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.     

副猪嗜血杆菌人工感染猪的病理学观察

用副猪嗜血杆菌分离株感染断奶仔猪,发病仔猪临床表现为体温中度升高、精神不振、被毛粗乱、站立不稳、厌食、呼吸困难。尸体剖检,可见胸腹腔纤维素渗出严重,有大量的渗出液、肺出血及脑充血等;病理组织学表现为,肺脏、心脏、脾脏等脏器含有大量的中性粒细胞的纤维蛋白性多发性浆膜炎、脑膜炎及支气管肺炎。结果表明,此分离株为一高致病潜力的菌株。     

Agents of the "suis-ide diseases" of swine: Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis.

In recent years, Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis have emerged as important pathogens of swine, particularly in high health status herds. Their association with a wide range of serious clinical conditions and has given rise to the moniker "suis-ide diseases." These organisms are early colonizers and, for that reason, are difficult to control by management procedures such as segregated early weaning. Vaccination, serodiagnostic testing, and even serotyping are complicated by the presence of multiple serotypes, cross-reactive antigens, and the absence of clear markers for virulence. In this review, we discuss our current understanding of the pathogenesis, epidemiology, and management of the causative agents of the "suis-ide diseases" of swine.     

Serum-resistance in Haemophilus parasuis is associated with systemic disease in swine

Haemophilus parasuis can cause pneumonia and systemic disease in swine but it is also a coloniser of the upper respiratory tract of healthy pigs. These differences in pathogenicity are probably the result of diverse mechanisms of virulence in different strains. Since serum-resistance is a feature frequently found in systemic pathogens, 31 H. parasuis strains of different clinical origin were tested and a variety of serum susceptibility levels detected. Nasal strains from healthy piglets were sensitive to the bactericidal effect of the serum, while systemic strains were mainly resistant. The pulmonary strains included both serum-sensitive and serum-resistant strains. Interestingly, the serum-resistant pulmonary strains were isolated from animals with systemic lesions. Heat-treatment of the sera abolished the bactericidal activity, indicating that complement is a key factor in this effect. Equivalent susceptibility was observed with rabbit and porcine sera, and the presence of H. parasuis specific antibodies did not increase the killing of the strains by serum. In an attempt to associate serum-resistance to a surface determinant of the bacteria, agglutination in acriflavine was tested but no direct link with serum susceptibility was found. The results indicate that serum-resistance is a virulence mechanism in H. parasuis.     

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.     

副猪嗜血杆菌对部分常用实验动物的致病性试验

为筛选对副猪嗜血杆菌易感的实验动物,对部分常用的不同品种、不同品系实验动物进行了副猪嗜血杆菌致病性试验。用副猪嗜血杆菌血清型5型标准株（Strain Nagasaki）培养物,以2×10^8-2×10^10CFU的接种量,通过腹腔感染SPF级小鼠、大鼠、金黄地鼠、豚鼠以及清洁级豚鼠和兔。结果当接种量达到2×10^9CFU时,SPF豚鼠、清洁级豚鼠和SPF昆明小鼠出现死亡,在死亡豚鼠观察到典型的副猪嗜血杆菌病病理变化,而在死亡小鼠未见。其它各实验动物在上述剂量范围内均未发病或死亡,感染7d后剖检也未见任何异常。对死亡豚鼠和小鼠的各个组织器官进行副猪嗜血杆菌分离,结果在豚鼠的大脑、心血、肺、肝和腹水中分离到了副猪嗜血杆菌。提示豚鼠对副猪嗜血杆菌易感,可以做为建立副猪嗜血杆菌感染动物模型的候选实验动物。     

副猪嗜血杆菌病的诊断及其综合防治

近年来，笔者在辽宁、江西、湖南、河南、河北、天津等省(市)均发现一种以哺乳阶段、保育阶段、生长育肥阶段的猪群为发病主体，以咳嗽、呼吸困难、消瘦、关节肿胀、跛行为主要临床症状，以浆液-纤维素性多发性浆膜炎(胸膜炎、腹膜炎等)和关节炎为主要病变的疾病正在日趋流行，尤其是当猪群处在断奶、     

Testing meningeal strains of Streptococcus suis to detect M protein genes.

