Identification of genes associated with prophage-like gene transfer agents in the pathogenic intestinal spirochaetes Brachyspira hyodysenteriae, Brachyspira pilosicoli and Brachyspira intermedia

VSH-1 is an unusual prophage-like gene transfer agent (GTA) that has been described in the intestinal spirochaete Brachyspira hyodysenteriae. The GTA does not self-propagate, but it assembles into a virus-like particle and transfers random 7.5kb fragments of host DNA to other B. hyodysenteriae cells. To date the GTA VSH-1 has only been analysed in B. hyodysenteriae strain B204, in which 11 late function genes encoding prophage capsid, tail and lysis elements have been described. The aim of the current study was to look for these 11 genes in the near-complete genomes of B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. intermedia HB60. All 11 genes were found in the three new strains. The GTA genes in WA1 and 95/1000 were contiguous, whilst some of those in HB60 were not-although in all three strains some gene rearrangements were present. A new predicted open reading frame with potential functional importance was found in a consistent position associated with all four GTAs, located between the genes for head protein Hvp24 and tail protein Hvp53, overlapping with the hvp24 sequence. Differences in the nucleotide and predicted amino acid sequences of the GTA genes in the spirochaete strains were consistent with the overall genetic distances between the strains. Hence the GTAs in the two B. hyodysenteriae strains were considered to be strain specific variants, and were designated GTA/Bh-B204 and GTA/Bh-WA1 respectively. The GTAs in the strains of B. intermedia and B. pilosicoli were designated GTA/Bint-HB60 and GTA/Bp-95/1000 respectively. Further work is required to determine the extent to which these GTAs can transfer host genes between different Brachyspira species and strains.     

The major surface Vsp proteins of Brachyspira hyodysenteriae form antigenic protein complexes

The Vsp proteins are the major outer membrane proteins of Brachyspira hyodysenteriae, the causative agent of swine dysentery. Eight vsp genes have been identified in B. hyodysenteriae strain B204, arranged into two four-gene loci, and at least two of the corresponding proteins are produced in vitro. The aims of this study were to characterise the vsp genes of the virulent Australian B. hyodysenteriae strain X576 and their corresponding proteins, Genomic sequence comparison with strains B204 and WA1 demonstrated that the number of vsp genes varies between B. hyodysenteriae strains, although the chromosomal locations of the vsp gene loci are consistent. We identified two additional vsp-like genes, designated vspI and vspJ, in each of the three strains. Double SDS-PAGE was used to demonstrate that Vsp proteins of B. hyodysenteriae strain X576 form multimeric protein complexes in the outer membrane that are stable in 6M urea but dissociate after boiling. The Vsp complexes primarily consisted of VspF but also contain VspE and VspI. VspD was also found in a series of complexes slightly larger than the more abundant VspF complexes. Vsp proteins are purported to be antigenic; however little direct data are available to support this claim. In this study convalescent pig sera did not bind denatured Vsp proteins by Western blotting, but did bind the Vsp complexes on Western blots, showing that conformational epitopes may be important in immune recognition of these major outer membrane proteins. This is the first definitive demonstration of the antigenicity of these proteins in swine dysentery.     

Swine dysentery: protection of pigs by oral and parenteral immunisation with attenuated Treponema hyodysenteriae.

An attenuated strain of Treponema hyodysenteriae was used to immunise 18 pigs in three experiments. Live attenuated spirochaetes were dosed orally and injected intra-peritoneally, and killed spirochaetes were injected intramuscularly with adjuvant. The vaccinated pigs, which developed high serum agglutination titres against T hyodysenteriae, and 18 unvaccinated litter-mates were repeatedly challenged with virulent T hyodysenteriae. Nine vaccinated pigs and 16 control pigs developed typical swine dysentery.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.     

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.     

A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection

Glycoproteins B (gB), gC and gD of pseudorabies virus (PRV) have been implicated as important antigens in protective immunity against PRV infection. As cell-mediated immunity plays a major role in this protective immunity, we determined the significance of these glycoproteins in the actual induction of cell-mediated immunity. We vaccinated pigs with plasmid DNA constructs coding for gB, gC or gD and challenged them with the virulent NIA-3 strain of pseudorabies virus. Vaccination with plasmid DNA coding for gB induced the strongest cell-mediated immune responses including cytotoxic T cell responses, whereas plasmid DNA coding for gD induced the strongest virus neutralising antibody responses. Interestingly, vaccination with gB-DNA reduced virus excretion early after challenge infection while vaccination with gC-DNA or gD-DNA did not.This is the first study to demonstrate that DNA vaccination induces cytotoxic T cell responses in pigs and that cell-mediated immunity induced by vaccination with gB-DNA is important for the reduction of virus excretion early after challenge infection.     

