Cps7
Epicuro Kishore
Streptococcus (S.) suis is an important porcine pathogen causing meningitis, arthritis and septicemia. As cps7 emerged recently in Germany in association with severe herd problems, the objective of this study was to characterize the geno- and phenotype of invasive cps7 strains. Twenty cps7 strains were isolated from diseased pigs from different farms with S. suis herd problems due to meningitis and other pathologies. Eighteen of the cps7 isolates belonged to sequence type (ST) 29. Most of these cps7 strains secreted a short MRP variant in agreement with a premature stop codon. Expression of Ide Ssuis , an IgM specific protease, was variable in four further investigated cps7 ST29 isolates. Bactericidal assays revealed very high survival factors of these four cps7 ST29 strains in the blood of weaning piglets. In growing piglets, the increase of specific IgM led to efficient killing of cps7 ST29 as shown by addition of the IgM protease Ide Ssuis . Finally, virulence of a cps7 ST29 strain was confirmed in experimental infection of weaning piglets leading to meningitis and arthritis. In conclusion, this study characterizes cps7 ST29 as a distinct S. suis pathotype showing high survival factors in porcine blood after weaning, but IgM-mediated killing in the blood of older growing piglets. This underlines the relevance of IgM as an important host defense mechanism against S. suis.
For absorption of sera with S. suis 10cpsΔEF, 2 mL of overnight cultures were centrifuged. Pelleted bacteria were resuspended in 200 μL of serum and rotated for 30 min at 4 C. After centrifugation for 10 min at 10 000 g, 100 μL of the supernatant was used as absorbed serum in ELISA.
Bactericidal assays were conducted at the described time points of serum sampling and addition of rIde Ssuis was used as a tool to investigate the impact of IgM on bacterial survival. The four investigated S. suis cps7 ST29 strains, which were randomly selected from our collection of invasive cps7 ST29 strains, showed very high survival factors in the blood of 4.5 week old piglets with mean survival factors between 4 and 35, indicating proliferation of the streptococci. The strains were efficiently killed in the blood of the same piglets 2 weeks later (Figure 4B), at a time when the piglets had just undergone a pronounced increase of specific IgM (Figure 4A). Addition of rIde Ssuis , a highly specific protease cleaving porcine IgM but not IgG [20], to the bactericidal assays of the 6.5 week old piglets in amounts sufficient to lead to complete cleavage of IgM (Figure 4C), resulted in a pronounced increase in survival of the cps7 strains. This increase in bacterial survival through addition of rIde Ssuis was statistically significant in 2 of 4 investigated strains and also seen in 8.5 and 10.5 week old piglets, but the effect was smaller in these older piglets. In conclusion, S. suis cps7 ST29 showed high proliferation in the blood of weaning piglets with low specific IgM titers but was restricted very much in survival as IgM increased in these piglets.
Next, we investigated the survival pattern of S. suis cps7 ST29 strains in the blood of piglets from a herd known to be free of S. suis cps7 and cps9 strains to find out if the described IgM-mediated killing observed in growing piglets is related to cps7 S. suis infection and not induced by infection with other serotypes. As shown in Figure 5A, survival of S. suis cps7 also decreased as weaning spf piglets became older. In this case, killing of the bacteria, which is indicated by survival factors smaller 1, set in at 8.5 weeks of age, 2 weeks later compared to piglets from the infected herd. In contrast to cps7, we observed only marginal changes in bacterial survival of a cps9 strain in the blood drawn from the same spf weaning and growing piglets as these animals grew older. In contrast to cps7 ST29, the bacterial survival factor of the cps9 strain was above 1 in the blood of 8.5 week old piglets.
Previous research on cps distribution of S. suis revealed the highest prevalences for cps9 and cps2, followed by other cps types such as cps1 and cps7 in Europe [12, 19]. Invasive cps2 strains mainly belong to ST1 but might also occur in ST25 and 28. ST16 is also important in Europe and associated with invasive isolates, mainly belonging to cps9 [6, 11,12,13, 30]. Recently, cps7 strains were detected in a number of cases with severe herd problems in Germany. All but two of our investigated invasive cps7 strains belong to ST29 suggesting that ST29 cps7 strains are emerging in Europe. Schultsz et al. describe a clonal complex 25 with ST29 as secondary founder based on the analysis of isolates obtained in the Netherlands [11], whilst ST29 is the primary founder of clonal complex 29 in the recent analysis of isolates from around the world conducted by Goyette-Desjardins et al. [6]. Cps7 is dominating in this clonal complex [6], which is supported by the MLST database and our results.
