigens (SPOMA).
Animals↗
Motility-regulated mucin association of Serpulina pilosicoli, the agent of colonic spirochetosis of humans and animals.
Colonic spirochetosis is a disease of humans and animals characterized by colonization of the colonic mucus gel and intimate attachment of Serpulina pilosicoli to the apical membrane of enterocytes. Motility-regulated mucin association plays a key role in colonic infection by the related spirochete Serpulina hyodysenteriae, the cause of swine dysentery. In this study the chemotaxis of Serpulina pilosicoli porcine isolate P43/6/78, human isolate SP16, and canine isolate 16242-94 was examined by anaerobic incubation of each spirochete in control medium or medium containing increasing concentrations of D-L serine or porcine gastric mucin (PGM). The porcine isolate had a chemotactic response towards 10 mM D-L serine, but not towards PGM. By contrast, the human and canine isolates were attracted towards 0.1% PGM, but not towards DL-serine. The composition of the growth medium appeared to modulate the chemotactic response of S. pilosicoli towards PGM; the loss of a chemotactic response of spirochetes grown in medium without pig fecal extract was restored by growing the spirochetes in medium containing 0.1% PGM. Serpulina pilosicoli displays a chemotactic response towards PGM which is modulated by the presence of certain substrate during the growth phase of the spirochete.
Animals↗
Coiling phagocytosis is the predominant mechanism for uptake of the colonic spirochetosis bacterium Serpulina pilosicoli by human monocytes.
Serpulina pilosicoli is a newly identified pathogenic spirochete that establishes persistent colonic infections in human beings and animals. Macrophages are one of the key defenses against invasion of mucosal surfaces by bacterial pathogens. Macrophages engulf many bacteria by conventional phagocytosis; however recent studies indicate coiling phagocytosis as a new and important mechanism for internalization of Legionella pneumophila and spirochetes of the genus Borrelia, Leptospira, and Treponema. In this study, THP-1 human monocytic cells were incubated with the human S. pilosicoli strain SP16 and the contribution of coiling and conventional phagocytosis to the total number of phagocytic events were determined by sequential ultrastructural examination between 5 and 45 minutes. The frequency of phagocytosis increased over time from 5.1% after 5 minutes up to 21.9% after 45 minutes with greater than 70% of the events involving coiling phagocytosis. The data indicate that coiling phagocytosis may be a universal mechanism for uptake of pathogenic spirochetes.
Brachyspira↗
Comparative pathology of bacterial enteric diseases of swine.
Enteric bacterial infections are among the most common and economically significant diseases affecting swine production worldwide. Clinical signs of these infections include diarrhea, reduced growth rate, weight loss, and death of preweaned, weanling, grower-finisher, young and adult age breeding animals. The most common etiological agents include Escherichia coli, Clostridium perfringens, Lawsonia intracellularis, Salmonella enterica, and Brachyspira (Serpulina) spp. With the exception of Brachyspira (Serpulina) hyodysenteriae, the cause of swine dysentery, and Lawsonia intracellularis, the cause of proliferative enteropathy, the pathological changes seen with these agents closely resemble the diseases occurring in human beings. Histological changes in the intestines of swine with enteric bacterial infections include bacterial colonization without significant damage (e.g., certain enterotoxigenic E. coli and C. perfringens type A), attaching and effacing lesions with enteropathogenic E. coli and Brachyspira pilosicoli, the cause of colonic spirochetosis, inflammation with S. enterica, and necrotizing and hemorrhagic lesions with certain C. perfringens. Extraintestinal spread of bacteria and/or toxins occurs with some serotypes of E. coli and most serotypes of S. enterica. Enteric bacterial diseases of swine have been used as models to study the pathogenesis of similar diseases of human beings. Several of these pathogens are also important causes of food-borne disease in humans.
Animals↗
Fatal outbreaks of proliferative enteritis caused by Lawsonia intracellularis in young colony-raised rhesus macaques.
Ten juvenile rhesus monkeys (Macaca mulatta) died acutely in two separate disease outbreaks. The animals had segmental thickening of the distal ileum with associated proliferative, rugous appearing mucosae. Microscopically, necrosis, exudative inflammation, mucosal ulceration, and crypt hyperplasia were present. Intracellular organisms were seen histochemically and ultrastructurally, and were confirmed to be Lawsonia intracellularis using a specific immunohistochemical method. Proliferative enteropathic conditions caused by L. intracellularis are reported in an ever-increasing range of hosts, suggesting that the infection may exist unrecognized in an even greater array of species, possibly including man.
Animals↗