Studies ofStreptococcus suis type 2 infection in pigs

Investigations into the immunology, pathogenesis and epidemiology ofStreptococcus suis type 2 infections were carried out in experimental pigs and in naturally-occurring field outbreaks of disease.The capsular polysaccharide fromStr. suis type 2 was shown to induce opsonic antibodies in pigs when injected with Freund's incomplete adjuvant, but difficulties encountered in experimental production of the disease prevented a study of their protective effects.Problems with the bactericidal tests led to an investigation of other assays for antibodies againstStr. suis type 2, namely, a phagocytic test with pig neutrophils, a mixed reverse passive antiglobulin haemagglutination test and an indirect haemagglutination test. There was evidence that with modifications both the latter tests would be useful.Transmission studies in 39 conventionally-reared and 7 hysterectomy-derived, colostrum-deprived pigs yielded interesting results with regard to the distribution of the organism in relation to the disease process.Tonsil carriage in clinically-healthy pigs was demonstrated after experimental and natural infection. Detectable carrier rates varied between 0 and 59%. The organism was shown to persist in the presence of circulating opsonic antibodies and in pigs on penicillin-medicated feed. Attempts to isolate the organism from the genital tract were unsuccessful.Medicated early weaning and classical SPF techniques applied to infected herds appeared to be effective in producing pigs free fromStr. suis type 2 infection.     

Prevalence of Streptococcus suis types 1 and 2 in domestic pigs in Australia and New Zealand

Using an indirect fluorescent antibody test, 54 per cent of 734 palatine tonsils of conventional pigs slaughtered in Australia and New Zealand were found to be infected with Streptococcus suis type 1 and 73 per cent of 959 were infected with S suis type 2. Variations in the prevalence of infection in pigs from different herds were thought to be due to differences in the sample sizes rather than to real differences in the prevalence between herds. The prevalence of infection with S suis was similar in pigs of either sex and in different age groups. Streptococcus suis type 2 was detected in the blood of 3 per cent of apparently normal pigs slaughtered at a meat processing plant. The presence of this organism in edible tissue may pose a health risk to consumers and meat-workers. Both S suis types 1 and 2 were detected in the vaginas and uteri of slaughtered pigs and the female reproductive tract could be another site for the carriage of infection. Piglets from sows with vaginas infected with S suis type 2 became infected earlier than piglets from sows with uninfected vaginas. No infected male reproductive tracts were detected and venereal transmission of S suis therefore appears unlikely. Three specific pathogen free herds were found to be free from infection with both S suis types 1 and 2. It is concluded that hysterectomy derived piglets are delivered free from infection, whereas some piglets born to sows with uterine and vaginal infections are either born infected or become infected at, or soon after, birth.     

Immunisation of pigs with killed cultures of Streptococcus suis type 2

Ten intravenous inoculations of 10(10) formalin-killed pathogenic Streptococcus suis type 2 stimulated detectable levels of opsonic antibody and a strong protective response against intravenous challenge with 10(9) viable S suis type 2 of the same strain. Heat-killed organisms did not stimulate a protective response, even though the type-specific antigen was intact and opsonic antibodies were induced. When the number of formalin-killed organisms in the inoculum was increased 10-fold, one inoculation was insufficient to stimulate a protective response, but two inoculations protected pigs against intravenous challenge with live, pathogenic organisms. Four-hour and overnight cultures were equally immunogenic, and the response was not enhanced by either Freund's incomplete adjuvant or aluminium hydroxide gel adjuvant.     

A murine model of Streptococcus suis type 2 meningitis in the pig

When young or adult mice were infected experimentally with isolates of Streptococcus suis type 2 of known pathogenicity for pigs, the organisms produced disease, young mice being more susceptible than adults. An isolate of S suis type 2 less pathogenic for pigs also appeared less pathogenic in mice. The organism could be reisolated from infected mice for up to 42 days. Inoculum size was one factor influencing the likelihood of an individual mouse developing meningitis following intravenous inoculation. Transmission of the organism from inoculated to in-contact mice occurred in young mice after intravenous or oral administration. These studies indicate that the behaviour of S suis type 2 in mice resembles its behaviour in pigs.     

Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O:9 in pigs

Due to almost identical lipopolysaccharide (LPS) O-antigens, infections with Yersinia enterocolitica serotype O:9 (YeO:9) cause false positive serological reactions (FPSR) in tests for Brucella and thus cause problems in National Brucella surveillance programs. As LPS are strong inducers of antibody responses it was hypothesized that cell-mediated immune responses to non-LPS antigens of the two bacteria can be used to separate immune responses to these two biologically very different infections. Following subclinical experimental infections with Brucella suis biovar 2, high interferon-gamma (IFN-gamma) assay responses with a commercial Brucella melitensis antigen preparation (Brucellergene OCB) preceded the development of antibodies. High IFN-gamma responses in the seven B. suis inoculated pigs with serological evidence of infection were consistent throughout a 20-week post-inoculation observation period. In contrast, IFN-gamma responses in two B. suis inoculated pigs without bacteriological or serological evidence of infection were below a cut-point of 25pg/ml at all samplings. IFN-gamma responses in repeated samplings from 5 uninfected control pigs and 18 pigs experimentally infected with YeO:9 were all negative, except for solitary false positives in 3.7% of the samples from both the experimentally YeO:9 infected pigs and control pigs. Skin tests using the same commercial Brucella antigen confirmed the ability of cell-mediated immune responses to differentiate between the two infections. In addition, a field evaluation of the diagnostic use of cell-mediated immune responses by IFN-gamma assay and skin test to resolve serological suspicions of Brucella was conducted in an YeO:9 infected pig herd. Following a screening of 200 pigs 39 pigs were identified with false positive serological Brucellosis reactions. While 36 of the 39 FPSR pigs were also FPSR in a second test, none of the pigs were test positive in whole blood IFN-gamma assay or Brucellergene OCB skin test. In conclusion, use of IFN-gamma assay and skin test as measurements of cell-mediated immune responses to non-LPS Brucella antigens were specific and sensitive in discriminating subclinical experimental infections with B. suis from both natural and experimental infections with YeO:9.     

The development of morphologic changes and the antibody response in guinea pigs experimentally infected with Brucella suis, biotype 2 bacteria

Sixty-two guinea pigs were infected vaginally, perorally, intranasally, conjunctivally and subcutaneously with the bacteria B. suis, biotype 2. The inoculum contained 13.8.10(9) of brucellas in 1 ml of physiological solution. Subcutaneous infection lasted 151 days, the other types of infection 56 days. All types of infections resulted in brucellotic changes developing in relation to the type and duration of infection. The least marked changes were observed after peroral infection. Brucellosis was chronical and the changes in liver, spleen, lungs, and after subcutaneous infection also in regional lymphoglandulae, were found out incessantly. Microscopically these changes were histiocytic granulomas. The pathogenesis of brucellosis process in guinea pig organism is influenced primarily by haematogenous propagation. The highest serological reactivity was caused by subcutaneous infection, the lowest by peroral infection. In the course of different types of infection the titres of agglutination and complement-fixation antibodies varied and the highest values were recorded between the 21st and 56th day. The diagnostic effectiveness of the surface fixation test was verified and no decrease in its sensitivity, not even in the chronic phase of infection, was observed. An incomplete correlation between morphological changes and serological reactivity in guinea pigs was detected. The highest number of Brucella infections was identified by histopathological examination which helped to reveal the initial stages of granulomas at the time when specific antibodies were not yet determined by serological tests.     

