Abstract

Intestinal spirochetosis (IS) is a disease of humans and animals that has potential public health significance. Two spirochetes have been proposed as etiologic agents of IS, Serpulina pilosicoli and Brachyspira aalborgi. The purpose of this study was to determine the identity of spirochetes present in North American opossums (Didelphis virginiana) with IS. Histopathologic changes indicative of IS were present in the ceca of 17 opossums obtained from California (n=4), Connecticut (n=9), Nebraska (n=2), and New York (n=2). Ultrastructural examination of cecal tissue obtained from ten opossums revealed spirochetes only in six specimens and spirochetes together with flagellated bacteria in four others. Spirochetes were isolated in pure culture from ten specimens, and total DNA from nine isolates was amplified using 16S ribosomal RNA (rRNA) polymerase chain reaction (PCR) assays specific for either B. aalborgi or S. pilosicoli. Seven isolates yielded products with both 16S rRNA PCR assays, whereas two isolates were negative for B. aalborgi, but positive for S. pilosicoli using the 16S rRNA PCR and a 23S rRNA PCR. The presence of IS in opossums obtained from eastern, central and western states suggested that the disease may be widespread in the United States. These findings help define the epidemiology of IS and provide evidence that the opossum is a natural reservoir for the agent(s) and potentially act as a biologic vector for transmission to mammals and birds.

Introduction

Serpulina pilosicoli is the etiologic agent of a diarrheal disease of humans, non-human primates, swine, dogs and birds that has been variously referred to as intestinal spirochetosis (IS), colonic spirochetosis, colorectal spirochetosis, rectal spirochetosis or cecal spirochetosis.^{1,3-5,8,10,11,13} Spirochetes of unknown taxonomic classification also have been seen in the cecum and colon of opossums and laboratory guinea pigs.^3,12 The disease caused by S. pilosicoli is characterized by intimate attachment of spirochetes to the apical surface of the cecal and colonic enterocytes with or without invasion into the gut wall.^{1,3-5,8,12,13} Also, spirochetes structurally and phenotypically different from S. pilosicoli, and called Brachyspira aalborgi, as well as some unclassified flagellated bacteria have been seen attached to the cecal and colonic enterocytes of humans and rhesus macaques.^1,3,4,6 The purpose of this study was to determine the identity of intestinal spirochetes of North American opossums (Didelphis virginiana) with IS. To ascertain the presence of B. aalborgi and S. pilosicoli in IS of opossums, pure cultures of intestinal spirochetes were isolated from opossums affected with the disease. Then the spirochetes were examined for their growth characteristics on artificial medium and for the presence of spirochete-specific 16S ribosomal RNA (rRNA) and 23S rRNA gene sequences by polymerase chain reaction (PCR) amplification assays.

Methods

Sample Collection

Cecal tissues and contents from 17 mature opossums obtained from four states were processed for microbiologic, light and ultrastructural examinations (Table 1). Opossums from California were captured alive as part of epidemiologic studies at two dairy farms in San Bernardino (numbers 1, 2 and 3) and an aviary near Concord (number 4). Opossums number 6 and number 8 were found dead on roads at various times after being hit by cars in Connecticut. Opossum number 14 and number 15 were found dead in residential areas in Lincoln, Nebraska, one with heavy parasitism and septicemic salmonellosis, and the other after being hit by a car, respectively. The remaining opossums were caught live in traps either by wildlife control servicemen in residential areas in Connecticut, or by a commercial wildlife supplier in New York. Live opossums were euthanatized by administration of an overdose of barbiturate.

Table 1. Laboratory findings in wild-caught North American opossums with intestinal spirochetosis

^aPresence (+) or absence (-) of spirochetes or flagellated bacteria attached to the enterocyte brush border of the cecum, as determined by transmission electron microscopic (TEM) examination; ND=not determined.
^bSpirochete isolated (+), not isolated (-), or overgrown (over.) by contaminants after anaerobic incubation of cecal scrapings on selective agar medium for intestinal spirochetes at 37°C for 21 days.
^cPresence (+) or absence (-) of products after amplification of purified DNA from pure cultures using polymerase chain reaction (PCR) of 16S rRNA gene sequences of Serpulina pilosicoli (Sp) or Brachyspira aalborgi (Ba); NA=not available; ND=not determined.

