Ultrastructural aspects of experimental cryptosporidiosis in pigs

Aspectos ultraestruturais da criptosporidiose experimental em suínos

Ultrastructural aspects of experimental cryptosporidiosis in pigs

(Aspectos ultraestruturais da criptosporidiose experimental em suínos)

A.P.F.R.L. Bracarense¹, A.C.F. Reis¹, I.L. Sinhorini²

¹Universidade Estadual de Londrina

Caixa Postal 6001

CEP 86051-990, Londrina, PR

²Faculdade de Medicina Veterinária da Universidade de São Paulo

Recebido para publicação, após modificação, em 16 de junho de 1999.

E-mail: [e-mail]

ABSTRACT

The aim of this work was to determine ultrastructural changes on the intestinal mucosa and associated lymphoid tissues after an experimental infection with Cryptosporidium sp. Twelve piglets dosed orally with 1´10⁶ oocysts were slaughtered on days 3, 6, 9 and 12 after inoculation. The ultrastructural lesions in the intestinal cells were irregular with thickened microvilli, cytoplasmic protrusions and vacuolation, swollen mitochondria, hypertrophic organelles and nuclei. The lymphocytes of the Peyer’s patches occasionally were mitotic with a larger number of lymphoblasts in the inoculated animals.

Keywords: Pig, cryptosporidiosis, ultrastructure, intestine

RESUMO

O presente estudo teve por objetivo investigar as alterações ultraestruturais da mucosa intestinal e do tecido linfóide associado na inoculação experimental de Cryptosporidium sp. Doze leitões foram inoculados experimentalmente por via oral com 1´10⁶ oocistos e sacrificados 3, 6, 9 e 12 dias depois. Ao exame ultraestrutural de células intestinais observou-se espessamento e irregularidade de microvilosidades, citoplasma vacuolizado e com protrusões, edema mitocondrial, hipertrofia de organelas citoplasmáticas e do núcleo. Nas placas de Peyer observou-se ocasionalmente mitose de células linfóides, verificando-se maior número de células blásticas.

Palavras-Chave: Suíno, criptosporidiose, ultraestrutura, intestino

INTRODUCTION

Cryptosporidiosis is a disease caused by a protozoan of the phylum Apicomplexa that affects a wide variety of vertebrates, including man. The parasite infects epithelial surfaces, especially the intestine, leading to diarrhea both in immunocompromised and immunocompetent animals. The disease affects mostly young individuals soon after birth. Protection given by colostrum does not seem to be effective in preventing cryptosporidiosis. Previous studies had focused mainly in investigating the life cycle and the taxonomy (Tzipori, 1988; Levine, 1993) of cryptosporidia. Histopathological lesions in the intestine are described, but little is known regarding the agent’s pathogeny. Full details of the clinical findings and histopathological lesions have been reported elsewhere (Bracarense & Sinhorini, 1997). In addition, previous ultrastructural studies had focused the evolutionary cycle of the parasite, supplying scarce details regarding the alterations found in the cells. The main objective of this study was to investigate ultrastructural lesions in the intestinal cells associated with experimental inoculation of Cryptosporidium sp. in pigs.

MATERIALS AND METHODS

Twenty-four crossbred Large-White ´ Landrace pigs, male and females, 3-4 days of age were used. Half of the animals were inoculated orally with 1ml saline containing 1´10⁶ oocysts of Cryptosporidium sp., whereas the other half received just 1ml of saline orally. The inoculum was obtained from feces from a calf previously inoculated with 1´10⁶ oocysts of Cryptosporidium sp. The preparation and purification of the oocysts followed a methodology previously described (Robert et al., 1990).

Three inoculated and three control animals were killed at 3, 6, 9 and 12 days after inoculation (pi). Samples for bacteriological and virological analysis were obtained before, halfway between inoculation and killing of the animals, and at the day the animals were killed. Virological exams consisted of nucleic acid extraction by the phenol-chloroform technique and polyacrylamide gel electrophoresis. Sterile swabs of the intestine were obtained and cultured onto blood and Mc Conkey agar media. Presence of Cryptosporidium oocysts was evaluated daily in fecal smears stained with modified Ziehl-Neelsen technique (Henriksen & Pohlenz, 1981). Oocysts shedding was assessed using previously described scores using 40x magnification (Robert et al., 1990).