猪链球菌病双重PCR诊断方法的建立

根据猪链球菌2型荚膜多糖和马链球菌兽疫亚种类M蛋白的保守区序列分别设计了2对简并引物,建立了一种能同时检测猪链球菌2型和马链球菌兽疫亚种的双重PCR方法。结果显示,该双重PCR能从100个细菌的混合纯培养物中扩增出2条目的片段。而且可以直接从病料组织中检测到相应的病原菌。用建立的双重PCR方法和细菌分离培养法平行检测人工感染的组织病料,PCR方法与细菌培养法的阳性检出率基本一致,但PCR方法的特异性好、敏感性高,简便易行,可以用于猪链球菌病的流行病学调查和实验室的快速鉴别诊断。     

Evaluation of a gel diffusion precipitin test for porcine parvovirus

The use of a gel diffusion precipitin (GDP) test for the detection of porcine parvovirus (PPV) infection in pigs is described. The close correlation between gel diffusion precipitin and haemagglutination inhibiting (HI) antibody titres indicates that, with careful standardisation, a high level of sensitivity can be achieved with the GDP test and that it is a simple and relatively inexpensive alternative to the more commonly used HI test. Experimental infection of 2 groups of pigs showed that GDP and HI antibody responses were closely correlated and that GDP antibodies to PPV persisted for at least 41 weeks after infection. In a commercial herd study, serological evidence of declining passive immunity and subsequent acquisition of active immunity was demonstrated by measuring the GDP and HI antibody titres in sequential serum samples of pigs from a known PPV endemic farm. The GDP test described was shown to be less sensitive than haemagglutination (HA) in the detection of viral antigen but was, nevertheless, considered useful as a simple screening test for the amounts of antigen usually present in PPV infected mummified foetuses.     

上海地区31株2型猪链球菌的分离鉴定及其耐药性分析

猪链球菌病是由猪链球菌引起的一种重要人畜共患病,尤其是猪链球菌2型,不仅对猪的致病性最强,而目可感染特定人群并致死,给人类公共卫生和养猪业带来巨大影响。本研究采集临床症状疑似2型猪链球菌感染猪的全血及内脏器官,通过观察菌落形态及培养特征,筛选出可疑样品,纯化培养并进行PCR鉴定,筛选到31株2型猪链球菌,最后用纸片扩散法对临床分离到的2型猪链球菌菌株测定其耐药性,结果显示对5种药物耐药率分别为：80%（AM）、68%（GM）、84%（CHL）、52%（SXT）、55%（CIP）。试验结果表明分离的31株2型猪源性链球菌,均为多重耐药株,且青霉素类药物的耐药性较为严重,这为临床在治疗2型猪链球菌病提供了合理的指导作用。     

Streptococcus suis type 2 infections in pigs in the Netherlands (Part two)

Since 1983 some pig breeding and fattening farms in the Netherlands have been faced with a considerable mortality in pigs due to Streptococcus suis type 2 infections. The most predominant clinical feature of S. suis type 2 infection is meningitis, although sudden deaths often occur. It was noted that some affected farms had imported breeding stock from the United Kingdom. Tonsils of slaughter pigs were collected from herds with and without a history of S. suis type 2 infections. Bacteriological examination was done by using an elective-selective medium. No significant difference was found in carrier rates of S. suis type 2 between clinically healthy and affected herds (38% vs. 45%). A cohort study was carried out by regular bacteriological examination of tonsil biopsies on a farm with a high incidence of streptococcal meningitis. Twenty-seven percent of the pigs were carriers of S. suis type 2 at nine weeks of age. Possible methods for disease control are discussed.     

Passive immunization of pigs against experimental infection with Streptococcus suis serotype 2

The safety and protective efficacy of a horse antiserum raised against inactivated whole cell preparations of Streptococcus suis serotype 2 was investigated in pigs by experimental challenge. The antiserum was evaluated in two similar experiments each comprising 12 4-week-old pigs treated with 6 ml of antiserum the day before challenge and four pigs used as challenge controls. Pigs were infected by subcutaneous injection with approximately 10(11) colony forming units of S. suis serotype 2. Clinical disease in the pigs that could be attributed to infection with S. suis was reduced from 88 to 35% (P = 0.015). The percentage of pigs with lesions that could be associated with S. suis was reduced from 88 to 22% (P = 0.002) and isolation of S. suis serotype 2 was reduced from five (63%) out of eight pigs in the combined challenge control groups to 3 (13%) out of 23 pigs in the combined treatment groups. These results indicate that passive immunization of pigs may be a way to reduce or control S. suis serotype 2 infections in pigs.     