Importantly, survival of S. suis cps7 ST29 strains in porcine blood showed an opposing trend to this increase of IgM. Though specific IgG also increased from week 4 to 10 (Figure 3), the sharp decline in bacterial survival from 4.5 to 6.5 weeks of age was mainly IgM-mediated as addition of Ide Ssuis to the blood of 6.5-week-old piglets resulted in survival factors comparable to the ones in blood of 4.5 week old piglets and Ide Ssuis is known to cleave only IgM and not IgG [20]. The increase in bacterial survival through addition of Ide Ssuis was lessened at an age of 8.5 and 10.5 weeks, probably due to further increasing, most likely opsonizing, specific IgG.
Ide Ssuis has been described to be expressed by all investigated S. suis strains [20]. One cps7 strain (V2310/1) investigated here expressed comparatively small amounts of Ide Ssuis , not sufficient to allow detection of IgM cleavage products. Our results indicated that the expression of Ide Ssuis by S. suis, at least in the case of cps7 ST29, is not sufficient to evade IgM-mediated killing in porcine blood with high IgM titers. However, S. suis cps9 did not show this trend as survival factors were similar at 8.5 and 4.5 weeks of age. Our results suggested that S. suis cps7 ST29 infections but not cps9 infections should occur more often in young weaning piglets up to 6/7 weeks rather than in older growing piglets. Years ago, this was also observed in Denmark where field isolates of cps7 were mostly isolated from piglets under 3 weeks of age [35]. Wisselink et al. observed no age-related difference in susceptibility to S. suis serotypes 2, 7 and 9 [12]. However, serotype 1 strains were mostly isolated from 3-week-old piglets, indicating that there are differences between serotypes regarding susceptibility at different age classes. Unfortunately, the age of the piglets the investigated cps7 strains were originally isolated from, is not well documented. Future epidemiological studies are needed to document the prevalences of cps7 infections at different age classes.
KR designed and conducted experiments. Furthermore, KR analysed the data and drafted the manuscript. AS, VR and KK conducted experiments. The experimental infection and necropsies were performed by KR, AS and CGB. KS did the histopathological screenings. CGB conceived the study and designed experiments. All authors read and approved the final manuscript.
All animal experiments or sampling were conducted by veterinarians and in accordance with the principles outlined in the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and the German Animal Protection Law (Tierschutzgesetz). The animal experiment of this study was approved by the Landesdirektion Sachsen (Permit No. TVV26/15), which includes approval through the registered committee for animal experiments. The collection of blood samples was approved by the Landesdirektion Sachsen (Permit Nos. N01/16 and N19/14, respectively).
Titers of serum IgM antibodies binding to the unencapsulated strain 10cpsΔEF increase in the five investigated cps7 free piglets from 4.5 to 6.5 weeks of age. The unencapsulated mutant 10cpsΔEF of cps2 strain 10 was used as antigen in an ELISA for determination of IgM antibodies binding to other S. suis antigens but capsular polysaccharides in the 5 piglets investigated for the data presented in Figure 5, which were from a herd considered free of cps7 and cps9. Means and standard deviations are indicated by horizontal lines and error bars, respectively. Each symbol represents a different animal. Differences were not significant using a two-tailed paired t-test.
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Streptococcus (S.) suis is an important porcine pathogen causing meningitis, arthritis and septicemia. As cps7 emerged recently in Germany in association with severe herd problems, the objective of this study was to characterize the geno- and phenotype of invasive cps7 strains. Twenty cps7 strains were isolated from diseased pigs from different farms with S. suis herd problems due to meningitis and other pathologies. Eighteen of the cps7 isolates belonged to sequence type (ST) 29. Most of these cps7 strains secreted a short MRP variant in agreement with a premature stop codon. Expression of Ide Ssuis , an IgM specific protease, was variable in four further investigated cps7 ST29 isolates. Bactericidal assays revealed very high survival factors of these four cps7 ST29 strains in the blood of weaning piglets. In growing piglets, the increase of specific IgM led to efficient killing of cps7 ST29 as shown by addition of the IgM protease Ide Ssuis . Finally, virulence of a cps7 ST29 strain was confirmed in experimental infection of weaning piglets leading to meningitis and arthritis. In conclusion, this study characterizes cps7 ST29 as a distinct S. suis pathotype showing high survival factors in porcine blood after weaning, but IgM-mediated killing in the blood of older growing piglets. This underlines the relevance of IgM as an important host defense mechanism against S. suis.
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