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.     

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.     

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.     

Evaluation of methods used for detecting Streptococcus suis type 2 in tonsils, and investigation of the carrier state in pigs

Streptococcus suis type 2 was isolated from the tonsils of 143 (76 per cent) of 188 pigs from three herds by culturing on media containing a specific antiserum. Culture of frozen sections of tonsillar tissue was more sensitive than culture of swabs or deep scrapes. The specificity of an indirect fluorescent antibody test for identifying S suis type 2 in mixed cultures was conservatively estimated to be 0.89. The level of infection in one herd without a history of meningitis was lower than in two herds which had experienced repeated outbreaks. Differences between herds in the level of infection were due to differences in the number of organisms carried by pigs, more so than differences in the detectable carrier rate.     

Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR.

We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP+EF+) and highly virulent S. suis type 1 strains (MRP(s)EF+) and of the EF protein of weakly virulent S. suis type 2 strains (MRP+EF). The latter strains give rise to larger PCR products than the virulent strains of S. suis type 1 and 2. A positive control template was included in the assay to identify false negative results. The PCR was evaluated using tonsillar specimens from herds known (or suspected) to be infected and herds without an S. suis history. The results obtained with the PCR assay were compared with the results obtained with a newly developed bacteriological examination. In this bacteriological examination we were able to identify the EF-positive strains directly in the tonsillar specimens. From the 99 tonsils examined, 48 were positive in the PCR and 51 negative. All specimens from which EF-positive S. suis strains were isolated were also positive in the PCR assay. Three samples were positive in the PCR, but negative by bacteriological examination. The results demonstrated that the PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying virulent strains of S. suis type 2 and highly virulent strains of type 1. Application of the assay may contribute to the control of S. suis infections.     

Experimental infections of mice and pigs with Streptococcus suis type 2.

Five inbred strains of mice were tested for their susceptibility to Streptococcus suis type 2 including the type strain, two isolates from meningitis in pigs and two isolates from tonsils of clinically healthy pigs. C57BL/6, ICR and ddY strain mice showed lower susceptibility to all strains of S. suis type 2 than BALB/c and SS strain mice. The type strain and the isolates from diseased pigs produced septicaemia and meningitis in BALB/c and SS mice inoculated with 10(8) colony forming unit of the bacteria and 60 to 100% of these infected mice died. On the other hand, mice inoculated with the isolates from healthy pigs showed mild clinical signs but none of them died. In BALB/c mice which died or developed nervous signs, the purulent meningo-encephalitis, myocarditis, ophthalmitis, labyrinthitis and otitis media were observed. S. suis type 2 antigen was demonstrated in these lesions by immunoperoxidase staining using rabbit S. suis type 2 antiserum. These results were similar to those in the experimentally infected pigs with these virulent and avirulent strains against mice. These results indicate that BALB/c and SS strains of mice are useful as an experimental model of S. suis type 2 infections in pigs, and that there are virulent and avirulent strains against mice and pigs among the strains of S. suis type 2.     

Streptococcus suis isolated from pigs in Finland

A total of 58 Streptococcus suis strains were isolated from deceased pigs submitted to the National Veterinary Institute, Regional Laboratory in Kuopio, Finland, over a 3 1/2 year period, most frequently from cases of pneumonia. The bacteria were isolated from cases of meningitis, sepsis, rhinitis, endocarditis and abortion. S. suis was also isolated from nasal cavity, lung and brain of some sick piglets without signs of inflammation. Further S. suis was detected in 12 out of 107 tonsils of healthy fatteners tested. S. suis strains were identified by biochemical methods followed by typing. The most common capsular types were 7, 3 and 2, respectively. Only one type 1 strain and no types 6 and 9 strains were found. All S. suis strains tested were sensitive to penicillin and ampicillin.S. suis is not uncommon in Finnish pig herds. S. suis may be regarded as a potentially pathogenic organism which under certain predisposing conditions may cause serious disease.     

Prevalence of pseudorabies virus infection and associated infections in six large swine herds in Illinois.