Swine dysentery: protection against experimental challenge following single dose parenteral immunisation with inactivated Treponema hyodysenteriae

Groups of five pigs were vaccinated at three to four weeks old with either formolised Treponema hyodysenteriae in oil adjuvant alone, formolised T hyodysenteriae in oil adjuvant plus formolised Campylobacter coli in oil adjuvant, or sterile medium in oil adjuvant (as a control). Each group was challenged four weeks after vaccination by oral dosing on two consecutive occasions with pure cultures of the homologous strain of T hyodysenteriae plus direct contact with two pigs exhibiting severe swine dysentery. The disease was observed in two of five pigs immunised with T hyodysenteriae alone, three of five pigs immunised with T hyodysenteriae plus C coli and all five controls; haemorrhagic diarrhoea was exhibited only by the control group. Each pig immunised with T hyodysenteriae (alone or with C coli) recovered spontaneously, whereas four controls died. Vaccination with T hyodysenteriae also markedly reduced the severity and duration of clinical signs and of weight loss. No differences were observed in response to challenge between pigs immunised with T hyodysenteriae alone and pigs immunised with T hyodysenteriae plus C coli.     

Detection of Brachyspira hyodysenteriae, Lawsonia intracellularis and Brachyspira pilosicoli in feral pigs

Feral pigs are recognized as being a potential reservoir of pathogenic microorganisms that can infect domestic pigs and other species. The aim of this study was to investigate whether feral pigs in Western Australia were colonized by the pathogenic enteric bacteria Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli. A total of 222 feral pigs from three study-populations were sampled. DNA was extracted from faeces or colonic contents and subjected to a previously described multiplex PCR for the three pathogenic bacterial species. A subset of 61 samples was cultured for Brachyspira species. A total of 42 (18.9%) of the 222 samples were PCR positive for L. intracellularis, 18 (8.1%) for B. hyodysenteriae and 1 (0.45%) for B. pilosicoli. Four samples were positive for both L. intracellularis and B. hyodysenteriae. Samples positive for the latter two pathogens were found in pigs from all three study-sites. A strongly haemolytic B. hyodysenteriae isolate was recovered from one of the 61 cultured samples. Comparison of a 1250-base pair region of the 16S rRNA gene amplified from DNA extracted from the isolate and five of the B. hyodysenteriae PCR positive faecal samples helped confirm these as being from B. hyodysenteriae. This is the first time that B. hyodysenteriae has been detected in feral pigs. As these animals range over considerable distances, they present a potential source of B. hyodysenteriae for any domesticated pigs with which they may come into contact.     

3-Acetyl-4'-isovaleryl tylosin for prevention of swine dysentery

The 21 field isolates of Treponema hyodysenteriae which were tested were sensitive to 3-acetyl-4'-isovaleryl tylosin (AIV); the minimal inhibitory concentration was 0.25 to 16 micrograms/ml. 3-Acetyl-4'-isovaleryl tylosin administered prophylactically to pigs at concentrations of 5 to 100 mg/kg of feed and tylosin at 110 mg/kg of feed for 28 or 31 days prevented swine dysentery induced by tylosin-sensitive T hyodysenteriae strain SQ2; 15 nonmedicated, inoculated control pigs had bloody diarrhea, and 9 pigs died. In 2 additional trials, AIV administered prophylactically for 28 days at 55 or 110 mg/kg of feed prevented swine dysentery induced by tylosin-insensitive T hyodysenteriae strain B204. All of the inoculated principal pigs medicated with AIV at 55 or 110 mg/kg of feed or carbadox at 55 mg/kg of feed and the noninoculated sentinel pigs for each group had solid feces throughout the 56-day trial. In the nonmedicated, inoculated control groups, bloody diarrhea began at 4 to 5 days after inoculation was done, and 9 of 10 principal pigs and 6 of 9 sentinel pigs had dysentery; 2 pigs died. In the groups medicated with AIV at 27.5 or 5.5 mg/kg of feed, all 5 principal pigs and 3 or 4 sentinel pigs in each group had dysentery; 3 or 4 pigs in each group died. In the group medicated with tylosin at 110 mg/kg of feed, 7 of 10 principal pigs and all 9 sentinel pigs had dysentery; 1 pig died.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of an experimental model allowing discrimination between virulent and avirulent isolates of Serpulina (Treponema) hyodysenteriae.

