Experimental enteric infection of gnotobiotic piglets with Chlamydia suis strain S45

https://doi.org/10.1016/j.vetmic.2008.09.038Get rights and content

Abstract

Enteric chlamydial infections of pigs with Chlamydia (C.) suis are frequent and often subclinical. The enteric pathogenicity of C. suis strain S45 was investigated in gnotobiotic piglets. Piglets from three litters (n = 31) were inoculated with egg-grown chlamydiae at 2–3 days of age (n = 17) or used as controls (n = 14). They were observed for clinical signs, killed and necropsied sequentially at 2–13 days postinoculation (DPI). Feces were collected daily and investigated with an ELISA for chlamydial antigen. At necropsy, specimens were collected for histopathology and for immunohistochemical, PCR-based, and serological (complement fixation test, ELISA) detection of chlamydiae.

Chlamydial replication and associated symptoms and lesions were observed from 2 to 13 DPI and were particularly pronounced within the first week PI. Clinical symptoms consisted of moderate-to-severe diarrhea, slight and transient anorexia, weakness and body weight loss. Immunohistochemistry and ELISA revealed that chlamydial replication was particularly marked at 2–4 DPI and primarily located in the small intestinal villus enterocytes. Further sites of replication included large intestinal enterocytes, the lamina propria and Tunica submucosa, and the mesenteric lymphnodes. Histopathological changes included moderate-to-severe villus atrophy with flattened enterocytes and focal villus tip erosions, and moderate mucosal inflammatory cell infiltrates and lymphangitis in the small intestine. PCR of spleen tissue and blood was mostly negative for chlamydiae, indicating that they did not substantially disseminate into the host up to 13 DPI. All sera were negative for anti-chlamydial antibodies. In conclusion, C. suis strain S45 elicited significant enteric disease and lesions in gnotobiotic piglets indicating its pathogenic potential for swine.

Introduction

Chlamydiae are obligate intracellular pathogens with a broad host spectrum and they have been isolated from a great variety of organs and diseases in swine (Szeredi et al., 1996). Chlamydial species detected in natural infections in swine include Chlamydophila (Cp.) abortus, Cp. pecorum and a recently proposed species, Chlamydia (C.) suis, which consists of porcine strains phylogenetically related to human C. trachomatis (Kaltenboeck et al., 1993, Everett et al., 1999). In previous immunohistochemical and molecular biological studies in Switzerland, involvement of C. suis was reported in the vast majority of chlamydial intestinal infections in swine, while Cp. abortus was rarely detected (Zahn et al., 1995, Szeredi et al., 1996, Schiller et al., 1997a). A study in Germany reported a high prevalence of mixed infections with C. suis and Cp. abortus in the lung and gut of pigs (Hoelzle et al., 2000). Another investigation revealed that C. suis was the most prevalent chlamydial species in intestinal samples from boars, while Cp. abortus was less prevalent and Cp. pecorum occurred only sporadically (Kauffold et al., 2006).

Prevalences of enteric chlamydial infections ranging from 15% in suckling pigs to 30% in finishing pigs have been reported (Zahn et al., 1995, Szeredi et al., 1996, Nietfeld et al., 1997). Nearly the same prevalence of diarrhea was found in infected and non-infected animals, suggesting that chlamydiae do not always cause diarrhea (Zahn et al., 1995, Nietfeld et al., 1997). However, in some cases no other enteropathogenic organisms were detected (Nietfeld et al., 1993, Nietfeld et al., 1997). Variations in virulence between strains, in particular between C. suis strains, cannot be excluded as a reason for this incongruence, especially considering the relatively high degree of genetic diversity observed in this chlamydial species when compared to other chlamydial species (Everett et al., 1999, Bush and Everett, 2001). A synergistic effect of chlamydiae with other enteropathogenic organisms has been suggested (Pospischil and Wood, 1987), and can be regarded as a further possible explanation for the inconsistent association of enteric chlamydiae with diarrhea.

Experimental infections have so far indicated that two porcine isolates classified as C. suis (Everett and Andersen, 1997), originating from diarrheic pigs, were enteropathogenic for gnotobiotic piglets (Rogers and Andersen, 1996). In contrast, a Cp. abortus strain was virtually apathogenic for the gastrointestinal tract of gnotobiotic piglets (Guscetti et al., 1998), while a Cp. psittaci isolate of avian origin elicited only mild intestinal lesions and symptoms (Guscetti et al., 2000). The aim of this study was to investigate enteric pathogenicity and dynamics of replication of the porcine C. suis strain S45 in gnotobiotic piglets. This isolate was harvested in the late 1960s from feces of an asymptomatic pig in Austria (Koelbl, 1969) and has been designated as the C. suis type strain (Everett et al., 1999). C. suis strains previously used in experimental infections of gnotobiotic piglets have been shown to significantly differ from strain S45 in the intergenic spacer region, the 23S and the 16S rRNA gene regions (Everett and Andersen, 1997, Everett et al., 1999). Moreover, they apparently belong to different serovars (Everett and Andersen, 1997). In the present investigation, pathogenicity was assessed based on clinical and histological observations, while the dynamics of chlamydial replication was studied by monitoring chlamydial antigen excretion in the feces using an ELISA, and by immunohistochemically labeling chlamydial antigen in the tissues of the experimental animals.

