Transformation-mediated serogroup conversion of Dichelobacter nodosus

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The type IV fimbriae of D. nodosus are required for virulence, are highly immunogenic and immunoprotective, and can be divided into 10 major serogroups. Fimbrial variation has been postulated to have arisen because of genetic recombination within the fimbrial gene region perhaps as a means of evading the immune response invoked by infection. To show that antigenic variation in these fimbriae could occur after natural transformation and subsequent homologous recombination, a suicide plasmid containing the fimbrial subunit gene, fimA, of a serogroup G strain was used to convert a serogroup I strain to serogroup G. The resultant mutants were shown by Western blotting and slide agglutination to produce serogroup G fimbriae, but by two independent methods to still have the genotype of the parent type I strain. These data have significant implications for the use of fimbrial vaccines for the control of ovine footrot and suggest that benign strains of D. nodosus could play an important role as a reservoir of alternative fimbrial antigens.     

Dichelobacter nodosus serotype M fimbrial subunit gene: implications for serological classification

Dichelobacter nodosus fimbrial subunit gene (fimA) from a serotype M strain (M-SPAHL) was investigated in this study. A primer set targeting the relatively conserved fimA regions and based on the published sequence from Nepalese serogroup M isolates (Nepalese M), failed to amplify the fimA of M-SPAHL. However, when the downstream primer was substituted with a primer that is specific for other serogroups of D. nodosus, the fimA was successfully amplified. Cloning followed by DNA sequencing, revealed that the M-SPAHL fimA was different to the Nepalese M fimA. The predicted amino acid sequence of the M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not Nepalese M. The consequences of serological relatedness and sequence dissimilarity are discussed.     

Update on ovine footrot in New Zealand: isolation, identification, and characterization of Dichelobacter nodosus strains [corrected

Dichelobacter nodosus, a Gram-negative strict anaerobe, is the essential causative agent of ovine footrot. Despite its worldwide presence, the disease has significant economic impact in those sheep-farming countries with a temperate climate and moderate to high rainfall, such as New Zealand (NZ) and Australia. In this study, we aimed to isolate, identify, and characterize as many D. nodosus strains as possible from NZ farms by using polymerase chain reaction (PCR)-based technology. Understanding the virulence of this bacterium and showing extensive genomic variation in the fimbrial subunit gene (fimA) in different D. nodosus strains was very important to produce serogroup specific and effective vaccine for NZ. More than 100 footrot samples were collected from four different farming regions in NZ. Thousands of primary plates were cultured anaerobically and examined with Gram-staining in order to detect single colonies of D. nodosus. Approximately 500 plates that had potential D. nodosus colonies were subcultured several times to eliminate contaminating colonies until single colonies were obtained. Variable and a part of the conserved regions of the fimbrial subunit gene (fimA) were amplified directly from bacterial DNA extracted from footrot lesions and also from cultured NZ D. nodosus isolates, using the polymerase chain reaction. Different fimA amplimers were analyzed by DNA sequencing. On the basis of DNA sequence analysis, 16 new D. nodosus isolates belonging to eight different serogroups were identified from NZ. These new D. nodosus fimA sequences from NZ were different to previously reported strains and strains used in a commercial vaccine.     

Extensive diversity in New Zealand Dichelobacter nodosus strains from infected sheep and goats

Footrot is a contagious bacterial disease of ruminants spread by the Gram-negative, anaerobic organism, Dichelobacter nodosus. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. Using the polymerase chain reaction (PCR) to amplify fragments of the fimbrial gene (fimA), D. nodosus was detected in 14 hoof scrapings, sampled from six farming regions within New Zealand. DNA sequencing revealed 15 strains covering eight serogroups on the New Zealand farms. The predominant serogroup was B which contained six strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Eleven out of the 15 D. nodosus strains had fimbriae sequences different to those previously reported and the presence of multiple strains on a single hoof was common (86% samples). Individual sheep from the same farm, or the same paddock, were often infected by a different range of strains, which suggests a host role in mediating footrot infection.     

Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation

Here we describe an approach to genotyping D. nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping).The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP). The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing [Vet. Microbiol. 71 (2000) 113], was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus strains present in a footrot sample can be accurately identified in less than 2 days.     

