Pathogenicity of Isospora suis in gnotobiotic and conventionalised piglets

Isospora suis is unequivocally a primary pathogen of swine. Inoculation of I suis in conventionalised and germ-free piglets caused a biphasic disease course with marked diarrhoea, villous atrophy and necrosis of the intestinal epithelium at four to six and eight to 10 days after inoculation. The presence of a normal bacterial flora markedly (P less than 0.05) influenced the survival rate of piglets but did not appear to affect the histopathological changes observed. Mild limited focal necrosis and bile stasis were present in the liver at eight to 10 days after inoculation. In this period there was also ectasia of lymph vessels in the intestinal lymph nodes.     

2株猪繁殖与呼吸综合征病毒毒株对仔猪致病性的比较

为了比较最新分离的猪繁殖与呼吸综合征病毒(PRRSV)变异分离株SY0608和传统毒株S1对仔猪的致病性,本研究选择9头30日龄商品仔猪,随机分为2组,分别接种2株病毒,即S1株感染组(n=5头),SY0608株感染组(n=4头),接种后隔离饲养观察2周。经临床症状观察、病理学、病原学和血常规学检查,结果:SY0608毒株感染组仔猪表现明显临床症状,接种3 d后体温急剧升高至41.8℃;白细胞数急剧减少(降低了45%);病理学变化严重,肺泡膈增宽,大部分肺组织肺泡不张;而S1株感染组仔猪仅出现轻微临床症状和病理变化。SY0608毒株感染组仔猪病毒血症持续时间较S1毒株感染组长,病毒在脏器中的分布更为广泛。SY0608株感染组血清ELISA抗体水平明显高于S1株感染组。结果表明,SY0608毒株对仔猪致病作用明显强于S1毒株。     

Pathogenicity of Vietnamese enterotoxigenic Escherichia coli strains in colostrum-deprived one-day-old piglets

Preweaning colibacillosis is a major cause of economic loss to the swine industry in Vietnam. The aim of this study was to examine the enteropathogenicity of representative enterotoxigenic Escherichia coli (ETEC) strains obtained during an earlier epidemiologic survey conducted in five provinces in North Vietnam. This included isolates belonging to serotype O8 that produced heat-stable and heat-labile enterotoxins but did not produce any of the recognized fimbriae (F4, F5, F6, F41, F18). In vitro hemagglutination (unique mannose-resistant hemagglutination activity with guinea pig, sheep, human, and chicken red blood cells at 37 degrees C, but not at 18 degrees C) and enterocyte brush border attachment assays suggested that the F- ETEC strains produced an unidentified colonization factor that promoted adherence to the intestinal epithelium. Colostrum-deprived 1-day-old piglets challenged with an F- strain (1-2 x 10(9) bacteria) developed acute watery diarrhea within 4 hours of inoculation and suffered up to 20% weight loss, with comparable severity to piglets challenged with conventional F4 and F5 strains. At necropsy, viable counts and histopathologic examination of intestinal sections demonstrated colonization of the duodenum, jejunum, and ileum by F4-positive strains. In comparison, the F- and F5-positive strains attached exclusively to the ileum. Transmission electron micrographs of negatively stained F- cells grown at 37 degrees C demonstrated the presence of fimbriae. These results confirm the presence of a potentially new pathogenic ETEC fimbrial type in piggeries in Vietnam, with a unique hemagglutination property and attachment characteristics similar to ETEC bearing F5 fimbriae.     

Acquisition of haemoglobin-bound iron by strains of the Actinobacillus minor/"porcitonsillarum" complex

Members of the Actinobacillus minor/"porcitonsillarum" complex are common inhabitants of the swine respiratory tract. Although avirulent or of low virulence for pigs, these organisms, like pathogens, do grow in vivo and must, therefore, be able to acquire iron within the host. Here, we investigated the abilities of six members of the A. minor/"porcitonsillarum" complex to acquire iron from transferrin and various haemoglobins. Using growth assays, all six strains were shown to acquire iron from porcine, bovine and human haemoglobins but not from porcine transferrin. Analyses of whole genome sequences revealed that A. minor strains NM305(T) and 202, unlike the swine-pathogenic actinobacilli, A. pleuropneumoniae and A. suis, lack not only the transferrin-binding protein genes, tbpA and tbpB, but also the haemoglobin-binding protein gene, hgbA. Strains NM305(T) and 202, however, were found to possess other putative haemin/haemoglobin-binding protein genes that were predicted to encode mature proteins of ～ 72 and ～ 75 kDa, respectively. An affinity procedure based on haemin-agarose allowed the isolation of ～ 65 and ～ 67 kDa iron-repressible outer membrane polypeptides from membranes derived from strains NM305(T) and 202, respectively, and mass spectrometry revealed that these polypeptides were the products of the putative haemin/haemoglobin-binding protein genes. PCR approaches allowed the amplification and sequencing of homologues of both haemin/haemoglobin-binding protein genes from each of the other four strains, strains 33PN and 7ATS of the A. minor/"porcitonsillarum" complex and "A. porcitonsillarum" strains 9953L55 and 0347, suggesting that such proteins are involved in the utilization of haemoglobin-bound iron, presumably as surface receptors, by all six strains investigated.     