The pigs were tranquilized with 1% acepromazine and maintained in surgical plane of inhalatory anesthesia with ether until the intestinal tract had been completely removed. They were killed by saturation with ethyl ether. Samples were obtained from mesenteric lymph nodes. Duodenum, jejunum, ileum and colon tissue samples were maintained in 2,5% glutaraldehyde and later processed and included in Araldite 502. Ultrathin sections stained with saturated aqueous uranyl acetate followed by lead citrate were examined in transmission electron microscope (Philips EM 201).

RESULTS

All the animals that were inoculated with Cryptosporidium sp. showed the same ultrastructural lesions. These lesions were most severe in animals killed later in the experiment. Microvilli became sparce, irregular, thick and decreased in size in a large number of cells (Fig. 1). Small numbers of cytoplasmic protrusions were observed in the apex of the cells. The continuity of the epithelium was preserved by the presence of cellular junctions (mainly desmosomes) where alterations were not verified. The cytoplasm showed vacuoles of variable diameters that contained a single membrane, and a non-electron dense content (Fig. 2). These lesions were more pronounced in the ileum of animals killed 6 and 9 days after inoculation. Swelling and occasionally loss of cristae were observed in the mitochondria (Fig. 3). Absorptive cells had a high number of free ribosomes in the apical and basal portions of the cells. Golgi complexes were better developed and more numerous in inoculated animals than in control animals. The nuclei of the enterocytes were located at the base of the cells and showed invagination of the nuclear membrane. Most nucleoli were hypertrophic with deposition of an electron dense material with a reticular pattern in the center of the nuclei (Fig. 3). The follicular epithelial cells had similar lesions. The lamina propria of the intestinal villi and the submucosa were infiltrated by plasma cells containing a large amount of rough endoplasmic reticulum. These areas were also infiltrated by macrophages that contained phagocytic particles. The lymphocytes of the Peyer’s patches occasionally were mitotic with a larger number of lymphoblasts in the inoculated animals. A few immature parasites were found associated to the intestinal cells (Fig. 4). Hemolytic and non hemolytic Escherichia coli were detected in all samples. No intestinal virus were detected. No parasites were found in the control animals. The absorptive cells of control animals showed no abnormalities.

DISCUSSION

Some of the ultrastructural lesions present in this study have been previously found by other authors (Pohlenz et al., 1978; Tadeja-Simborio et al., 1993). The shortening, thickening, or absence of microvilli probably result from a direct action of the parasite upon the intestinal cells. These changes cause a decrease and even loss of the absorptive capacity of the cells. The ultrastructural mitochondrial lesions have been shown before in cryptosporidiosis (Tadeja-Simborio et al., 1993), but was not correlated with changes in cell function. The mitochondrial edema observed in this study suggests that cryptosporidiosis may lead to abnormalities in cell respiration. The mitochondrial edema may result from a direct action of cytotoxins produced by the parasites upon these organeles. The atypical nuclei and the prominent nucleoli indicate enhanced RNA synthesis that is necessary for the increased protein synthesis in the cytoplasm (Junqueira & Carneiro, 1995). The alterations in the rough endoplasmic reticulum, ribosomes and Golgi complex also suggest an increase in protein synthesis in the cells. It has been suggested (Tzipori, 1985) that absorptive cells respond to cryptosporidium infection by increasing protein synthesis. The increase in protein synthesis is probably necessary to compensate for the loss of brush border digestive enzymes. The increase in protein synthesis may also result from a direct stimulation by the parasites which would then use the proteins produced by the cell to complete their life cycle.

CONCLUSIONS

The ultrastrucutural changes of the intestinal cells of the inoculated animals suggest that edema and hipertrophy of nuclear and cytoplasmic organeles and degeneration of cytoplasmic organeles occur in response to cryptosporidium infection in pigs. The severity of the lesions in the cytoplasmic organeles and cell membrane is likely responsible for the death of the absorptive cells. These changes may be due to direct physical damage or due to production of cytotoxic factors by the parasite.

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Publication Dates

• Publication in this collection
  05 Apr 2001
• Date of issue
  Oct 1999

History

• Received
  16 June 1999