Section snippets

Inocula

The inocula for the piglets were prepared from a lyophilized preparation of the porcine C. suis S45 strain (kindly donated by Prof. J. Storz, Baton Rouge, Louisiana). The isolate had been passaged in chicken eggs between 25 and 35 times. The lyophilisate was resuspended in sucrose–phosphate–glutamate (SPG) medium and propagated in the yolk sac of embryonated specific-pathogen-free chicken eggs as previously described (Guscetti et al., 1998). All procedures for harvesting the inocula,

Microbiological sterility monitoring

Group 1: Staphylococcus sp. was isolated from the gut of the principal piglet killed at 5 DPI. Group 2: Lactobacillus sp. was isolated from the gut of all principals and controls. Group 3: Bacillus sp. was isolated from the gut of the principals killed at 12 and 13 DPI. Staphylococcus epidermidis was isolated from the gut of the control piglet killed at 9 DPI. No contaminant bacterial or fungal microorganisms were isolated from the intestines of any of the remaining animals.

Clinical and necropsy findings

All inoculated

Discussion

The data presented in this study indicate that the porcine C. suis strain S45, although originating from a pig without clinical disease, is enteropathogenic for gnotobiotic piglets. In a previous study, diarrhea and similar histopathologic lesions had been reported upon inoculation of gnotobiotic piglets with two further C. suis strains isolated from pigs with diarrhea (Rogers and Andersen, 1996, Everett and Andersen, 1997). In addition to confirming the findings of these previous experiments,

Conflict of interest statement

None of the authors has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the present paper.

Acknowledgements

The authors wish to thank Prof. J. Storz for kindly donating the initial S45 isolate, Prof. Dr. R.K. Hoop (Institute of Veterinary Bacteriology, Vetsuisse Faculty Zurich) for growing and harvesting the inoculum, and Dr. L. Corboz (Institute of Veterinary Bacteriology, Vetsuisse Faculty Zurich) for performing the sterility monitoring and the complement fixation test. The skilful technical assistance of the laboratory technical staff of the Institute of Veterinary Pathology is greatly

References (24)

  • L.E. Hoelzle et al.

    PCR-based detection of chlamydial infection in swine and subsequent PCR-coupled genotyping of chlamydial omp1-gene amplicons by DNA-hybridization, RFLP-analysis, and nucleotide sequence analysis

    Epidemiol. Infect.

    (2000)
  • B. Kaltenboeck et al.

    Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species

    J. Bacteriol.

    (1993)
  • Cited by (32)

    • Prevalence and molecular characterization of C. pecorum detected in Swiss fattening pigs

      2021, Veterinary Microbiology
      Citation Excerpt :

      Only few studies have so far investigated the correlation between clinical signs and the chlamydial burden. One study found that higher bacterial loads of C. suis (Ct-value < 36) were more likely to correlate with conjunctivitis than lower bacterial loads (Ct < 38) (Englund et al., 2012) and experimental studies demonstrated that high infectious doses had an impact on the severity of disease (Guscetti et al., 2009). However, there has been no in-depth study comparing the chlamydial loads of pigs from farms with Chlamydia-related problems such as conjunctivitis (Schautteet et al., 2010; Unterweger et al., 2020) with farms without (Hoffmann et al., 2015) in order to confirm the interplay between bacterial load and clinical signs.

    • Veterinary Medicine, Eleventh Edition

      2016, Veterinary Medicine, Eleventh Edition
    • Alimentary System

      2016, Jubb, Kennedy and Palmer's Pathology of Domestic Animals: Sixth Edition
    • Missing the Target to Treat in Crohn's Disease

      2015, Clinical Gastroenterology and Hepatology
    • Selection for tetracycline-resistant Chlamydia suis in treated pigs

      2012, Veterinary Microbiology
      Citation Excerpt :

      Chlamydiae are commonly found in the intestine of pigs mostly associated with subclinical infections (Nietfeld et al., 1997). However, C. suis caused enteritis when inoculated in gnotobiotic piglets (Rogers and Andersen, 1996; Guscetti et al., 2009) or caused histologically intestinal lesions in asymptomatic young weanling pigs (Rogers and Andersen, 2000). Tetracycline-resistant C. suis strains have been described in the US (Lenart et al., 2001) and in Italy (Di Francesco et al., 2008).

    View all citing articles on Scopus
    1

    Present address: Prionics AG, Wagistrasse 27A, CH-8952 Schlieren, Switzerland.

    View full text