Sera were collected from 6 large farrow-to-finish swine herds infected with pseudorabies virus (PRV) in Illinois. All herds were participating in the Large Herd Cleanup Study, a USDA-initiated project to evaluate the feasibility of eradicating pseudorabies from large farms (greater than 400 sows) by use of a combination of vaccination and management changes. Herd size ranged between 425 and 1,500 breeding females. Between April and July 1990, sera for measurement of PRV antibodies were obtained from 113 to 156 sows and 112 to 162 finishing pigs (body weight greater than 70 kg)/herd. Duplicate sera from 30 sows and 30 market-weight pigs/herd were obtained for measurement of serum antibodies to the following associated organisms: swine influenza virus, transmissible gastroenteritis virus, encephalomyocarditis virus, Actinobacillus pleuropneumoniae, Eperythrozoon suis, and 6 serovars of Leptospira interrogans. Prevalence of PRV antibodies attributable to field virus infection ranged between 53.8 and 100% for sows and between 0.7 and 97.3% for finishing pigs, as determined by the appropriate differential test for the vaccine being used on each farm. In only 1 herd, PRV seroprevalence was increased with higher sow parity. For associated infections, the risk of seropositivity attributable to PRV was not significant (for most infections) on all farms and varied among farms. Thus, pseudorabies did not appear, in general, to increase susceptibility to infection with other disease agents.     

Streptococcus suis serotypes associated with disease in weaned pigs

Streptococcus suis was recovered from 9 outbreaks of septicaemia and meningitis in weaned pigs between 1979 and 1983. Fifteen isolates from 7 outbreaks were identified as S. suis type 9, and 3 isolates from 2 outbreaks as S. suis type 2. Three further isolates of S. suis type 2 and an isolate of S. suis type 3 were recovered from cases of bronchopneumonia in weaned pigs from 4 other piggeries.     

Streptococcus suis infections in pigs in the Netherlands (Part I)

Data are presented on the incidence of various streptococcal infections in pigs in the Netherlands. 314 Strains isolated in the course of routine post-mortem diagnosis were examined. The most frequently occurring streptococcus was S. subacidus (bio) type II which was isolated in 31.2% of the cases. S. suis type 2 (Serogroup R) and S. equisimilis (Serogroup C) constituted 16.2% and 13.7% of the isolates respectively. Besides meningitis, endocarditis and polyserositis S. suis type 2 infections may frequently be associated with pneumonia (42%). The biochemical profiles of the various S. suis and S. subacidus (bio) types are presented. The profile of both species is almost identical. It seems justified to use the name S. suis for strains with this characteristic profile and to abandon the name S. subacidus. Haemolysis does not appear to be a suitable characteristic to screen for S. subacidus/S. suis types. In comparing three serological methods for typing S. suis type 2, gel precipitation using Fuller's extract and slide agglutination give an almost 100% correlation. These two methods are recommended for serotyping.     

Prevalence of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella multocida, and Streptococcus suis in representative Ontario swine herds

Tonsillar and nasal swabs were collected from weanling pigs in 50 representative Ontario swine herds and tested for the presence of 5 important bacterial upper respiratory tract pathogens. All but 1 herd (2%) tested positive for Streptococcus suis by polymerase chain reaction (PCR); 48% of herds were S. suis serovar 2, 1/2 positive. In all but 2 herds there was evidence of Haemophilus parasuis infection. In contrast, toxigenic strains of Pasteurella multocida were detected by a P. multocida--enzyme-linked immunosorbant assay (PMT-ELISA) in only one herd. Seventy-eight percent of the herds were diagnosed positive for Actinobacillus pleuropneumoniae by apxIV PCR. Sera from finishing pigs on the same farms were also collected and tested by ELISA for the presence of A. pleuropneumoniae antibodies. Seventy percent of the herds tested had evidence of antibodies to A. pleuropneumoniae including serovars 1-9-11 (2%), 2 (4%), 3-6-8-15 (15%), 5 (6%), 4-7 (26%), and 12 (17%). This likely represents a shift from previous years when infection with A. pleuropneumoniae serovars 1, 5, and 7 predominated. At least 16% and possibly as many as 94% of the herds tested were Actinobacillus suis positive; only 3 of the 50 herds were both A. pleuropneumoniae and A. suis negative as judged by the absence of a positive PCR test for apxII. Taken together, these data suggest that over the past 10 years, there has been a shift in the presence of pathogenic bacteria carried by healthy Ontario swine with the virtual elimination of toxigenic strains of P. multocida and a move to less virulent A. pleuropneumoniae serovars. As well, there appears to be an increase in prevalence of S. suis serovar 2, 1/2, but this may be a reflection of the use of a more sensitive detection method.     

