Analysis of the outer membrane proteins of Bacteroides nodosus, the causal organism of ovine footrot

Examination by SDS-PAGE of lithium acetate extracts of several strains of depiliated Bacteroides nodosus revealed 6 major outer membrane proteins (including pilin). The 5 membrane proteins exhibited approximate molecular weights of 75000, 50000, 38000, 34500 and 26500 whereas pilin had a MW of 17500 for the majority of strains. All proteins were accessible to lactoperoxidase-catalysed iodination and proteins 1, 2 and 5 were shown to be glycoproteins. Several attempts to isolate individual OMC proteins in pure form by selective solubilization and gel filtration were unsuccessful, but electroelution of individual outer membrane complex proteins resolved by SDS-PAGE provided sufficient quantities of antigen for immunization of sheep and for immunochemical analysis.     

Characteristics of Bacteroides nodosus isolated from cattle

Fourteen isolates of Bacteroides nodosus were cultured from cattle on six farms and examined for colony morphology, pilation, agglutinability, proteolytic activity and pathogenicity for sheep. Where sheep were also present on the farms, isolates from these were compared with those from cattle.Colonies of the bovine isolates were moderately fimbriate. Cells from these colonies were pilated, but not to the extent observed on virulent sheep isolates. The proteolytic activity of the isolates was also less than that described for virulent ovine isolates. When inoculated into sheep, B. nodosus from cattle produced only mild interdigital inflammation. There was no separation of horn characteristic of virulent foot-rot. Preliminary studies, based on agglutination tests, suggest that B. nodosus with different surface antigens may occur in cattle in the same herd and in cattle and sheep on the same farm. On one of six farms isolates from cattle and sheep were indistinguishable by the agglutination test.     

Phagocytosis of Bacteroides nodosus by ovine peripheral blood leucocytes

Phagocytosis of Bacteroides nodosus by ovine peripheral blood leucocytes (PBL) was examined after organisms had been opsonized in sera from normal sheep, or from animals immune to, or infected with ovine footrot. Ingestion of bacteria, as assessed microscopically or by counting isotopically-labelled organisms spectrometrically was effected in suspensions by polymorphonuclear leucocytes (PMN). Opsonization of bacteria in immune serum, particularly its IgG₂ isotype, enhanced the rate of phagocytosis by PMN compared with that promoted by normal serum or medium alone. Whereas IgG₂ from immune serum also increased the rate of ingestion of B. nodosus by adherent PMN, IgM and IgG₁ from immune serum also initiated phagocytosis of bacteria by adherent ovine monocytes. Leucocytes from normal, immune or infected sheep of different breeds ingested B. nodosus with equal facility.     

Role of outer membrane proteins in immunity against murine salmonellosis--1. Antibody response to crude outer membrane proteins of Salmonella typhimurium

The humoral immune response to crude outer membrane proteins (comp) of S. typhimurium in mice has been characterized. Maximal and quicker antibody response was observed when 50 micrograms of comp was injected intraperitoneally. The comp of smooth C5 strain of S. typhimurium evoked antibody response to both lipopolysaccharide (LPS) and proteins. Absorption of these sera with LPS coated erythrocytes eliminated the antibodies to LPS completely, while the antibody level to protein was left unaltered. The comp from rough mutant (lacking O-specific chain of LPS) of S. typhimurium elicited antibodies to proteins but not to LPS. These results indicate the concomitant production of antibodies to Salmonella outer membrane proteins also. The significance of such antibodies in protection and diagnosis has been discussed.     

Expression of iron-regulated outer membrane proteins by porcine strains of Pasteurella multocida.

The outer membrane protein (OMP) profiles of two strains of capsular type A Pasteurella multocida isolated from the lungs of pigs with enzootic pneumonia were studied. Sarkosyl extracted OMPs from P. multocida grown under iron-restricted and iron-replete conditions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 74 kDa, 94 kDa, 99 kDa and 109 kDa were expressed by strain A52, while 74 kDa, 82 kDa, 94 kDa and 99 kDa IROMPs were expressed by strain B80. Swine immune sera, obtained from pigs which were first immunized with a polyvalent P. multocida type A and type D bacterin and subsequently challenged with type A strain of P. multocida, contained antibodies against the IROMPs. These antibodies cross-reacted with the IROMPs expressed by avian strain P1059 of P. multocida. Convalescent-phase serum obtained from turkeys which survived fowl cholera, also cross-reacted with the IROMPs from porcine strains of P. multocida. These results suggested that IROMPs from porcine and avian strains of P. multocida may share common epitopes that were recognized by swine immune serum as well as turkey convalescent-phase serum.     

Comparison of antibody responses in cattle to outer membrane proteins from Pasteurella haemolytica serotype 1 and from eight untypeable strains.