Pathologic Examination

Blocks of cecal tissue were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin, cut at 5 µm, and stained with hematoxylin and eosin (HE) and Warthin-Starry (WS) stain. Additional formalin-fixed deparaffinized sections were processed for staining of spirochetes by immunohistochemistry using the Serpulina spp. periplasmic flagellar FlaB-specific mouse monoclonal antibody 7G2 and avidin biotin complex alkaline phosphatase (Dako, Carpinteria, CA).¹² For transmission electron microscopy, fresh (numbers 6–13), formalin-fixed (numbers 14–17), or formalin-fixed deparaffinized blocks (numbers 1–5) were immersed in 3% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2), embedded in araldite, processed for ultrathin sectioning, stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (Philips 201, Eindhoven, The Netherlands).

Bacteriologic Examination

Primary isolation of spirochetes was done by streaking cecal mucosal scrapings onto colistin, vancomycin, and spectinomycin selective agar medium and incubation at 37°C in a commercial anaerobic culture system (Gas Pak Anaerobic System, BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD) as previously described.^2,8,9 Pure cultures of spirochetes were propagated on trypticase soy agar with 5% citrated sheep blood (TSA). Isolates numbers 14 and 15 were examined for growth on TSA incubated at either 37°C or 42°C.

PCR Assays

Total DNA from pure cultures of spirochetes was isolated using a previously described method.⁹ Serpulina pilosicoli identification was done by PCR amplification with a 16S rRNA sequence-specific forward primer (5’-AGAGGAAAGTTTTTTCGCTTC-3’) and a conserved Serpulina spp. 16S rRNA reverse primer (5’-TCCGCCTACTCACCCTTTAC-3’) as previously described.^4,8,13 For PCR amplification of B. aalborgi, a 16S rRNA-specific forward primer (5’-GCCTGTTGGTGAGATAAAAG-3’) was used in combination with the conserved Serpulina spp. 16S rRNA reverse primer as previously described.⁴ The total DNA from each spirochete isolate was examined further using a S. pilosicoli 23S rRNA gene-specific forward (5’-AGGTGATGGTTATCCTC-3’) and reverse (5’-AACCTTAGGAATTATTTCTAA-3’) primer set and PCR amplification.⁷ Positive and negative controls consisted of S. pilosicoli porcine strain P43/6/78T DNA and no template DNA, respectively. The amplified products were visualized after electrophoresis in 1.5% agarose gels and staining with ethidium bromide.

Results

Light microscopic examination of HE-stained sections of ceca revealed a diffuse basophilic 4 µm layer on the brush border of enterocytes along the surface and extending about half-way into the crypts in 16 of 17 opossums. Opossum number 14 had large numbers of spirochetes in the mucin on the surface and in the crypt lumena with rare foci of surface attachment. The basophilic layer was dark brown to black with WS stain. Immunohistochemical staining confirmed the presence of a dense layer of spirochetes arranged in parallel arrays on the apical surface of cecal enterocytes in all of the opossums. Associated inflammatory infiltrates or exudates were minimal or non-existent. Ultrastructural examination revealed diffuse colonization by spirochetes attached perpendicularly by one pole to the apical membrane of enterocytes causing effacement of microvilli and disruption of the terminal web microfilaments (Table 1). Vesicles that contained electron-dense granular material were present in the apical cytoplasm of colonized cells. In some specimens, spirochetes were seen between epithelial cells or free in the lamina propria, and in opossum number 11, few spirochetes also were seen free in the cytoplasm of a goblet cell. In addition to the spirochetes, flagellated bacteria were attached perpendicularly by one pole to the apical membrane of enterocytes in the ceca of opossums from California and New York.