Rapid detection of Streptococcus suis serotype 2 in weaned pigs

A survey to detect Streptococcus suis serotype 2 in 1,716 weaned pigs was done in Quebec. Forty-nine sow herds were included in this survey: in 26 herds, S suis serotype 2 had been isolated during the preceding 12 months and in 23 herds (control), the organism had not been detected during a previous study. Swab specimens of the nasal cavity and tonsils of pigs were obtained for bacteriologic culture, and S suis serotype 2 was easily detected by the use of brain-heart infusion agar containing a Streptococcus-selective supplement and 5% goat antiserum raised against S suis serotype 2. After measurement of the diameter of the precipitation zone of 539 isolates, a slide agglutination test was performed to identify the S suis serotype 2 isolates. The mean precipitation zone diameter obtained for group S suis serotype 2 was larger (P less than 0.001) than that for the group designated as "others". With slide agglutination test results as reference and on the basis of discriminant analysis to stimulate detection of S suis serotype 2, 93.1% of all isolates were correctly classified, using the precipitation zone diameter as unique classification criterion. Relative specificity was 94.5% and relative sensitivity was 88.7%. Use of the precipitation zone diameter on a quantitative basis led to the proposal of a simple and reliable technique to screen swine herds for S suis serotype 2 in weaned pigs. Nasal and tonsillar swab specimens were obtained and analyzed concurrently for S suis serotype 2. The organism was found in both sites in only 20.4% of 103 carrier pigs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intestinal translocation of Streptococcus suis type 2 EF+ in pigs

Sepsis with subsequent multisystem organ failure after translocation of bacteria from the gut is a serious risk associated with stress situations. We showed that intestinal bacterial translocation could be one of the pathways for pathogenic Streptococcus suis infections in the pig. In 24 piglets weighing 10-14 kg, free of the extracellular factor (EF+) producing phenotype of S. suis serotype 2, a silicon canula was placed in the proximal jejunum to enable intestinal inoculation and bypassing the upper alimentary tract. The pigs were individually housed. After stress induction in 18 pigs by means of a truck drive in individual cages for 1h, pigs were inoculated through the intestinal canula either with S. suis type 2 EF+ or with growth medium only, and put back in their original housing. The six not transported pigs were also inoculated with the same strain. To prevent oral self-infection, faeces were collected in a bag that was glued around the anus. Clinical and behavioral symptoms were recorded for 72 h post inoculation, and then the animals were sacrificed for pathological and bacteriological examination. In three animals, the inoculation strain was re-isolated from mesenterial lymph nodes and typically affected organs. No S. suis type 2 EF+ was detected by specific polymerase chain reaction (PCR) in any of the tonsil-swabs and -homogenates. We concluded that infection of the organs had taken place after bacterial translocation out of the gut and that the intestinal tract can be a porte d'entree for S. suis type 2 EF+.     

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.     

Investigation of a possible chain of Streptococcus suis infection in a pig breeding community using PCR-based genotyping

Streptococcus (S.) suis plays an important role in pig breeding causing invasive diseases such as meningitis and polyarthritis. Herd problems with Streptococcus suis are common in many pig farms and are frequently characterised by disease outbreaks. In this context, it is often important to identify chains of infection, e.g. between a farrow-to-wean and a grower farm. In the following case report a possible chain of infection among the different farms of a farrow-to-finish system was investigated. In two grower units herd problems with S. suis were present; the mortality was higher than 5 %. It appeared likely, that the streptococci causing these problems were transmitted from a single farrow-to-wean unit to the two different grower farms. In the respective farms swabs were taken from different healthy animals and, in the case of the grower farms, also from the infected animals. Genotypic profiling of strains by a multiplex-PCR and AFLP typing method revealed that two different S. suis pathotypes were responsible for the herd problems. Both pathotypes could not be detected in the farrow-to-wean farm.Thus, a chain of infection originating from the farrow-to-wean farm appeared unlikely. The multiplex PCR was in this case sufficient to elucidate the described problem.     