Membrane associated proteins from 8 untypeable Pasteurella haemolytica strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with those of P haemolytica serotypes 1 and 2. Cattle antisera obtained from P haemolytica serotype 1 vaccine trials were used in immunoblotting assays to compare the membrane proteins from the 8 untypeable strains with those from P haemolytica serotypes 1 and 2. Densitometry was used to identify bands, and using linear regression analyses, the peak area optical densities (measuring antibody response) were correlated to lesion scores from the vaccinated calves. Significant antibody responses to proteins of 99, 69, 60, 55, 47, 45, 39, 33, 30, 16, and 14.5 kDa were detected for 4 or more of the 8 P haemolytica untypeable strains. Serotypes 1 and 2 of P haemolytica contained a comigrating 30-kDa protein. Antibody responses to proteins of 39, 33, and 32.5 kDa were significant for 3 of the untypeable strains and had significant correlation to lesion scores. Antibody responses to various other proteins were significant for 2 untypeable strains each.     

Molecular genetic analysis of Dichelobacter nodosus proteases AprV2/B2, AprV5/B5 and BprV/B in clinical material from European sheep flocks

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.     

Development and duration of protection against salmonellosis in mice and sheep immunised with live aromatic-dependent Salmonella typhimurium.

The aim of this investigation was to determine the development and duration of protection in mice or sheep immunised with aromatic-dependent (aro-) Salmonella typhimurium strain CS332, by either parenteral or oral routes. Immunisation of mice by the intraperitoneal or sheep by the intramuscular routes was found to impart protection against oral challenge with the virulent parent S typhimurium strain CS94 as early as seven days after immunisation. In contrast, when immunisation was carried out by the oral route, protection was not evident until three weeks after immunisation. Regardless of the route of immunisation, mice were still partially protected at three months and were fully susceptible at six months after immunisation. In sheep, protection persisted for six months but not 12 months after immunisation. Only parenterally immunised mice and sheep developed high ELISA and, or, agglutinating antibody titres, and cutaneous delayed-type hypersensitivity (DTH) at three weeks after immunisation. Although both antibody and DTH were detectable three months after immunisation of mice with aro- S typhimurium strain CS332, none was detected at six months. Antibody measured by agglutination and ELISA was detectable six months after immunisation in sheep, although no DTH was evident. At 12 months after immunisation low levels of anti-LPS antibody (measurable by ELISA only) were detected in sheep immunised by the intramuscular route.     

The isolation of multiple strains of Mycoplasma ovipneumoniae from individual pneumonic sheep lungs.

The heterogeneity of Mycoplasma ovipneumoniae isolates from the lungs of sheep with chronic non-progressive pneumonia (CNP) from the same flock raised the possibility that multiple isolates derived from one lung were not all identical. To test this hypothesis, thirty isolates were obtained from each of six pneumonic sheep lungs at slaughter. Four lungs had relatively severe lesions and from each of these, three or four strains of M. ovipneumonia, distinguishable by REA and in most cases by SDS-PAGE, were detected. From the lungs of each of two sheep with mild lesions, two strains of M. ovipneumoniae were detected. Four isolates from one lung were further examined by restriction endonuclease analysis (REA) using many restriction endonucleases. Those which differed with EcoRI also differed when other restriction endonucleases were used. However, partial digests occurred mainly with those restriction endonucleases which recognise cytosine-rich sequences. The presence of multiple strains of one species of microorganism in individual lesions is an unusual concept which may not be limited to one disease or to one host.     

Effect of adjuvants on antibody responses of sheep immunised with recombinant pili from Dichelobacter nodosus

Objective To compare the effects of two oil emulsion adjuvants (incomplete Freunds adjuvant and a proprietary oil adjuvant), DEAE-dextran, L-tyrosine particles and Quil A on the humoral immune responses of sheep immunised with recombinant pili of Dichelobacter nodosus (strain A).
Procedure Antibody titres were studied for up to 32 weeks and were measured by bacterial agglutination and ELISA. The relative avidity of antibodies for pili was determined and the incidence and severity of adverse reactions at the site of injection of vaccines were recorded.
Results The oil emulsion adjuvants and Quil A were more effective than either DEAE-dextran or L-tyrosine at stimulating antibodies in sheep. The incidence and severity of adverse reactions was lower in sheep which received vaccines containing either Quil A or DEAE-dextran than in sheep which received vaccines containing oil emulsion adjuvants. L-tyrosine had no adverse effects.
Conclusion Quil A was as effective as oil adjuvants at stimulating high levels of antibodies against recombinant pili in sheep and had the significant advantage of being less irritant after subcutaneous injection.     