Serological cross-reactivity between a porcine Actinobacillus strain and Haemophilus pleuropneumoniae.

During serological screening of a closed SPF-herd free of pleuropneumonia, more than half of the pigs were positive for complement-fixing antibodies to Haemophilus pleuropneumoniae. Actinobacillus bacteria closely related to A. suis were isolated from tonsillar tissue of 14 out of 20 slaughtered pigs submitted for pathological and bacteriological evaluation. None of the pigs had evidence of respiratory disease. Two pigs inoculated endobronchially with a selected Actinobacillus strain developed mild focal pneumonia and complement-fixing antibodies cross-reacting with H. pleuropneumoniae. Five pigs exposed and vaccinated with the Actinobacillus strain and five pigs spontaneously infected with the strain also developed complement-fixing antibodies against H. pleuropneumoniae and appeared to be less susceptible to experimental Haemophilus pleuropneumonia than pigs not exposed to the Actinobacillus infection. The agglutination test applied on serum treated with 2-mercaptoethanol detected antibodies against H. pleuropneumoniae serotype 5 but not against serotype 1 in pigs exposed to the Actinobacillus strain. Antibodies reactive with the Actinobacillus strain were also found in pigs hyperimmunized against H. pleuropneumoniae serotypes 1-5 in 2-mercaptoethanol tube agglutination test and rabbits hyperimmunized against serotypes 1,2 and 7, and strain 73567 in the immunodiffusion test. Conversely rabbits immunized against the Actinobacillus strain had antibodies against H. pleuropneumoniae serotypes 1, 3, 4, 5 and 6. It is concluded that pigs infected with Actinobacillus organisms may become false positive reactors against H. pleuropneumoniae.     

Experimental airborne transmission of Streptococcus suis capsular type 2 in pigs.

Experimental airborne transmission of Streptococcus suis type 2 was studied in specific pathogen free piglets. Forty piglets were allotted to five groups of eight 7-week-old animals and housed in three separated units. Negative control pigs (group 1) were housed in unit A and infected batches were housed in units B (group 2) and C (groups 4). In units B and C, non-inoculated groups (groups 3 and 5, respectively), 40 cm distant from the respective inoculated group and without any physical contact between them, also took place. Six animals of groups 2 and 4 were inoculated intravenously with 2 x 10(8) colony forming units (cfu) of a mild and a high virulent S. suis strains, respectively. The remaining animals in these groups and pigs from groups 1, 3, 5 received broth medium in the same way. Differences among virulence of S. suis capsular type 2 were observed in inoculated pigs of groups 2 and 4. Pigs from group 2 became carriers, showing only mild symptoms. By contrast, animals from group 4 presented an acute form of the disease. All the indirect contact pigs in groups 3 and 5 had S. suis in palatine tonsils from day 6 after the infection and they presented clinical manifestations similar to those observed in experimentally infected pigs. Two direct contact animals were also contaminated in the upper respiratory tract but surprisingly they did not show any symptoms. Airborne transmission of S. suis in experimentally pigs was demonstrated in the present study. Indirect infections, as described in this study, are a more realistic way to infect pigs than other experimental procedures and may be used to further study the pathogenesis of the infection caused by this important pathogen.     

Experimental infection of specific pathogen free piglets with French strains of Streptococcus suis capsular type 2.

