A critical review on experimental Streptococcus suis infection in pigs with a focus on clinical monitoring and refinement strategies.

Streptococcus suis (S. suis) is a major pig pathogen worldwide with zoonotic potential. Though different research groups have contributed to a better understanding of the pathogenesis of S. suis infections in recent years, there are still numerous neglected research topics requiring animal infection trials. Of note, animal experiments are crucial to develop a cross-protective vaccine which is highly needed in the field. Due to the severe clinical signs associated with S. suis pathologies such as meningitis and arthritis, implementation of refinement is very important to reduce pain and distress of experimentally infected pigs. This review highlights the great diversity of clinical signs and courses of disease after experimental S. suis pig infections. We review clinical read out parameters and refinement strategies in experimental S. suis pig infections published between 2000 and 2021. Currently, substantial differences exist in describing clinical monitoring and humane endpoints. Most of the reviewed studies set the body temperature threshold of fever as high as 40.5°C. Monitoring intervals vary mainly between daily, twice a day and three times a day. Only a few studies apply scoring systems. Published scoring systems are inconsistent in their inclusion of parameters such as body temperature, feeding behavior, and respiratory signs. Locomotion and central nervous system signs are more common clinical scoring parameters in different studies by various research groups. As the heterogenicity in clinical monitoring limits the comparability between studies we hope to initiate a discussion with this review leading to an agreement on clinical read out parameters and monitoring intervals among S. suis research groups.

A new S. suis serotype 3 infection model in pigs: lack of effect of buprenorphine treatment to reduce distress.

BACKGROUND: Streptoccocus suis (S. suis) is a major porcine pathogen causing meningitis, septicemia, arthritis and endocarditis. These diseases severely impair welfare of pigs. Experimental studies in pigs are important to better understand the pathogenesis and to identify protective antigens, as so far there is no vaccine available protecting against various serotypes (cps). Due to the severity of disease, application of appropriate refinement strategies in experimental S. suis infections is essential to reduce distress imposed on the piglets without jeopardizing the scientific output. The objectives of this study were to evaluate buprenorphine treatment as a refinement measure and serum cortisol levels as a distress read out parameter in a new S. suis cps3 infection model in pigs. RESULTS: Intravenous application of 2 × 108 CFU of S. suis cps3 (sly+, mrp+) to 6-week-old piglets led to severe morbidity in approximately 50% of the animals. Main pathological findings included suppurative meningoencephalitis and arthritis as well as fibrinosuppurative endocarditis. Buprenorphine treatment (0.05 mg/kg every 8 h) did not prevent signs of severe pain, high clinical scores, moderate to severe pathologies or high levels of serum cortisol in single severely affected piglets. Significant differences in the course of leukocytosis, induction of specific antibodies and bactericidal immunity were not recorded between groups with or w/o buprenorphine treatment. Of note, clinically unobtrusive piglets showed serum cortisol levels at 2 and 5 days post infectionem (dpi) comparable to the levels prior to infection with cps3. Cortisol levels in serum were significantly increased in piglets euthanized due to severe disease in comparison to clinically unobtrusive pigs. CONCLUSIONS: Different clinical courses and pathologies are induced after intravenous challenge of piglets with 2 × 108 CFU of this S. suis cps3 strain. The chosen protocol of buprenorphine application does not prevent severe distress in this infection model. Important parameters of the humoral immune response, such as the level of IgM binding to S. suis cps3, do not appear to be affected by buprenorphine treatment. Serum cortisol is a meaningful parameter to measure distress in piglets experimentally infected with S. suis and to evaluate refinement strategies. In this intravenous model, which includes close clinical monitoring and different humane endpoints, clinics and cortisol levels suggest convalescence in surviving piglets within 5 days following experimental infection.

Streptococcus suis cps7: an emerging virulent sequence type (ST29) shows a distinct, IgM-determined pattern of bacterial survival in blood of piglets during the early adaptive immune response after weaning.

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.

Porcine iucA+ but rmpA- Klebsiella pneumoniae strains proliferate in blood of young piglets but are killed by IgM and complement dependent opsonophagocytosis when these piglets get older.

Klebsiella (K.) pneumoniae causes different diseases in humans and animals including the life-threatening liver abscess syndrome and septicemia, respectively. However, host-pathogen interactions of K. pneumoniae in porcine blood have not been studied. We investigated the working hypothesis that only distinct K. pneumoniae strains have the capacity to survive in porcine blood and that this feature is associated with specific molecular markers such as sequence type, profile of siderophore genes and the regulator of the mucoid phenotype (rmp). Furthermore, we characterize the immune response in growing piglets leading to killing of an invasive K. pneumoniae strain. The veterinary isolates showed great diversity in sequence types and profile of siderophore genes. Porcine isolates were mainly positive for the aerobactin gene iucA but did not carry rmpA and this genotype was associated with proliferation in blood of 4-week-old piglets. Supernatants of an iucA+ but not an iucA- strain boosted growth in porcine serum. Between four and eight weeks of age, piglets showed a prominent increase of IgM binding to K. pneumoniae. Immunglobulin M and complement were crucial for killing of a serum-resistant iucA+ porcine K. pneumoniae strain at eight weeks of age. Flow cytometry analysis confirmed induction of phagocytosis and oxidative burst mediated by serum samples of 8-week-old piglets. Based on our in vitro findings we propose that many porcine iucA+ rmpA- K. pneumoniae strains have the ability to cause bacteremia in young piglets in association with aerobactin-mediated iron acquisition and that this phenotype is lost as specific IgM increases after weaning.

Proline-rich antimicrobial peptide Api137 is bactericidal in porcine blood infected ex vivo with a porcine or human Klebsiella pneumoniae strain.