抗鸭多杀性巴氏杆菌外膜蛋白H单克隆抗体的制备及鉴定

为制备抗鸭多杀性巴氏杆菌(P.multocida)外膜蛋白H(OmpH)单克隆抗体(MAb),本研究以原核表达并纯化的OmpH重组蛋白免疫BALB/c小鼠,将其脾淋巴细胞与SP2/0进行融合,并以重组OmpH蛋白为抗原,通过间接ELISA方法筛选出3株能够稳定分泌抗OmpH的杂交瘤细胞株(1A9、1E9、3G7)。MAb亚类鉴定表明,1A9、1E9和3G7均为IgG1亚类,轻链均为κ链。经western blot和间接免疫荧光检测证明3株MAb均具有良好的特异性。这些抗OmpH MAb的获得,为鸭P.multocida病的快速、有效、敏感的诊断方法建立奠定了基础。     

Agglutination of Brucella abortus cells by sera from cattle experimentally infected with Escherichia coli

Infection of normal adult cattle and sporadic serological Brucella abortus reactors, which no longer had diagnostic titers to B. abortus, with a culture of Escherichia coli isolated from a cow (or its environment) resulted in the production of a primary IgM serum antibody response to both B. abortus and E. coli. Cattle injected with heat killed E. coli and guinea pigs or young calves lacking natural agglutinins to B. abortus injected with live E. coli, produced serum antibody only to E. coli. The subsequent reinjections of live E. coli into the former two groups of cattle resulted in all but one animal in each group producing IgM ‘secondary’ responses to B. abortus of decreasing magnitude, while the anti-E. coli responses increased and eventually switched to synthesis of IgG class antibody. The remaining two animals produced a series of ‘secondary’ responses of IgM antibody to B. abortus similar in amplitude and duration to the primary immune response. The anti-E. coli agglutinins of these two animals increased in titer and IgG class antibody to E. coli was evident after several injections of E. coli. These results indicate that infection of cattle by E. coli can cause a problem in the serological fiagnosis of B. abortus infection. Speculations on the cause of this cross-reaction and ways of minimizing misdiagnosis are discussed.     

Babesia argentina: Immunization of cattle with a killed antigen against infection with a heterologous strain

Cattle were immunized againts infection with a heterogologous strain of Babesia argentina by the subcuntaneous inoculation of killed antigen mixed with Freund's complete adjuvant. The most effective antigen, infectted erythrocyte antigen (IEA), which confered protection comparable with that conferred by the presence of subclinical infection, was prepared by the disruption of parasitized erythrocytes in a French pressure cell. In contrast, antigen prepared from the plasma of infected animals was weakly immunogenic. The inoculation of IEA by the intravenous route without adjuvant did not confer protection but demonstrated the presence of a “kallikrein activating” substances(s) in the contents of the parasitized erythrocytes.     

Evaluation of protection against experimental salmonellosis in sheep immunised with 1 or 2 doses of live aromatic-dependent Salmonella typhimurium

The minimum number of doses of a live aromatic dependent (aro-) Salmonella typhimurium vaccine strain (SL1479), given by the intramuscular, oral or subcutaneous route required to protect sheep from experimentally-induced clinical salmonellosis, was determined. A significant reduction in mortalities and diarrhoea occurred in those sheep immunised with one or 2 intramuscular doses or 2 subcutaneous doses. On the other hand, sheep immunised with one subcutaneous dose were not protected. Immunisation with one or 2 oral doses also resulted in a significant reduction in mortality, although reduction in the prevalence of severe diarrhoea was less consistent. Sheep immunised with a single intramuscular dose of aro- S. typhimurium developed high levels of serum antibodies and significant delayed-type cutaneous hypersensitivity response to homologous Salmonella lipopolysaccharide and flagellin, whereas those with a single oral dose did not. It was concluded that immunisation of sheep with a single oral or intramuscular dose of live aro- S. typhimurium reduced mortalities and the prevalence of diarrhoea in sheep due to infection with virulent S. typhimurium.     

Immunological features of LPS from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower fecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-γ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P < 0.05) in the T group at 4 wpi. The IFN-γ production was higher (P < 0.01) at 12 wpi as well as its level of expression at 4 wpi (P < 0.05) in the T group. We found a higher expression level of TGF-β at 4 wpi in the T group (P < 0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P < 0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.     

布鲁菌外膜蛋白的糖基化及免疫效果检测

为研制安全、有效、新型靶向化布鲁菌外膜蛋白疫苗,采用去污剂连续抽提与电洗脱法分离纯化出布鲁菌外膜蛋白,并进行α-D-甘露吡喃异硫氰酸苯酯糖基修饰、佐剂乳化.依据统计学中完全随机实验设计试验,免疫BALB/c小鼠,收集不同时间点血液样品,ELISA法检测血清中IgG和IFN-γ含量.结果显示分离纯化出37.5 kD与47.2 kD的目的蛋白并成功糖基化修饰;纳米颗粒佐剂乳化后的试验用疫苗免疫小鼠,血清IFN-γ水平呈显著升高趋势(P＜0.05);抗体水平略显优势,但差异不显著(P＞0.05).表明试验用疫苗能激发机体的细胞免疫应答,且产生了免疫记忆.本研究为研制布鲁菌新型靶向化疫苗的研发和推广提供了研究依据.     

Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens

A simple procedure for obtaining surface exposed antigens of Salmonella Enteritidis is described. A heat treatment of whole bacteria in saline solution induced the release of small membrane vesicles containing outer membrane components as well as surface appendage components, such as fimbriae and flagellin. The characterization of the structural components of this extract, called HE, was established by SDS-PAGE and immunoblotting using polyclonal and monoclonal specific antibodies. Five major groups of proteins were identified: flagellin, porins, OmpA, SEF21 and SEF14 fimbriae. The immunogenicity of these proteins was studied by immunoblotting with serum samples from naturally infected hens. Flagellin, porins, OmpA, SEF14 and SEF21 fimbriae were immunogenic in the S. Enteritidis infected hens (frequency of reactants: 47.3, 97.3, 64.7, 50.0 and 60.8%, respectively); porins also reacted with sera from non infected hens (66.7%). The immunogenicity of these antigens in infected birds provide promise that they may serve as components of an effective subcellular vaccine for poultry salmonellosis.     

Anthelmintic activity of artesunate against Fasciola hepatica in naturally infected sheep

In light of rapidly spreading triclabendazole resistance alternative fasciocidal drugs are urgently needed. Following up on promising results obtained with artemether in Fasciola hepatica infected sheep, we here report the efficacy and safety of artesunate in sheep with a natural F. hepatica infection. Artesunate was administered intravenously and intramuscularly, adverse events were monitored and drug efficacy was elucidated by means of faecal egg and worm burden reductions. A single 40 mg/kg intravenous dose of artesunate induced an egg count reduction of 68.9% and a worm burden reduction of 77.4%. Intramuscular artesunate at 40 mg/kg reduced faecal egg count and worm burden by 97.6% and 91.9%, respectively; whereas at 60 mg/kg it caused 93.2% and 87.1% reduction in faecal egg count and worm burden, respectively. Three sheep died 24-72 h post-treatment with a double dose of 40 mg/kg intramuscular artesunate, showing lethargy, sialorrhoea, reduced rumination and tremors. Egg and worm burden reductions of 93.3% and 83.9%, respectively, were calculated in the three surviving sheep. In conclusion, the interesting fasciocidal properties of artesunate in sheep warrant further investigations with an emphasis on toxicity studies.     

Anthelmintic activity of Cocos nucifera L. against sheep gastrointestinal nematodes

The development of anthelmintic resistance has made the search for alternatives to control gastrointestinal nematodes of small ruminants imperative. Among these alternatives are several medicinal plants traditionally used as anthelmintics. This work evaluated the efficacy of Cocos nucifera fruit on sheep gastrointestinal parasites. The ethyl acetate extract obtained from the liquid of green coconut husk fiber (LGCHF) was submitted to in vitro and in vivo tests. The in vitro assay was based on egg hatching (EHT) and larval development tests (LDT) with Haemonchus contortus. The concentrations tested in the EHT were 0.31, 0.62, 1.25, 2.5 and 5 mg ml⁻¹, while in the LDT they were 5, 10, 20, 40 and 80 mg ml⁻¹. The in vivo assay was a controlled test. In this experiment, 18 sheep infected with gastrointestinal nematodes were divided into three groups (n = 6), with the following doses administered: G1—400 mg kg⁻¹ LGCHF ethyl acetate extract, G2—0.2 mg kg⁻¹ moxidectin (Cydectin^®) and G3—3% DMSO. The worm burden was analyzed. The results of the in vitro and in vivo tests were submitted to ANOVA and analyzed by the Tukey and Kruskal–Wallis tests, respectively. The extract efficacy in the EHT and LDT, at the highest concentrations tested, was 100% on egg hatching and 99.77% on larval development. The parameters evaluated in the controlled test were not statistically different, showing that despite the significant results of the in vitro tests, the LGCHF ethyl acetate extract showed no activity against sheep gastrointestinal nematodes.     

Precipitating antibodies against excretory/secretory antigens of Fasciola hepatica in sheep serum

Gel diffusion techniques were used to study antigen-antibody reactions in precipitates forming around juvenile, semi-mature and adult Fasciola hepatica cultured in serum from infected sheep. The number of reactions was analysed in both primary and challenge infections. The number of antigens shared by the various developmental stages of the fluke was also examined.At least 2 antigens were involved in precipitate formation around juvenile flukes. These antigens were also produced by the later stages of development. Two additional antigens were produced by semi-mature flukes and these, together with two others, were produced by adult flukes.Challenge infections had little effect on the number of antigens or antibodies in the precipitate.