A standardized model of Streptococcus suis type 2 infection in specific-pathogen-free piglets, housed in high-security barns, was used to compare the virulence of 3 French field strains of S. suis serotype 2 isolated from tonsils of a healthy pig (strain 65) or from diseased pigs (meningitis, strain 166', or septicemia, strain 24). In one of the 2 trials, 7-week-old pigs, in 3 groups of 8, were inoculated intravenously with 2 x 10(8) colony-forming units of S. suis type 2. In each group, 1 uninfected animal was a sentinel. Eight animals were also used as negative control group. The experiment was repeated under similar conditions with strains 65 and 166'. Virulence differed markedly among these S. suis strains when clinical signs, zootechnical performances, lesions, and bacteriological data were analyzed. Strain 65 did not induce clinical signs in inoculated pigs. In contrast, pigs infected with the other 2 strains exhibited clinical signs and typical lesions of S. suis type 2 infections. Differences in virulence were also observed between the 2 virulent strains. Sentinel animals exhibited the same manifestations as those recorded in inoculated piglets. Results were similar in the second trial, indicating that under the present experimental conditions, results were reproducible. The standardized conditions described in this study could be a useful tool to further study about the S. suis infection.     

Paramyxovirus type 1 infections of racing pigeons: 1 characterisation of isolated viruses

Viruses isolated from field outbreaks of disease in racing pigeons in continental Europe and Great Britain were shown to be identical by serological tests using conventional chicken antisera and mouse monoclonal antibodies. The pigeon viruses showed high levels of cross-reaction to Newcastle disease virus (NDV) in haemagglutination inhibition tests and Madin-Darby bovine kidney cells infected with pigeon virus isolates bound three out of nine mouse monoclonal antibodies prepared against NDV Ulster 2C. These results confirm their classification in the paramyxovirus type 1 serotype of avian paramyxoviruses. However, the pigeon viruses could be distinguished from more classical paramyxovirus type 1 viruses by the significantly different titres obtained in haemagglutination inhibition tests, the failure of mouse monoclonal antibodies directed against the HN1 epitope of NDV Ulster 2C to inhibit their haemagglutinating activity and a unique binding pattern seen with the nine mouse monoclonal antibodies.     

Serologic profile of a cohort of pigs and antibody response to an autogenous vaccine for Actinobacillus suis

Actinobacillus suis is a commensal opportunistic pathogen in swine. However, in recent years, an increasing prevalence of clinical signs associated with A. suis has been observed in high health status herds in North America. The objectives of the study were to assess the kinetics of antibodies to A. suis in pigs from a herd showing clinical signs of A. suis infection and, to evaluate the antibody response in gilts following vaccination with an autogenous vaccine. An enzyme-linked immunosorbent assay (ELISA) using a saline extract of boiled-formalinized whole cells of a field strain as the coating antigen was standardized. This ELISA was used as a tool for monitoring, in a comparative way, the variations in A. suis antibody levels. The herd selected for the serologic profile was negative for Actinobacillus pleuropneumoniae infection and showed clinical signs of A. suis infection in 16 to 19-week-old pigs. A cohort of 20 pigs was blood sampled at 5, 8, 12, and 16 weeks of age. The lowest level of serum antibodies was observed between weeks 8 and 12, this probably corresponding to a decrease in maternal immunity. A marked increase in the antibody response was seen at 16-week of age, at the approximate time of onset of A. suis clinical signs in the herd. The evaluation of serum antibody responses to an autogenous vaccine revealed that the humoral immunity of gilts further increased following vaccination although the level of antibodies was already high prior to vaccination. The magnitude of the response to vaccination was higher when the level of antibodies was low prior to the first injection. The ELISA test seems to detect antibodies against the O-chain LPS.     

猪链球菌2型的分离鉴定及致病性试验

从全国部分猪场采集疑似猪链球菌感染病例脑样品20份进行细菌分离,成功分离到10株细菌,通过猪链球菌及血清类型鉴定,最终确定其中一株为2型猪链球菌。应用猪链球菌7种主要毒力因子特异性基因扩增检测方法检测所分离到的2型猪链球菌的毒力因子分布情况,并应用小鼠攻毒试验对其致病性进行观察研究。结果表明:分离的2型猪链球菌具备7种毒力因子;动物试验表明SS2能引起小鼠的急性败血症及脑膜炎;细菌回归试验结果表明,试验组死亡小鼠的脑、心脏、肝脏、脾脏、肺脏、肾脏均有细菌定植,且能分离出攻毒菌。此分离菌株的研究为研制2型猪链球菌病疫苗奠定了基础。     