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.     

Dynamics of Campylobacter jejuni invasion in gnotobiotic piglets

The study deals with the results of the investigation of C. jejuni populations in the intestines of gnotobiotic piglets after oral infection with C. jejuni in the span of one to five days after infection (DAI). The infection of the whole large intestine was revealed, with the onset of pathological lesions 3rd DAI. C. jejuni was isolated from liver, gallbladder, ileum, rectum, colon, mesenterial lymphatic node, rectal smears and blood. From 1st to 5th DAI C. jejuni was demonstrated in liver, gallbladder and posterior part of ileum. In the cecum and rectum C. jejuni was detected as late as on 2nd-3rd DAI to 5th DAI and in mesenterial lymphatic nodes from 2nd to 4th DAI. In rectal smears C. jejuni was found out regularly during the whole period of experiment. C. jejuni was also isolated from inflammatory infiltrate. Histological examination revealed C. jejuni between the villi and in the contents of cup-shaped cells from 2nd to 5th DAI.     

Pathogenesis of intestinal cryptosporidiosis in conventional and gnotobiotic piglets.

The pathogenesis of intestinal cryptosporidiosis was studied in 52 conventionally reared and 20 gnotobiotically reared piglets by inoculation with different doses of Cryptosporidium parvum oocysts. The prepatent period of C. parvum in both groups of animals were variable, depending on the number of oocysts administered. The patent period of C. parvum in conventionally reared piglets was 8 or 9 days; in gnotobiotic piglets cryptosporidia were found in feces until Day post infection (DPI) 16, when the last piglet was necropsied. Cryptosporidiosis in conventionally reared piglets is a self-limited diarrheal disease associated with morphological changes within the intestine. The most severe lesion was seen in the posterior jejunum and ileum from DPI 3 to DPI 7, and consisted of villous atrophy, crypt hyperplasia and inflammatory infiltration in the lamina propria. In gnotobiotic piglets cryptosporidia induced severe enterocolitis which occurred at least until DPI 16. The characteristics of enteric lesions were similar to those found in conventionally reared piglets. Intestinal cryptosporidiosis in both groups of animals shifted in the course of infection in the caudal direction and terminated in the large intestine. Examination by scanning electron microscope showed that infected absorptive cells had thicker and longer microvilli than those on non-infected cells; neighboring non-infected cells were hypertrophic, bulbously protuberant with minute microvilli with no distinct intercellular borders. Numerous cryptosporidia in the heterotopic glandular epithelium in the submucosa of cecum and colon on DPI 9 and 10 were found. No differences in the location and degree of cryptosporidial infection between colostrum-fed and colostrum-deprived conventionally reared piglets were found. Sow's colostrum does not appear to protect piglets from C. parvum infection. The role of intestinal microflora in the pathogenesis of cryptosporidiosis in piglets is discussed.     

猪源隐孢子虫对仔猪致病性研究

为确定我国猪源隐孢子虫的危害性，采用光镜、扫描电镜和透射电镜等技术研究了猪源隐孢子虫sucp株对5日龄仔猪的致病性。结果：感染仔猪出现水样腹泻和脱水，粪便中混有肠黏膜碎片，潜伏期为3～5d，排卵囊持续期33-43d，虫体寄生于盲肠、结肠和直肠，并且盲肠和结肠杯状细胞增生，有炎性细胞浸润，结肠和直肠黏膜微绒毛萎缩、倒伏和脱落。结果表明sucp株对新生仔猪有一定致病性。     

Studies on the occurrence of Peptococcus indolicus and Corynebacterium pyogenes in abscesses in swine, and on the occurrence of Pc. indolicus in apparently normal skin and mucous membranes of piglets.

Pc. indolicus was isolated from 42.7% and Cb. pyogenes from 48.7% of 150 pus-samples from abscesses in pigs (slaughter-house material, Table I). In 41 specimens the two organisms were found together. Further, Pc. indolicus was demonstrated in 22.4% of 290 swabs from apparently normal skin and mucous membranes of piglets (autopsy material, Table II). By gel diffusion analysis the strains of Pc. indolicus were referred to the serotypes B, C, D, E, or F. The type distribution (Table III) possibly reflects a type-related variation in the invasive properties of Pc. indolicus.     