Previous reports have suggested that the surface proteins found in meningeal strains of Streptococcus suis might be similar to the M protein of group A streptococci. Fifty-five strains of S suis, including human and swine meningeal and pneumonic isolates, were tested for M protein genes by DNA probes representing the constant domain of the 3' end of the group A, M protein gene. None of the S suis strains examined was positive, indicating that these organisms either lack M protein genes or harbour different genes, not expressing the constant domains of protein M from group A.     

Prevalence of Streptococcus suis in four to eight week old clinically healthy piglets.

The study was undertaken to determine the prevalence, in the nasal cavities, of Streptococcus suis in four to eight week old clinically healthy piglets. Streptococci biochemically compatible with S. suis were isolated from 94% of piglets and 98% of farms. Of the 782 isolates submitted to serotyping, only 164 (21%) were included in the nine official serotypes. These 164 typable isolates originated from 121 (31%) of the 388 piglets and from 36 (73%) of the 49 farms included in the study. The most frequent serotypes found in piglets were, in decreasing order, 3, 4, 8 and 2. Serotype 1 was not detected in this survey. As many as 32% of piglets were found positive for two different serotypes and three different serotypes were found in 1%.     

Contribution to the experimental hemophilus infection (Haemophilus parahaemolyticus, Haemophilus parasuis) in SPF piglets. 2. Comparative pathology and histology

The pathologico-anatomic and even more the histological pattern of experimental Haemophilus parahaemolyticus pleuropneumonia in specific-pathogen-free piglets was found to be characteristic in the peracute, acute, and subacute-chronic stages of the disease. It is an infection which can be morphologically differentiated from other forms of pneumonia in swine primarily by three factors, localisation of the changes, inflammation, and a typical cell response. Changes following experimental Haemophilus parasuis infection were found to occur irregularly on serous skins and, in continuation, in the lung and joints. These were not characteristic and failed to exhibit morphological peculiarities.     

副猪嗜血杆菌的分离鉴定及药敏试验

从病猪中分离到4株细菌,经染色镜检、生化试验以及PCR等方法鉴定为副猪嗜血杆菌,。通过药敏试验确定其对药物的敏感性,以期对临床用药有一定的指导作用。     

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.     

副猪嗜血杆菌流行优势菌株调查和耐药性分析

为调查研究河南规模化猪场副猪嗜血杆菌(HPS)流行优势菌株及其耐药性情况,2017-2018年从河南规模化猪场分离到30株HPS,根据菌株分离部位统计,肺脏、气管是HPS分离的首要组织部位,肺脏分离14株,占46.67%;气管分离11株,占36.67%。通过PCR分子血清型鉴定,河南流行的优势菌株为血清5,7,4型,其中血清型5型有9株,占30%;血清型7型有5株,占16.67%;血清型4型有4株,占13.33%。通过K-B纸片琼脂法药敏试验,30株分离菌株除对头孢噻呋100%敏感外,对其他17种常用抗生素都有不同程度的耐药现象,多重耐药现象突出,预防控制HPS病选药、用药方面更需慎重和规范。     

Biofilm formation by field isolates and reference strains of Haemophilus parasuis

The ability to form biofilms for a total of 80 field isolates and 15 reference strains of Haemophilus parasuis, the etiological agent of Glasser's disease, was tested by glass tube and polystyrene microtiter plate assays. A total 43% of field isolates, including strains representing 13 serovars (except serovars 3 and 8) and non-typable strains, exhibited the ability to form biofilms at different levels via polystyrene microtiter plate assays. Among the reference strains representing 15 serovars, only serovars 2, 9, 12, 13 and 15 could not form biofilms on the polystyrene surface. A total of 85% of the strains forming biofilms at air-liquid interfaces in glass tubes also formed biofilms on polystyrene surfaces. Generally, non-virulent serovars showed a higher degree of biofilm formation than virulent serovars. The biofilm formation phenotype of most strains was maintained when cultures were passaged on agar and in broth. H. parasuis from the nasal cavities of pigs experimentally infected with biofilm-positive bacteria maintained the biofilm formation phenotype, whereas bacteria recovered from the lung and brain lost the ability to form biofilms. The biofilm-negative strains did not recover the ability to form biofilms via experimental infection. Our data indicate that most serovars of H. parasuis could form biofilms in vitro, and the biofilm formation phenotype is associated with the recovery site of the strains and is maintained when bacteria are passaged in vitro and in the upper respiratory tract.     

