Cryptosporidium sp. Infections in Chickens, Produced by Intra-Cloacal Inoculation of Oocysts

Abstract

Clinical disease in chickens caused by Cryptosporidium sp. has been reported to occur when the parasites are located in the respiratory tract (Dhillon et al., 1981, Avian Diseases 25: 747751; Itakura et al., 1984, Avian Pathology 13: 487-499). However, overt clinical disease does not appear to occur when the parasites are confined to the digestive tract (Fletcher et al., 1975, Avian Diseases 19: 630-639; Randall, 1982, Avian Pathology 11: 95-102). The present authors characterized tissue specificities of an avian isolate of Cryptosporidium sp. in chicks inoculated orally or intra-tracheally (IT) with oocysts (Lindsay et al., 1986, American Journal of Veterinary Research 47: 876-879). The distribution of Cryptosporidium sp. within the digestive tract was not influenced by the mode of inoculation; parasites were present in the cecum (4.3%), terminal colon (26.1%), bursa of Fabricius (BF) (95.7%) and cloaca (100%) of chicks that were positive for developmental stages. However, extensive respiratory involvement occurred only in chicks inoculated IT, with resultant infections of the trachea, primary and secondary bronchi, air sacs, nasal turbinates and the ducts of the salivary glands. In addition, 4 of 21 (19.0%) chicks inoculated orally and examined 7, 18, 21 and 24 days postinoculation (PI) had cryptosporidia in the trachea. Although accidental intra-tracheal inoculation of these 4 chicks with oocysts could not be ruled out, it was also possible that developmental stages of Cryptosporidium sp. migrated or were transported to the trachea from the digestive tract. Because oocysts of Cryptosporidium sp. are fully sporulated when passed in the feces, naturally acquired respiratory infections are also possible beginning 5 days PI when chicks begin passing oocysts in their feces (Lindsay et al., 1986, loc. cit.). The present study was conducted in order to determine if chicks could be infected with Cryptosporidium sp. by intra-cloacal inoculation of oocysts, and whether respiratory infections would result. If respiratory infections occurred in a majority of these chicks, it would suggest that developmental stages actively migrated, or were transported, to the respiratory tract. In the present study, Cryptosporidium sp. oocysts, originally obtained from the bursae of naturally infected broiler chickens (AU-B 1 isolate), were collected from the feces of experimentally infected chicks that were coccidia-free at the time of inoculation. Feces were mixed with a 2.5% (w/v) aqueous potassium dichromate (K2Cr207) solution, passed through a series of graded sieves (final exclusion 45 tm) and stored a 4 C in 2.5% K2Cr207 solution. Oocysts from this mixture were concentrated by flotation using Sheather's sugar solution, washed in distilled water, and suspended in Hanks' balanced salt solution (HBSS) (Gibco Laboratories, Grand Island, New York). The number of oocysts in this stock inoculum was determined using a hemacytometer and Nomarski interference-contrast (NIC) microscopy. Concentrated oocysts were stored in HBSS at 4 C and were less than 30 days old when used for experimental inoculations. Thirty-four 2-day-old chicks were each inoculated intra-cloacally (IC) with 0.1 ml of HBSS containing 300,000 oocysts of Cryptosporidium sp. Five 2-day-old chicks were not inoculated and served as controls. Inoculated and control chicks were housed separately as groups in wirebottom cages raised over excreta pans, and were fed chick starter and water ad libitum. Pairs of inoculated chicks were each examined at necropsy on days 5-21 postinoculation (PI). A single control chick was examined at necropsy 5, 12, 13, 14 and 17 days PI. Portions of the BF, cloaca, terminal colon, ileum, both ceca, lung, and 4 segments of trachea were obtained from each chick, fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned at 8 min, and stained with hematoxylin and eosin. Mucosal smears were made from the BF and trachea of each chick at the time of necropsy and examined