OBJECTIVES: Klebsiella pneumoniae is an emerging invasive pathogen in humans and pigs. Resistance against multiple antibiotics in this species is a major health concern and the development of new antibiotics is urgently needed. The objective of this study was to investigate the effects of proline-rich antimicrobial peptides (PrAMPs) on the survival of K. pneumoniae strains in porcine blood. METHODS: We established a bactericidal assay with K. pneumoniae in fresh blood drawn from 4-week-old piglets. PrAMPs, namely the apidaecins Api137 and Api802 as well as the oncocin Onc112, were added to ex vivo-infected whole blood samples in order to study their bactericidal effects and, in the case of Api137, also immune responses. RESULTS: A porcine invasive and a human iucA+rmpA+ K. pneumoniae strain showed prominent proliferation in porcine blood. Application of Api137 resulted in a dose-dependent prominent bactericidal effect killing the invasive porcine K. pneumoniae strain. Addition of 8 µg/mL Api137 also resulted in complete killing of the human iucA+rmpA+ strain. Cytotoxicity, haemolysis and induction of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNFα) in K. pneumoniae-infected porcine blood treated with Api137 was comparable with values obtained after application of 10 µg/mL cefquinome. CONCLUSION: We describe a new non-rodent model for invasive K. pneumoniae bacteraemia and present promising data for the PrAMP Api137 for the control of infection with hypervirulent K. pneumoniae strains.

A critical review speculating on the protective efficacies of autogenous Streptococcus suis bacterins as used in Europe.

BACKGROUND: Streptococcus (S.) suis is a major porcine pathogen causing high morbidity worldwide. This includes well-managed herds with high hygiene standards. In Europe, no licensed vaccine is available. As practitioners are obliged to reduce the use of antibiotics, autogenous S. suis vaccines have become very popular in Europe. MAIN BODY: Autogenous vaccines (AV) are generally neither tested for safety, immunogenicity nor protective efficacy, which leads to substantial uncertainties regarding control of disease and return on investment. Here, S. suis publications are reviewed that include important data on epidemiology, pathologies and bacterin vaccination relevant for the use of AV in the field. Differences between herds such as the porcine reproductive and respiratory syndrome virus infection status and the impact of specific S. suis pathotypes are probably highly relevant for the outcome of immunoprophylaxis using autogenous S. suis bacterins. Thus, a profound diagnosis of the herd status is crucial for management of expectations and successful implementation of AV as a tool to control S. suis disease. Induction of opsonizing antibodies is an in vitro correlate of protective immunity elicited by S. suis bacterins. However, opsonophagocytosis assays are difficult to include in the portfolio of diagnostic services. CONCLUSION: Autogenous S. suis bacterins are associated with limitations and risks of failure, which can partly be managed through improvement of diagnostics.

Vaccination with the immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis, leads to protection against a highly virulent serotype 9 strain.

Vaccination of weaning piglets with the recombinant IgM degrading enzyme of Streptococcus suis (S. suis), rIde Ssuis, elicits protection against disease caused by serotype (cps) 2 infection. In Europe, S. suis cps9 is at least as important as cps2 in causing severe herd problems associated with meningitis, septicemia and arthritis. The objective of this study was to determine humoral and cellular immunogenicities of rIde Ssuis suckling piglet vaccination and to investigate protection against a virulent cps9 strain. Vaccination in the 2nd and 4th week of life with rIde Ssuis and an oil-in-water adjuvant induced seroconversion against Ide Ssuis in 13 of 20 vaccinated piglets. In the 5th week, survival of the S. suis cps9 strain was significantly reduced in the blood of prime-booster vaccinated piglets. After a 2nd booster vaccination Ide Ssuis -reactive T helper (Th) cells partially producing TNF-α, IL-17A or IFN-É£ were detectable in rIde Ssuis -vaccinated but not in placebo-treated piglets and frequencies of Ide Ssuis -reactive Th cells correlated with α-Ide Ssuis-IgG levels. An intravenous challenge, conducted with a cps9 strain of sequence type (ST) 94, led to 89% mortality in placebo-treated piglets due to septicemia and meningitis. In contrast, all rIde Ssuis prime-booster-booster vaccinated littermates survived the challenge despite signs of disease such as fever and lameness. In conclusion, the described rIde Ssuis vaccination induces humoral and detectable Ide Ssuis -reactive Th cell responses and leads to protection against a highly virulent cps9 strain.

Streptococcus suis serotype 9 endocarditis and subsequent severe meningitis in a growing pig despite specific bactericidal humoral immunity.

INTRODUCTION: . Meningitis and endocarditis are common pathologies of Streptococcussuis infections in pigs and humans. S. suis serotype 9 strains contribute substantially to health problems in European pig production, and immune prophylaxis against this serotype is very difficult. CASE PRESENTATION: . We report the clinical course and histopathological picture of a 10-week-old growing pig following experimental intravenous infection with S. suis serotype 9. The piglet showed rapid onset of severe clinical signs of meningitis 11 days post-intravenous challenge following prime-booster vaccination. Histopathological findings revealed a diffuse fibrinosuppurative leptomeningitis. Additionally, a polyphasic endocarditis valvularis thromboticans with numerous bacterial colonies was diagnosed. Bacteriological culture of the brain and the mitral valve confirmed association with the challenge strain. However, virulent serotype 2 and 9 strains were killed in the blood of this piglet ex vivo prior experimental infection. CONCLUSION: . Endocarditis induced by S. suis infection might develop and persist despite the presence of high specific bactericidal activity in the blood. Severe leptomeningitis is a putative sequela of such an endocarditis.