Host Response to Porcine Strains of Escherichia coli in a Novel Pyelonephritis Model

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Summary

The initial pathology and pathogenesis of pyelonephritis and the influence of different strains of Escherichia coli were investigated in a novel porcine model. Nine female pigs were divided into three groups (A, B and C) and inoculated repeatedly into one renal pelvis with porcine pyelonephritis E. coli strain LK67 (P fimbriae PapGI), LK76 (type 1 fimbriae) or LK82 (type 1 fimbriae and P fimbriae PapGII/III), respectively. The contralateral kidneys were inoculated with saline and served as controls. Pigs were killed 6 h post-inoculation (hpi). Differential leucocyte counts, serum biochemical analyses and measurement of serum concentrations of proinflammatory cytokines and acute phase proteins were carried out at 0, 3 and 6 hpi. Bacteriological evaluation of urine, kidneys, spleen, liver, abdominal swabs and blood samples and gross and histopathological evaluation of kidneys, renal lymph nodes, liver and spleen were performed by quantitative, semiquantitative and/or descriptive methods. Immunohistochemistry was used to identify cells expressing L1 antigen, CD3ɛ, CD4, CD8, CD79αcy and lysozyme, and to identify E. coli and Tamm-Horsfall protein (THP). E. coli was re-isolated from all inoculated kidneys. Gross and microscopical lesions of acute pyelonephritis were demonstrated in all but one kidney inoculated with E. coli, but in none of the control kidneys. Renal parenchymal infiltration with both neutrophils and mononuclear cells, primarily CD3+ T lymphocytes, was observed at 6 hpi. Most T lymphocytes were CD8+. Pigs in group C had the highest mean pathology scores. Neutrophils were the dominant renal leucocyte in this group, while the number of mononuclear cells was at least equal to the number of neutrophils in the lesions of pigs from groups A and B. Kidneys with a high number of E. coli had severe lesions. Systemic spread of E. coli was observed in five pigs. THP was observed interstitially in 89% of the E. coli-inoculated kidneys. In all groups, increased numbers of neutrophils and decreased numbers of lymphocytes and monocytes were shown by differential leucocyte count at 6 hpi, and from 3 to 6 hpi there was a significant increase in C-reactive protein concentration.

Introduction

Pyelonephritis, an ascending bacterial infection of the kidney, is a common reason for sows to be culled and a cause of reduced animal welfare and considerable economic loss due to morbidity and mortality (Carr, 1990, D’Allaire et al., 1991, Wanyoike and Bilkei, 2006). Escherichia coli and Actinobaculum suis are two of the most commonly isolated bacteria from cases of pyelonephritis in sows. As well as being isolated from cases of pyelonephritis, E. coli has, in contrast to A. suis, also frequently been isolated from sows with asymptomatic bacteriuria, and in cases of pyelonephritis other pathogenic bacteria have often been found in co-infection with E. coli (D’Allaire et al., 1991, Carr and Walton, 1993, Almanjd and Bilkei, 2008). Consequently, the influence of E. coli in porcine pyelonephritis requires further investigation.

In man, the ability of uropathogenic E. coli (UPEC) to cause symptomatic urinary tract infection (UTI) is associated with the expression of a variety of virulence factors such as adhesins. Two important fimbrial adhesins associated with human UPEC strains are type 1 fimbriae, which are mainly associated with cystitis, and P fimbriae that are primarily linked to human pyelonephritic strains (Domingue et al., 1985, Wu et al., 1996, Mulvey, 2002). As studies concerning the expression and influence of E. coli virulence factors in pigs with pyelonephritis are sparse (Carr and Walton, 1993, de Brito et al., 1999, Krag et al., 2009), the influence of P fimbriae and type 1 fimbriae in the pathogenesis of pyelonephritis in pigs is yet to be elucidated.

Most previous studies of pyelonephritis in pigs focus on bacteriological findings and do not describe histological lesions in detail (D’Allaire et al., 1991, Carr and Walton, 1993). In addition, existing investigations using an established porcine model of reflux pyelonephritis concentrate on the influence of human E. coli strains and their virulence factors on the formation of scar tissue (Torres et al., 1985, Arnold et al., 1993). Consequently, mainly chronic pyelonephritis lesions have been described previously and the local and systemic acute inflammatory response is yet to be clarified. Additionally, the role of extratubular Tamm-Horsfall protein (THP) should be investigated, as this has previously been a matter of dispute (Ransley and Risdon, 1981, Mayrer et al., 1983, Serafini-Cessi et al., 2003).

The aim of the present study was to investigate the initial pathology and pathogenesis of porcine pyelonephritis and the influence of E. coli strains expressing different virulence factors. This was accomplished by studying the host response to repeated intrapelvic inoculation of three different strains of E. coli associated with porcine pyelonephritis. This novel porcine model is better suited to investigation of the acute inflammatory response.

Section snippets

Animals

Nine female specific pathogen-free, Danish landrace/large white crossbred pigs (Erik Mølbak, Denmark) with a mean body weight of 19 kg were randomly allocated into three groups (A, B and C) of three pigs each (AI–III, BI–III and CI–III). The pigs were treated in accordance with the Danish Animal Experimentation Act, which is in accordance with the Council of Europe Convention ETS 123. The National Animal Experimentation Board licensed the study.

Escherichia coli Strains used for Inoculation

The three E. coli strains used (LK67, LK76 and

Differential Leucocyte Counts and Serum Biochemistry

Fig. 2 shows the results of the differential leucocyte counts. At 6 hpi the number of neutrophils was above, and the number of monocytes below, the reference intervals in all groups. Although the number of lymphocytes decreased from 0 to 6 hpi, the number at 6 hpi was still within the reference interval in all groups. Creatinine, carbamide, globulin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, bilirubin, serum protein, albumin,

Discussion

A porcine model of chronic pyelonephritis was described by Ransley and Risdon (1981) in which vesicoureteral reflux was induced surgically and, subsequently, E. coli was inoculated into the bladder together with paraffin. This model does not allow investigation of the acute inflammatory response, as the dose of bacteria and the exact infection time are difficult to control because these parameters depend on urine flow rate and the time interval before bladder emptying. Consequently, only half

Acknowledgments

The authors would like to acknowledge B. Andersen, L. Kioerboe, H. H. Moeller and M. Carlsen for excellent technical assistance.

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