Variation in virulence among different strains of Serpulina hyodysenteriae was studied by oral inoculation of specific pathogen free piglets and CD-1 mice. Piglets infected with serotype 2 reference strain B204 and an untypable field strain LHV-90-9-I had severe diarrhea tainted intermittently with mucus and fresh blood. The piglets inoculated with B169, B8044, B6933, and ACK300-8 reference strains representing serotypes 3, 5, 6, and 7 respectively developed moderate diarrhea. However, reference strains B234 and A-1 of serotypes 1 and 4, respectively, failed to cause any diarrhea. None of the S. hyodysenteriae strains caused diarrhea in mice. The results indicate a great variation in virulence among strains of different serotypes of S. hyodysenteriae. Mice were less susceptible to infection with S. hyodysenteriae.     

Evaluation of a live avirulent Escherichia coli vaccine for K88+, LT+ enterotoxigenic colibacillosis in weaned pigs.

Live, avirulent Escherichia coli vaccine strains were constructed and tested for efficacy in preventing colibacillosis in 4-week-old pigs. Either or both of 2 plasmids were inserted into avirulent E coli strain G58-1 (0101:NM). These plasmids were pPMC4, which encodes for LTb subunits of heat-labile enterotoxin, and pDHF1, which encodes for K88ac fimbriae. Litter- and weight-matched pigs were removed from sows when they were 10 days old and vaccinated orally with the constructed strains or with G58-1 (negative control vaccine) when they were 2 weeks old and 5 days later. All pigs were challenge-inoculated with virulent E coli strain 3030-2 (O157:K88, LT+, STb+) 2 weeks after the first vaccination. Only 1 pig vaccinated with G58-1/pPMC4/pDHF1 developed diarrhea and none died following challenge inoculation. Seventeen of 31 control pigs developed diarrhea and 11 died. Of 18 pigs vaccinated with G58-1/pDHF1 then challenge-inoculated with the virulent strain, 5 developed diarrhea and 2 died. Fifteen of 18 litter- and weight-matched controls developed diarrhea and 8 died. When compared with G58-1 (negative control), G58-1/pPMC4 afforded no protection to pigs challenge-inoculated with 3030-2.     

Parenteral immunization of pigs against infection with Treponema hyodysenteriae.

Six intravenous injections of formalin-inactivated Treponema hyodysenteriae were given to 8 specific-pathogen-free pigs at 6-day intervals. The 8 vaccinated and 8 control pigs were challenged intragastrically with pure cultures of T hyodysenteriae 7 and 8 days after the last intravenous injection. Clinical signs of swine dysentery were observed in all 8 control pigs, but was observed in only 1 of the immunized pigs. Three control pigs died. These findings suggest that parenteral immunization with T hyodysenteriae provided a marked degree of protection against subsequent intragastric challenge exposure with the homologous isolate of T hyodysenteriae.     

Evidence for plasmid DNA in Alcaligenes faecalis

A 10.6 megadalton plasmid was isolated from a virulent strain (NC-D) of Alcaligenes faecalis. Virulence and antibiotic sensitivity of this strain were compared with those characteristics of a mutant plasmid-free derivative, strain NC-D1. Strain NC-D1 was avirulent and lacked the streptomycin and sulfonamide resistances of the parent strain.     

副猪嗜血杆菌OMP5的克隆表达及其对豚鼠免疫保护作用

根据GenBank中登录的副猪嗜血杆菌外膜蛋白P5(outer membrane protein P5,OMP5)基因序列设计1对特异性引物,以江西分离株NC0807基因组DNA为模板,扩增出OMP5基因。将其克隆到pET-28a(+)中,构建重组表达质粒pET-28a-OMP5,质粒转化大肠杆菌BL21(DE3),通过SDS-PAGE和Western blotting分析重组蛋白的表达情况和反应原性。重组蛋白经镍柱亲和层析纯化后免疫豚鼠,测定其免疫原性和保护效率。结果表明,重组蛋白在大肠杆菌中获得了高效表达。表达的蛋白分子质量约为43 ku,能被副猪嗜血杆菌阳性血清识别。动物试验结果表明,重组蛋白免疫后能诱导产生高水平的OMP5特异性抗体,并可显著保护豚鼠抵抗副猪嗜血杆菌强毒菌株的攻击,提示OMP5是副猪嗜血杆菌的保护性抗原。     