副猪嗜血杆菌感染豚鼠试验及在其体内的分布

为了评估副猪嗜血杆菌(Haemophilus parasuis)对豚鼠的毒力以及在豚鼠组织器官的分布,作者采用改进的寇氏法测定了H.parasuis血清5型标准株对豚鼠的LD50,以1 LD50剂量经鼻腔、气管、肺、腹腔、肌肉和皮下接种感染豚鼠,比较不同途径感染H.parasuis对豚鼠致病性的差异;以1 LD50剂量气管接种豚鼠,接种后在不同时间取豚鼠的脑、心、肝、肺、脾和肾组织,制作石蜡切片,用免疫组化技术检测H.parasuis在豚鼠上述组织的分布.结果显示该H.parasuis血清5型标准株的LD50为1.33×109 CFU,LD50的95%可信限范围为1.35×109～1.30×109 CFU;感染试验结果显示,气管、肺和腹腔3个接种途径试验组豚鼠出现了4/8、2/8和5/8的死亡;在气管感染豚鼠6 h后,首先在肺脏组织呈现阳性结果,在感染8 h后,上述组织中都出现了阳性结果.结果表明气管、肺、腹腔和肌肉4个途径都可以使H.parasuis对豚鼠致病,免疫组化检测并结合细菌分离结果表明H.parasuis感染后能侵染肺、心、肝、脾和肾组织,并能突破血一脑屏障而侵染脑组织.     

Colonization of suckling pigs by Streptococcus suis with particular reference to pathogenic serotype 2 strains.

Three swine commercial farms with high mortality rates in nursery pigs due to Streptococcus suis serotype 2 were studied. Brain samples from diseased animals were collected for a period of 6 to 10 mo and used to isolate the strain that was responsible for the mortality (virulent strain) in each farm. Tonsil swabs from piglets at 5, 10 and 15 d were taken to assess both total colonization and colonization by the virulent strain. The effect of sow vaccination against S. suis on colonization was evaluated in 1 of the farms. All suspect tonsil isolates were identified biochemically and then tested against serotype 2. The genomic patterns of serotype 2 isolates were compared to that of the virulent strain using Rep-PCR. Results showed that total colonization by S. suis occurred very early in the pigs' life, with most animals being colonized by weaning age. Prevalence of colonization by serotype 2 strains was much lower than total colonization. After comparing serotype 2 isolates with the virulent strains, only 1 tonsillar isolate had the same genomic pattern as the virulent strain and it belonged to a 4-week-old weaned pig. The genomic pattern of the virulent strain was not found in any tonsillar isolate from 15-day-old or younger pigs. Although limited by sample size, sow vaccination against S. suis increased total colonization at the same time significantly decreasing colonization by serotype 2 strains. Even though most pigs are colonized early in age by S. suis, colonization by the virulent strain is of low prevalence and delayed in time. This could constitute a risk factor for developing the disease later in time, because animals would be colonized when maternal immunity is no longer present, allowing the organism to become systemic.     

Genomic relatedness among reference strains of different Streptococcus suis serotypes.

Hybridization studies using genomic DNA and a rDNA probe revealed genetic relatedness among reference strains of different Streptococcus suis serotypes. Although most serotype 22 isolates are biochemically atypical, the reference strain of capsular type 22 is genetically related to other S. suis serotypes, but not to Streptococcus pneumoniae. Using DNA digested with BamHI and BglII for ribotyping, some S. suis reference strains had common patterns, but this analysis mainly revealed variations in patterns of S. suis strains of different serotypes.     

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.