Production of muraminidase-released protein (MRP), extracellular factor (EF) and suilysin by field isolates of Streptococcus suis capsular types 2, 1/2, 9, 7 and 3 isolated from swine in France

A total of 323 isolates of Streptococcus suis recovered from diseased or healthy pigs in France were serotyped. The presence of virulence-related proteins, Muraminidase-Released Protein (MRP), Extracellular Factor (EF) and Suilysin was also studied in 122 isolates of capsular types 2, 1/2, 9, 7 and 3 to evaluate their implication in virulence of S. suis. Capsular types 2, 1/2, 9, 7 and 3 were the most frequently detected (93%), with 69% for the capsular type 2 alone. Capsular types 2, 1/2, 9, 7, 3, 1, 4, 8, 18, 10 and 12 were isolated from diseased pigs, whereas types 2, 7, 9, 1/2, and 3 originated from the nasal cavities or tonsils of healthy animals. Most of the S. suis type 2 isolates recovered from diseased pigs carried MRP+ EF- Suilysin- (46%) or MRP+ EF+ Suilysin+ (28%) phenotypes. The MRP+ EF- Suilysin- phenotype was also detected in 67% of S. suis type 2 strains isolated from healthy pigs. The production of the virulence-related proteins was less frequently found in S. suis types 1/2, 9, 7 and 3 recovered either from diseased or healthy pigs. In this study, all the capsular type 1/2 strains were MRP+ EF- Suilysin- and all the S. suis type 7 harboured an MRP- EF- Suilysin- phenotype. The MRP- EF- Suilysin- phenotype was found in S. suis types 2, 3, 7 and 9 isolated from septicaemia, meningitis, pneumonia, and pleurisy. These results suggest that the presence of these proteins should not be used as a single condition for classifying the virulence of a field isolate in France.     

Studies of Streptococcus suis type 2 infection in pigs

Investigations into the immunology, pathogenesis and epidemiology of Streptococcus suis type 2 infections were carried out in experimental pigs and in naturally-occurring field outbreaks of disease. The capsular polysaccharide from Str. 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 against Str. 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 from Str. suis type 2 infection.     

Actinobacillus suis infection in pigs in Australia

This is the first report of the isolation of Actinobacillus suis in association with significant disease among preweaned pigs in Australia. Sudden deaths occurred in preweaned piglets at one facility and enlarged joints, particularly the stifles and tarsi, occurred at another. Isolates with the biochemical phenotype and apx genotype profile of A suis were cultured from affected piglets. Both facilities were of high health status and one had undergone a recent depopulation, disinfection and sow repopulation process. Reviewing initial reports of A suis disease overseas, it is apparent that outbreaks occurred sporadically in various locations, with disease occurring among the herds involved for some months only, before development of immunity. A suis disease is now considered an emerging disease on high health status farms.     

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.     

Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine

The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventing infection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suis serotype 2 strain (MRP+EF+) was compared with the efficacy of a vaccine containing formalin-killed bacterin of S. suis serotype 2 (MRP+EF+). The enhancement of the immune response by different adjuvants (a water-in-oil emulsion [WO] and an aluminium hydroxide-based adjuvant [AH]) and their side effects were also studied. The MRP and EF were purified by affinity chromatography. Pigs were vaccinated twice at three weeks and six weeks of age and challenged intravenously with virulent S. suis serotype 2 strains (MRP+EF+) at eight weeks of age. At challenge, the pigs vaccinated with MRP+EF/WO had high anti-MRP and anti-EF titres and were protected as effectively as pigs vaccinated with WO-formulated vaccines with bacterin. Eight of the nine pigs survived the challenge and almost no clinical signs of disease were observed. The titres obtained with the MRP+EF/AH vaccine were low and only two of the five pigs were protected. Pigs vaccinated with either MRP or EF were less well protected; three of the four pigs died after challenge but the clinical signs of disease were significantly less severe than those observed in the placebo-vaccinated pigs. The protective capacity of the bacterin/AH vaccine was very low, and the mortality among these pigs was as high as in the placebo-vaccinated pigs (80 per cent). Postmortem histological examination revealed meningitis, polyserositis and arthritis in the clinically affected pigs. The results demonstrate that a subunit vaccine containing both MRP and EF, formulated with the WO adjuvant, protected pigs against challenge with virulent S. suis type 2 strains.