Detection and molecular typing of Streptococcus suis in tonsils from live pigs in France

Streptococcus suis is an important pathogen of swine, causing meningitis, arthritis, polyserositis, septicemia, and sudden death in weaning piglets as well as fattening pigs. Recently, 3 molecular tests have been developed in our laboratory: a multiplex polymerase chain reaction (m-PCR) assay for the detection of S. suis species and serotypes 2 and 1/2, and 2 molecular typing methods, pulsed-field gel electrophoresis and an approach based on PCR amplification of a fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S rDNA intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis (ISR-RFLP). In the present study, we used these tests to analyze tonsil samples from clinically healthy pigs and to identify individual isolates of S. suis during epidemiologic investigations of 8 related herds with a history of septicemia caused by S. suis serotype 2. Capsular typing showed that 58% of the strains were nontypable. Of the 17 serotypes present, serotype 22 was the most prevalent. In the 7 farms without clinical signs on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, in less than 5% of the pigs by m-PCR or by bacteriologic culture. In the 8th farm, on which 2 pigs had clinical signs of septicemia on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, by m-PCR in the tonsils of 40% of fattening pigs (21 wk old) that lacked symptoms. Molecular typing of the serotype 2 strains showed a common origin of contamination in these herds, given that 1 pattern (C1) was detected in the isolates from 6 of the 8 herds. However, up to 4 patterns were associated with septicemia and sudden death. Several patterns of S. suis serotype 2 can be responsible for disease in the same herd. These molecular tools may be useful for confident studies of the transmission of S. suis, thereby contributing to the control of S. suis infection.     

Serological and immunological studies of pleuropneumonia of swine caused by Haemophilus parahaemolyticus

The development of circulating antibodies for H. parahaemolyticus was studied in experimentally infected SPF pigs and in-contact SPF pigs. Blood serum titers were determined by a modified complement fixation test with normal SPF swine serum as a source of supplementary complement factor, and by an indirect haemagglutination test.CF and IHA titers became positive within the first 2 weeks following exposure to H. parahaemolyticus, and reached peak values after 2 to 7 weeks (Figs. 1 to 3). The exposed pigs proved immune, in that they showed no clinical symptoms on challenge after resp. 6, 9 and 11 weeks.While distinct titers were thus obtained with both tests in SPF swine experimentally exposed to H. parahaemolyticus, the CF test proved more specific than the IHA test when the 2 tests were compared in a field outbreak of polyserositis (Glässers disease) caused by H. parasuis. The CF test would therefore seem to be preferable to the IHA test in field diagnostic work (Table 1).A noticeable finding was that challenge did not elicit an anamnestic antibody response in any of the immune pigs (Figs. 1 to 3). This fact together with negative bacteriological findings in the animals in question would seem to suggest that the challenge dose was unable to establish a permanent infection in the respiratory tract of the immune pigs.     

Streptococcus suis type 2 infections in pigs in the Netherlands (part two)

Summary

Since 1983 some pig breeding and fattening farms in the Netherlands have been faced with a considerable mortality in pigs due to Streptococcus suis type 2 infections. The most predominant clinical feature of S. suis type 2 infection is meningitis, although sudden deaths often occur. It was noted that some affected farms had imported breeding stock from the United Kingdom.

Tonsils of slaughter pigs were collected from herds with and without a history of S. suis type 2 infections. Bacteriological examination was done by using an elective‐selective medium. No significant difference was found in carrier rates of S. suis type 2 between clinically healthy and affected herds (38% vs. 45%).

A cohort study was carried out by regular bacteriological examination of tonsil biopsies on a farm with a high incidence of streptococcal meningitis. Twenty‐seven percent of the pigs were carriers of S. suis type 2 at nine weeks of age. Possible methods for disease control are discussed.