Life cycle of Isospora suis in gnotobiotic and conventionalized piglets

Isospora suis had 3 asexual and 1 sexual intra-intestinal conventional life cycle. The first asexual generation was most prominent at 2 days p.i. (post inoculation) and produced 2–7 merozoites. The second-generation meronts were prevalent at 3–4 days p.i. and produced 2–12 large merozoites. At 4–5 days p.i. the third generation meronts were prominent and produced 4–24 small crescent shaped merozoites. Mature sexual stages were most prominent at 5–6 days p.i. The stages were most numeroous in the distal half of the small intestine. At 8–9 days p.i. stages morphologically similar to the second generation of meronts reappeared, followed by the further development into third generation merozoites and sexual stages. This was reflected in a prepatent period of 5 days and a biphasic patent period of 5–8 or 9, and 11–14 days p.i.Intraperitoneal injection of liver/spleen and intestinal lymph node homogenates, respectively, from piglets infected 24 and 48 h, previously with high doses of oocysts, resulted in a patent infection 10–12 days post inoculation of the donor piglets. No differences in the life cycle of I. suis were observed between conventionalized and germ-free piglets. An extra-intestinal life cycle of I. suis related to the second patent period was postulated.     

Actinobacillus suis-like organisms and evidence of hemolytic strains of Actinobacillus lignieresii in horses

Thirty-seven local isolates of Actinobacillus suis-like organisms from diseased and clinically normal horses and 1 llama were compared with reference strains of A suis, A lignieresii, A equuli, A capsulatus, A hominis, A (Pasteurella) ureae, and equine A suis-like organisms (ASLO) previously described in literature. Comparison was by cultural characteristics, carbohydrate fermentation, enzyme profiles, and whole-cell protein polyacrylamide gel electrophoresis. Carbohydrate fermentation, determined by API-CH gallery, divided 36 equine ASLO isolates into 6 API-CH biotypes. The llama isolate was an additional distinct biotype. The biochemical comparisons between A suis and ASLO did not reveal remarkable and consistent differences. Enzyme analysis revealed 5 API-ZYM biotypes, one of which included the same strains as one of the API-CH biotypes and consisted in both instances of 4 esculin-negative ASLO cultures and the reference strain of A lignieresii. We conclude that the 4 strains were hemolytic variants of A lignieresii. Protein electrophoresis disclosed 15 banding patterns, 10 of which represented equine ASLO strains. The reference strains of A suis shared the pattern predominant among equine ASLO. Four of the remaining reference strains of Actinobacillus species each had a unique profile, whereas the type strain of A capsulatus and the llama isolate had similar profiles. The groupings of cultures resulting from the different testing methods had little relation to each other and to the anatomic source of the strains except the strains comprising API-CH biotype II, which originated in the equine respiratory tract, and the A lignieressi cluster.     

Pathogenicity of some Mycoplasma and Acholeplasma species in the lungs of gnotobiotic calves

Cloned cultures of 16 strains, representing nine different species of Mycoplasma and Acholeplasma, were inoculated intratracheally into gnotobiotic calves. Strains of M bovirhinis, M canadense, M verecundum, A axanthum and A modicum did not produce visible pneumonic lesions and were not reisolated from the lungs. Strains of M alkalescens and M arginini colonised the lower respiratory tract but failed to produce visible pneumonia. M bovigenitalium (strain M991/70) and M dispar (strain Gri226) both colonised the respiratory tract and induced pneumonic lesions estimated to involve up to 8 per cent (M bovigenitalium) and 17 per cent (M dispar) of the lung. Histologically M bovigenitalium produced a cuffing pneumonia and M dispar produced a interstitial alveolitis.     

Early cytokine response of gnotobiotic piglets to Salmonella enterica serotype typhimurium

Cytokine response against Salmonella Typhimurium is traditionally studied in conventional animals. Germ-free animals, however, enable to study response against infection without background effect of other microorganisms. Plasma and ileal inflammatory cytokines in germ-free piglets orally infected with virulent LT2 strain or, with a non-virulent SF1591 rough mutant were quantified by ELISA. In plasma and ileal washes, IFN-gamma levels significantly increased in both infected groups. TNF-alpha and IL-18 were mostly missing in plasma 24 h after infection. In the ileum, IFN-gamma, TNF-alpha, and IL-1beta were induced mainly by the virulent strain, whereas IL-18 was induced in highest quantity by non-virulent Salmonella. These data confirmed an important role of IFN-gamma, as well as other inflammatory cytokines in early stage of salmonellosis.