Spirochetes were isolated from ten out of 17 intestinal specimens with IS; spirochetes were not isolated from specimen numbers 1, 2 and 3 which had been frozen, whereas specimens 6, 11, 12 and 13 had spirochetes in wet mounts of cecal contents examined by dark field microscopy that were overgrown by contaminants. All spirochete isolates grew very slowly taking up to 14 days to form a visible non hemolytic surface film that became weakly β-hemolytic after 21 days of incubation at 37°C. When examined for growth at 42°C, isolates number 14 and number 15 did not show any surface growth or hemolysis after 21 days. Nine of the isolates were examined using the 16S rRNA PCR assays, and with the exception of number 9 and number 10, all of the isolates yielded 314-bp B. aalborgi-specific and 361-bp S. pilosicoli-specific products (Table 1). The same isolates were negative for S. pilosicoli-specific 23S rRNA, whereas isolates number 9 and number 10 yielded S. pilosicoli-specific products with the 16S and the 23S rRNA gene sequence-specific PCR assays, and no products with the B. aalborgi 16S rRNA PCR assay.

Discussion

Intestinal spirochetosis of wild opossums was first reported by Turek and Meyers;¹² five opossums from Illinois had adherent spirochetes that caused disruption of the brush border of cecal enterocytes in a pattern similar to that seen in the present report. The spirochetes isolated from these opossums grew as a thin haze on the surface of blood agar, producing very slight or no hemolysis when incubated anaerobically at 37°C. Transmission electron microscopic examination of the cultured spirochetes revealed four to five subterminally inserted periplasmic flagella at each end of the protoplasmic cylinder. These morphologic and cultural characteristics were consistent with those of B. aalborgi, a spirochete that was later described in human beings with IS.⁶

Based on the pathology and microbiology findings, a diagnosis of IS was made in all 17 opossums. The ultrastructural data also indicated that flagellated bacteria often were present together with the spirochetes; a disease association similar to that seen in human beings and non-human primates^1,3,4, but different from swine, dogs and birds where flagellated bacteria have not been seen^3,5,8,12. Although there was evidence of massive mucosal involvement, the contents of the ceca were normal in consistency. Therefore, the clinical significance and pathogenetic mechanisms of IS in these opossums remains unknown. However, the data presented extended the observations made by Turek and Meyers,¹² and further indicated that IS was widespread among opossums in the United States.

With the advent of DNA-based typing methods, it is assumed that the identity of culturable intestinal spirochetes can be determine with some confidence. However, on the basis of PCR amplification of 16S rRNA gene sequences, the majority of the isolates gave positive amplification products with both PCR assays. Because the growth characteristics of S. pilosicoli are different from B. aalborgi; growth occurs within days and produces a clear zone of β-hemolysis on blood agar plates incubated at 37°C and 42°C,^6,8,11 the results of the S. pilosicoli 16S rRNA PCR assays might not be specific. This is supported by the absence of growth of two of these isolates at 42°C and the absence of specific products with the S. pilosicoli 23S rRNA PCR assay. Since not all of the isolates were examined for growth at 42°C, the possibility that some of the cultures contained S. pilosicoli and B. aalborgi cannot be ruled out completely. Conversely, the results of 16S and 23S rRNA PCR assays suggested that opossums number 9 and number 10 had only S. pilosicoli. Although additional work is needed to improve the specificity of DNA-based typing methods, spirochetes are genetically diverse, and it is possible that the opossum spirochetes belong to a group that shares characteristics of S. pilosicoli and B. aalborgi.

The data presented indicated that the North American opossum is a natural reservoir for IS. Because IS was found in opossums living in both rural and residential areas, it raised the possibility that the North American opossum may serve as a biologic vector for transmission of the disease to livestock and urban dwellers.

Acknowledgments

This work was supported by funds provided by the United States Department of Agriculture; Regional Research Project NC-62; Enteric Diseases of Swine and Cattle: Prevention, Control, and Food Safety.

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