The pathogenesis of ovine footrot

Ovine footrot is a contagious and debilitating disease that is of major economic significance to the sheep meat and wool industries. The causative bacterium is the gram negative anaerobe Dichelobacter nodosus. Research that has used a classical molecular genetics approach has led to major advances in our understanding of the role of the key virulence factors of D. nodosus in the disease process. D. nodosus strains produce polar type IV fimbriae and extracellular serine proteases. Mutagenesis of the fimbrial subunit gene fimA and the pilT gene, which is required for fimbrial retraction, and subsequent testing of these mutants in sheep virulence trials has shown that type IV fimbriae-mediated twitching motility is essential for virulence. The extracellular protease genes aprV2, aprV5 and bprV have also been mutated. Analysis of these mutants has shown that ArpV5 is the major extracellular protease and that AprV2 is the thermostable protease that is responsible for the extracellular elastase activity. Structural analysis of AprV2 has revealed that it contains several novel loops, one of which appears to act as an exosite that may modulate substrate accessibility. Finally, virulence experiments in sheep have shown that the AprV2 protease is required for virulence.     

Current understanding of the aetiology and laboratory diagnosis of footrot

Footrot is a highly contagious disease of the feet of ruminants caused by the synergistic action of certain bacterial species of which Dichelobacter nodosus (D. nodosus) is the main transmitting agent. The infection is specific to sheep and goats, although it has also been reported in cattle, horses, pigs, deer and mouflon. The antigenic diversity of D. nodosus is due to variations in the DNA sequence of its fimbrial subunit gene (fimA) and provides the basis for classification of the organism into at least 10 major serogroups (A-I and M), the distribution of which varies with different geographical locations. Host immune response to vaccination is serogroup specific. There are three different clinical forms of disease caused by virulent, intermediate and benign strains of D. nodosus, respectively. In order to facilitate rapid and reliable clinical diagnosis, virulence determination, strain differentiation and serogroup identification for effective control measures, immunological tests, DNA probes and PCR based techniques have been introduced. This review summarises the current understanding of the mechanisms of antigenic diversity of D. nodosus as well as advances made in its strain differentiation and diagnosis.     

Isolation and characterization of Dichelobacter nodosus from ovine and caprine footrot in Kashmir, India

Footrot is a highly contagious and economically important disease of sheep and goats, caused by Dichelobacter nodosus, a slow growing anaerobic Gram-negative rod. The current Australian antigenic classification system, based on variation in the fimbriae, classifies D. nodosus into at least 10 serogroups (A-I and M) and 18 serotypes. This investigation was intended to determine the serological diversity of D. nodosus in this region of Kashmir, India. Exudates of footrot lesions were collected from 24 naturally infected sheep and 42 goats located in the Kashmir valley. Of these 66 samples, 24 yielded evidence of D. nodosus by PCR using 16SrDNA specific primers. Multiplex PCR using serogroup specific primers revealed the presence of serogroup B in all the samples except two, which showed the presence of serogroup E D. nodosus. This study also documents the isolation of D. nodosus and detection of serogroup E for the first time in India.     

Identification and genetic mapping of proteins encoded in the fimbrial subunit gene region of Bacteroides nodosus

The fimbriae produced by the anaerobic bacterium Bacteroides nodosus are important in the pathogenesis of ovine foot rot. Studies on other microorganisms have shown that the genes coding for the production and assembly of fimbriae are often clustered. By the use of maxicells, transposon mutagenesis and expression vectors, we have identified several genes which are located in the fimbrial subunit gene region. Antiserum was prepared against one of the proteins (88 kDa) which we were able to overproduce in Escherichia coli. In Western blots, these antibodies reacted with an 88 kDa protein located in the B. nodosus cell membrane. However, they did not react with the putative basal protein which is found in fimbrial preparations. We concluded that in B. nodosus the genes involved in fimbrial assembly are not all localised to one small region of the genome. In addition, our studies showed that although the fimbrial subunits are not assembled into intact fimbriae, an N-terminal sequence is processed in E. coli.     

Novel fimbrial subunit genes of Dichelobacter nodosus: recombination in vivo or in vitro?

