广西、四川两地屠宰场猪链球菌致病特征分析

Pathogenic characteristics of Streptococcus suis isolated from slaughterhouses in Sichuan and Guangxi

摘要:

【目的】猪链球菌(Streptococcus suis)是猪的重要致病菌，同时也是一种人畜共患病原。屠宰场健康猪的猪链球菌感染率较高，血清型多而复杂，是人和猪的重要传染源。四川、广西两地都曾暴发猪链球菌疫情，大量生猪发病死亡的同时，也导致部分与病猪有密切接触的人群发病死亡。因此，对上述两地区屠宰场进行猪链球菌感染调查，明确其致病特征，具有重要公共卫生意义。【方法】本研究自2021-2022年采集广西、四川两地屠宰场健康猪扁桃体，分离鉴定猪链球菌并进行血清型分型，对分离株进行斑马鱼毒力、小鼠毒力试验，并对毒力株进行基因组测序，进行多序列位点分型(multilocus sequence typing, MLST)及毒力标志基因分析，探究其致病特征。【结果】广西57份健康猪扁桃体样品猪链球菌阳性率为84.21% (48/57)，分离鉴定出60株猪链球菌，其中分离率最高的是血清31型(16.67%, 10/60)，其次为9型(11.67%, 7/60)、4型(10.00%, 6/60)、12型(8.33%, 5/60)等，2型菌株仅分离出1株，含2种及以上不同血清型猪链球菌的扁桃体占33.33% (16/48)；四川250份健康猪扁桃体样品猪链球菌阳性率为10.8% (27/250)，分离鉴定出41株猪链球菌，其中分离率最高的血清型为31型(34.15%, 14/41)，其次为16型(17.07%, 7/41)、9型(12.20%, 5/41)、28型(9.76%, 4/41)等，2型菌株仅分离出1株，含有2种以上不同血清型的扁桃体占33.33% (9/27)。选择44株不同血清型的代表菌株(广西28株，四川16株)进行斑马鱼毒力试验，结果显示，5株菌株对斑马鱼致病性较强，攻毒计量在3×106 CFU/尾时，对斑马鱼的致死率高达70%-100%，其中广西3株分别为2型、9型和新荚膜基因簇(novel capsular polysaccharide loci, NCL)18型，四川2株分别为2型和9型。对上述5株毒力株进行小鼠毒力试验，结果显示4株菌株致死率高达80%-100%，1株致死率为60%，与斑马鱼试验较为相符。MLST及毒力标志基因分析表明，2株血清2型菌株均为ST1型及mrp+、epf+、sly+毒力型，为世界范围内广泛流行的具有人畜共患潜力的致病型菌株；2株血清9型菌株毒力型均为mrp-、epf-和sly-，ST型分别为ST1198和ST2104，1株NCL18型菌株为ST2102型和mrp+、epf-、sly-毒力型。【结论】上述结果表明，两地屠宰场猪链球菌的感染情况存在差异，广西屠宰场猪链球菌分离率较高，都存在世界范围内广泛流行的具有人畜共患潜能的血清2型ST1毒力株，公共卫生意义值得关注。

Abstract:

[Objective] Streptococcus suis is a zoonotic pathogen that can cause diseases in pigs and humans. Healthy pigs in slaughterhouses often carry diverse serotypes of S. suis, which can be a major source of infections for susceptible animals and humans. The outbreaks of S. suis occurred in Sichuan and Guangxi, resulting in severe illness and deaths of pigs and humans. Therefore, it is crucial to investigate the infection rate of S. suis in healthy pigs in slaughterhouses and unveil the pathogenic characteristics of S. suis in these two areas. [Methods] We isolated S. suis from the tonsils of healthy pigs collected from slaughterhouses in Guangxi and Sichuan during 2021–2022 and performed serotype-specific PCR for serotyping the isolates. Furthermore, we examined the virulence of the isolates in zebrafish and mice and performed genome sequencing and multilocus-sequence typing (MLST) on the virulent strains to explore their pathogenic characteristics. [Results] The positive rate of S. suis was 84.21% (48/57) in the 57 tonsil samples collected from Guangxi. Among the 60 strains isolated from Guangxi, serotype 31 (16.67%, 10/60) had the highest isolation rate, followed by serotype 9 (11.67%, 7/60), serotype 4 (10.00%, 6/60), and serotype 12 (8.33%, 5/60). The tonsil samples carrying at least two serotypes of strains accounted for 33.33% (16/48) in Guangxi. The positive rate of S. suis in 250 tonsil samples collected from Sichuan was 10.8% (27/250). Among the 41 strains isolated from Sichuan, serotype 31 (34.15%, 14/41) had the highest isolation rate, followed by serotype 16 (17.07%, 7/41), serotype 9 (12.20%, 5/41), and serotype 28 (9.76%, 4/41). The tonsil samples carrying at least 2 serotypes of strains accounted for 33.33% (9/27) in Sichuan. Zebrafish infection experiments with 28 strains from Guangxi and 16 strains from Sichuan showed that five strains were highly virulent, causing the mortality rates ranging from 70% to 100% in the zebrafish challenged with the dose of 3×10⁶ CFU/fish. The five strains included three strains from Guangxi (serotypes 2, 9 and NCL18) and two strains from Sichuan (serotypes 2 and 9). Furthermore, the 5 strains were used to infect mice, with four strains causing the mortality rates ranging from 80% to 100% and 1 strain causing the mortality rate of 60%, which were consistent with the results of the zebrafish infection experiments. MLST and virulence gene analysis revealed that the two strains of serotype 2 belonged to ST1 and exhibited the virulence genotypes mrp+, epf+, and sly+, showing the potential as zoonotic pathogens. The two strains of serotype 9 had the virulence genotypes mrp-, epf-, and sly- and belonged to ST1198 and ST2104, respectively. The NCL18 strain was typed as ST2102 and had the virulence genotypes mrp+, epf-, and sly-. [Conclusion] The S. suis infection rates differed between slaughterhouses in Guangxi and Sichuan, and the isolation rate was higher in Guangxi. The slaughterhouses in both Guangxi and Sichuan harbor serotype 2 ST1 strains with zoonotic potential that are widespread worldwide, warranting attention.