03型伪结核杆菌毒力基因的研究

用质粒消除技术,从03型伪结核杆菌毒力株中筛选出同宗的质粒治愈株,后者在缺失了一个分子量约为66kb(43.3MD)的大质粒的同时,丧失了对动物的侵袭性,细菌毒力消失。证实03型伪结核杆菌的毒力基因是位于染色体外的环状质粒DNA上,该质粒还编码了细菌的两个表型,1.外膜蛋白的产生;2.依赖钙离子生长。本文提示:要判定从临床上分离的03型伪结核杆菌是否具有毒力,应以检测其是否具有66kb的毒力质粒为标准。     

Protection of gnotobiotic pigs against Salmonella enterica serotype Typhimurium by rough mutant of the same serotype is accompanied by the change of local and systemic cytokine response

We have demonstrated that severe systemic disease caused by virulent LT2 strain Salmonella enterica serotype Typhimurium in gnotobiotic piglets can be alleviated by oral inoculation with an avirulent rough (R) mutant of the same serotype 24 h before challenge with the virulent strain. Protected piglets had no signs of enteritis. The concentrations of TNF-alpha, IL-1beta, IL-8 and IL-10 were measured by ELISA in ileal washings and plasma of uninfected and infected pigs. The cytokines were not detected in plasma of germ-free piglets, and low concentrations of IL-1beta and IL-8 were found in their ileal washings. The pre-inoculation of the rough mutant induced an increase in IL-8 and decrease in IL-1beta and IL-10 in plasma. The virulent LT2 strain induced very high TNF-alpha concentrations in the ileum which were reduced in the pigs pre-inoculated with the R mutant.     

Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs

Three strains of Bordetella bronchiseptica were compared for their ability to assist colonisation of the nasal cavity of gnotobiotic pigs by toxigenic Pasteurella multocida. Toxigenic P multocida (counted in nasal washings) colonised the cavity in large numbers in pigs previously infected with a cytotoxic phase I strain of B bronchiseptica (B58), whereas it colonised only in small numbers in those previously infected with B65, a phenotypic phase III variant of B58. Toxigenic P multocida colonised pigs infected with a non-cytotoxic phase I strain of B bronchiseptica (PV6) in fewer numbers than were seen in pigs infected with the cytotoxic phase I strain but in greater numbers than in pigs infected with the phase III strain. The turbinates of pigs infected with the cytotoxic phase I strain of B bronchiseptica and toxigenic P multocida were most severely affected and those in pigs infected with the non-cytotoxic phase I strain and toxigenic P multocida were moderately reduced in size. The turbinates of pigs infected with the phase III strain and toxigenic P multocida were slightly reduced in size except for one piglet whose turbinates were severely affected. Pigs infected with the non-cytotoxic phase I strain of B bronchiseptica alone showed no signs of atrophy and their turbinates were used to calculate reductions (per cent) in those infected with P multocida. The reduction (per cent) in size of turbinates and total numbers of P multocida isolated from the nasal washings of each pig were linearly related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky’s disease virus

This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky’s disease virus) DNA vaccine. Aujeszky’s disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-γ. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-γ). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-γ had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky’s disease.     

Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins

Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin.     

Identification and distribution of putative virulent genes in strains of Streptococcus suis serotype 2

In order to identify gene sequences unique to the virulent strains, suppression subtractive hybridization (SSH) was conducted using virulent Streptococcus suis type 2 (SS2) strain HA9801 and avirulent S. suis type 2 strain T15. Thirty genomic regions were absent in T15, and the DNA sequences of these regions in HA9801 were determined. These DNA fragments, containing putative virulence genes, encoded 28 proteins that were homologous to proteins involved in various aspects of cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems and others of unknown function. According to the published SS2 genomic sequence of the Chinese strain 98HAH33, PCR primers for 14 significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis strains from different sources, serotypes, regions, groups and times. The results showed that these 14 DNA fragments were widely distributed in 37 detected SS2 strains, yet were absent among the avirulent strain T15. Moreover, these fragments could be detected in other serotypes of S. suis, but each serotype had a different distribution of the fragments.