Polymerase chain reaction (PCR) was used to amplify the variable region of the fimbrial subunit encoding gene (fimA) of Dichelobacter nodosus from sheep and goats infected with footrot. Two amplimers (designated X and Y) generated single-strand conformation polymorphism (SSCP) patterns different to those of previously identified serogroups and serotypes. DNA sequencing revealed that these two fragments were novel. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1, whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype F1 and the upstream primer specific for serotype M or E1, generated PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artifacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It, therefore, seems more likely that fragments X and Y reflect genuine fimA genes of D. nodosus which have resulted from in vivo DNA recombination rather than from a PCR recombination artifact.     

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.     

Studies on the ultrastructural morphology of Bacteroides nodosus.

The morphology of Bacteroides nodosus was examined with the electron microscope. B nodosus stained with solium phosphotungstate and uranyl acetate possessed fimbriae and in addition organisms negatively stained with sodium phosphotungstate often possessed rings on their surface. Phage-like particles were also observed in negatively stained preparations. In thin sections, B nodosus had a multilayered cell envelope and the type of cell division characteristic of Gram-negative bacteria. The cytoplasmic region contained a diffuse nucleoid area, ribosomes and, sometimes, concentrically arranged membranous lamellae. Fimbriae and capsular material were also seen in sections of B nodosus fixed with glutaraldehyde-osmium. Their visualisation appeared to be enhanced when ruthenium red was incorporated n the glutaraldehyde-osmium fixative but only when sections were stained with heavy metal salts, indicating that the fimbriae and capsule were not predominantly polysaccharide in nature.     

腐蹄病节瘤拟杆菌的分子致病机制的研究进展

腐蹄病是反刍动物绵羊、山羊、鹿和奶牛等常见的一种高度接触性传染病。节瘤拟杆菌是致病作用的主要病菌之一,它是通过IV型纤毛和细胞外蛋白酶而产生作用。但从腐蹄病发病症状看,节瘤拟杆菌所致疾病的严重程度不是相同的,据此节瘤拟杆菌被分为毒性、弱毒性和良性菌,这种分类通过对该菌基因组毒性关联蛋白Vap(Virulence-associated protein)区域和毒性相关位点vrl(Virulence Related Locus)区域的研究,发现其致病特点与基因的顺序有很大联系。     

The detection and characterisation of Dichelobacter nodosus from cases of ovine footrot in England and Wales

Footrot, caused by the strictly anaerobic bacterium Dichelobacter nodosus, is the most common cause of lameness in sheep in Great Britain but problems exist in association with its diagnosis and control. The fastidious nature of D. nodosus means that complex media and several weeks are required for characterisation. An alternative method to simplify and enhance the detection of D. nodosus in clinical samples is therefore highly desirable. In terms of control, anecdotal evidence from the farming community suggests that the commercially available vaccine, based on Australian isolates of D. nodosus, is not widely employed in this country due to its perceived inefficacy. Seven hundred and six isolates, collected from outbreaks in England and Wales, were therefore used to investigate these issues. A 16S rRNA PCR was adapted to detect D. nodosus in clinical material within 1 day of sampling; a 15% increase in detection compared with culture and less than 1% false negatives were achieved. This represents a major advance in the rapid diagnosis of footrot and will be of great value to practitioners and diagnostic laboratories. Bacterial virulence was tested using protease thermostability and zymogram assays, whilst serogrouping was performed by slide agglutination. All isolates demonstrated virulence patterns previously recorded in Australia and all nine serogroups of D. nodosus (A-I) were represented. Serogroup H was predominant. There was, therefore, no evidence for the presence of novel strains of D. nodosus compared with Australia suggesting the need for further investigation into farmers' views on the use of the commercial vaccine in Great Britain.     

Impact of a mutator phenotype on motility and cell adherence in Salmonella Heidelberg

In this study, we investigated adherence and motility of the hypermutator Salmonella enterica Heidelberg B182 bovine strain related to a 12bp deletion in mutS. This mutator phenotype was associated with increased adherence to epithelial cells and with high expression of fimA as shown by real-time RT-PCR. Motility studies showed that fliC were up-regulated in the B182 strain, while fljA and fljB were down-regulated. In order to determine if mutated mutS is implicated in this genes expression, isogenic strains, derived from a WT strain, containing the 12bp deletion in mutS (Δ12bpmutS) or an inactivated mutS (ΔmutS) were generated. Δ12bpmutS and ΔmutS strains showed a spontaneous mutation rate similar to the environmental strain B182, but exhibited lower adherence capacity and fimA expression. In contrast to the fimbriae genes, in Δ12bpmutS, fliC expression was up-regulated, but fljA and fljB expression were decreased, as in the B182 strain. Only fljB expression was increased in ΔmutS mutants. Taken together, our data suggest that mutS alteration does not influence fimbriae expression but can impact flagella genes.     

奶牛源化脓隐秘杆菌的分离鉴定及其毒力基因的检测

本研究旨在分离鉴定来自北京某奶牛场死亡奶牛肺脏的1株疑似病原菌CVCC 3982,并测定其致病性。通过分离培养获得疑似病原菌,采用Biolog鉴定系统和16S rDNA序列分析对其进行了鉴定,人工接种CD-1小鼠测定了其致病性,合成引物对其主要毒力基因进行了检测。结果显示,该疑似病原菌为革兰氏阳性杆菌,β溶血,Biolog鉴定结果显示其为化脓隐秘杆菌,其16S rDNA序列与化脓隐秘杆菌模式菌株NCTC 5224的同源性达100%,系统发育分析显示其与化脓隐秘杆菌处于同一分支。腹腔注射该菌可致小鼠死亡。分离菌株基因组中含有溶血素(PLO)基因,神经氨酸酶H(NanH)基因,神经氨酸酶P(NanP)基因,菌毛基因(fimA、fimC和fimE),但缺失胶原结合蛋白(CbpA)基因和菌毛fimG基因。结果表明该分离菌株为化脓隐秘杆菌且具有致病性。     

Association between virulence factors of Escherichia coli, Fusobacterium necrophorum, and Arcanobacterium pyogenes and uterine diseases of dairy cows

The objective of this study was to evaluate the relationship between bacterial species-specific virulence factors (VFs) present in the uterus at 3 different stages of lactation (1-3, 8-10, and 34-36 days in milk (DIM)) and the incidence of metritis and clinical endometritis in dairy cows. The following VF genes were investigated: plo (pyolysin), cbpA (collagen-binding protein), and fimA (fimbriae expression) which are Arcanobacterium pyogenes specific; fimH (a type 1 pilus component), Escherichia coli specific; and lktA (leukotoxin), Fusobacterium necrophorum specific. Uterine swabs were collected from 111 postpartum dairy cows. PCR was used to detect the presence of plo, cbpA, fimA, fimH, and lktA genes. A. pyogenes cbpA was detected in only 5 samples and therefore was not subjected to further analysis. E. coli (fimH) was significantly associated with metritis and endometritis when detected at 1-3 DIM; F. necrophorum (lktA) was significantly associated with metritis when detected at 1-3 and 8-12 DIM and with endometritis when detected at 34-36 DIM; and A. pyogenes (fimA and plo) was associated with metritis (fimA) when detected at 1-3 DIM and endometritis (fimA and plo) when detected at 8-10 and 34-36 DIM.     

Occurrence of different strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections

OBJECTIVE: To investigate the occurrence of S1, U1 and T strains of Dichelobacter nodosus in new clinical lesions in sheep exposed to footrot associated with multi-strain infections. DESIGN: Seventy-seven donor sheep were grazed with 84 recipients for 33 weeks. The donor sheep were Merinos with a history of clinically virulent footrot associated with protease type S1, U1 and T strains of D nodosus that hybridised with gene sequences pJIR314B, pJIR318 and/or pB645-335. The recipient sheep were Merinos with no history of footrot. PROCEDURE: Each fortnight, all feet were examined, their lesion scores were recorded and samples of lesion material were taken for laboratory tests. RESULTS: Eighty-nine percent (299 of 336) of feet of recipient sheep developed new clinical lesions. S1, U1 and T strains of D nodosus were recovered from 58%, 22% and 18%, respectively, of these lesions at a ratio that remained constant during two apparent peaks in footrot transmission. Gene sequences homologous to pJIR314B and pB645-335 were detected in 56% (93 of 166) and 29% (48 of 166), respectively, of S1 strains of D nodosus at a ratio that was not constant during the experiment. CONCLUSIONS: S1 was the dominant protease type of D nodosus in new clinical lesions. The occurrence of S1 strains did not increase relative to U1 and T strains of D nodosus during the experiment. S1, U1 and T strains of D nodosus remained in equilibrium despite changes in environment, genetic types in the population of S1 strains, and host resistance to footrot.