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Clinical, Serological, and Parasitological Analysis of Snakes Veterinary Parasitology 198 (2013) 54–61 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Contents lists available at ScienceDirect provided by Elsevier - Publisher Connector Veterinary Parasitology jou rnal homepage: www.elsevier.com/locate/vetpar Clinical, serological, and parasitological analysis of snakes naturally infected with Cryptosporidium serpentis a,b b c Philipp Ricardo S.O. Paiva , Kathleen F. Grego , Valéria M.F. Lima , c c c,∗ Alex A. Nakamura , Deuvânia C. da Silva , Marcelo V. Meireles a Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, USP, Avenida Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, Brazil b Laboratório de Herpetologia, Instituto Butantan, Avenida Vital Brazil, 1500 São Paulo, Brazil c Faculdade de Medicina Veterinária, Univ Estadual Paulista, UNESP, Arac¸ atuba, Rua Clóvis Pestana, 793, Arac¸ atuba, SP, Brazil a r t i c l e i n f o a b s t r a c t Article history: Infection by Cryptosporidium serpentis is one of the most important diseases in reptiles and Received 21 June 2013 is characterized by chronic clinical or subclinical infection and the presence of hypertrophic Received in revised form 13 August 2013 gastritis, food regurgitation, progressive weight loss, mortality, and intermittent or contin- Accepted 16 August 2013 uous shedding of oocysts in the feces. The objectives of this study were to standardize an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against C. ser- Keywords: pentis and to evaluate the clinical, parasitological, and humoral immune response in snakes Cryptosporidium serpentis Snakes naturally infected with C. serpentis. Twenty-one snakes naturally infected with C. serpentis and housed at the Butantan Institute, São Paulo, Brazil, underwent clinical and parasitolo- Indirect ELISA Serology gical analyses for C. serpentis infection through daily records of clinical signs and a monthly Diagnostic survey of fecal shedding of oocysts using the Kinyoun’s acid-fast staining. The serologi- cal evaluation was performed monthly by indirect ELISA using crude total antigen from oocysts of C. serpentis to detect anti-C. serpentis antibodies. Clinical symptoms consisted of food regurgitation, inappetence, and progressive weight loss. The parasitological analysis revealed intermittent fecal shedding of a variable number of oocysts in all snakes, with positivity in 85.32% (157/184) of the samples. The indirect ELISA was positive in 68.25% (86/126) of the samples. A humoral immune response was observed in most animals; how- ever, fluctuating antibodies levels, leading to alternating positive and negative results, were observed in most snakes. © 2013 Elsevier B.V. All rights reserved. 1. Introduction (Cranfield and Graczyk, 1994). The most common clinical signs are inappetence, postprandial regurgitation, lethargy, Infection with Cryptosporidium serpentis in snakes is progressive weight loss, and midbody swelling (Godshalk characterized by chronic clinical or subclinical infection et al., 1986; Carmel and Groves, 1993; O’Donoghue, 1995; and the presence of hypertrophic gastritis, accompanied Ramirez et al., 2004; Xiao et al., 2004a). by weight loss and mortality (Brownstein et al., 1977). Cryptosporidiosis in snakes is of the utmost importance In chronic gastric cryptosporidiosis, there is continuous for ophidian collections because it can be lethal and deci- or intermittent shedding of oocysts, which can last for years mate the entire population. The lack of effective treatment for cryptosporidiosis almost always results in euthanasia of the infected snakes, which often leads to the loss of valuable ∗ animals in a collection (Carmel and Groves, 1993). Corresponding author. Tel.: +55 18 3636 1425; fax: +55 18 3636 1403. To develop measures to prevent and control infection E-mail addresses: [email protected], [email protected] (M.V. Meireles). by C. serpentis, information is needed on the evolution of 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.08.016 P.R.S.O. Paiva et al. / Veterinary Parasitology 198 (2013) 54–61 55 its clinical signs, shedding of oocysts, and the serological 2.3. Serological evaluation response in naturally infected snakes. Studies that evaluate the serological response of snakes facing infection by infec- The humoral immune response against C. serpentis was tious agents are rare; the only study on detecting antibodies evaluated in 12 snakes through an indirect enzyme-linked against C. serpentis in snakes was performed by Graczyk and immunosorbent assay (ELISA), with 126 serum samples Cranfield (1997). obtained after monthly blood collection for 12 months, The goal of the present project is to provide a clinical, except in the animals that died (Table 1). parasitological, and serological evaluation of snakes natu- The indirect ELISA was standardized using an anti- rally infected with C. serpentis, with the aim of providing gen produced from oocysts of C. serpentis, chicken IgY information for the diagnosis and control of cryptospori- anti-snake gamma globulins, and peroxidase-labeled rab- diosis in ophidian collections. bit anti-chicken IgY conjugate (Sigma, Saint Louis, MO, USA). To obtain the antigen, the stool samples or regurgitation content from the snakes naturally infected with C. serpentis 2. Materials and methods were collected and preserved in 2.5% potassium dichro- ◦ mate at 4 C. The species of Cryptosporidium was classified 2.1. Snakes using nested PCR (Xiao et al., 2000) and sequencing the amplified fragments. The samples were diluted in Tween Twenty-one snakes (six genera and six species) natu- 20 (0.1%), strained with sieves with decreasing porosity rally infected with C. serpentis, and donated to the Butantan up to 36 ␮m, purified by centrifugation in Percoll gradi- Institute in São Paulo, Brazil, were used (Tables 1 and 2). ents (Abassi et al., 2000), resuspended in 1.75% sodium Before being transferred to the Butantan Institute, they hypochlorite for 15 min, and centrifuged at 12,000 × g for were zoo-housed in an open enclosure. Snakes were known 3 min. The sediment was resuspended in distilled water, to be infected with C. serpentis by screening for oocysts in homogenized in a vortex, and centrifuged at 12,000 × g for fecal samples using the Kinyoun’s acid-fast staining fol- 3 min; this step was repeated five times to remove the lowed by nested polymerase chain reaction (nested PCR) sodium hypochlorite. ◦ to amplify a partial fragment of 18S rRNA gene (Xiao et al., The oocysts were diluted in PBS pH 7.2, stored at 4 C, 6 2000) and sequencing of amplified fragments. During the quantified in a Neubauer chamber (7 × 10 ), and were lysed experiment, snakes were kept individually in snake cages by sonication for five 3 min cycles in an ice bath. The total with corrugated cardboard and a pot of water, in a room protein in the solution containing the antigen derived from with artificial lighting and heating, and monthly fed with lysed oocysts was measured with a BCA1 kit (Sigma, Saint rats or mice provided by the Central Vivarium of the Butan- Louis, MO, USA). tan Institute. The snake gamma globulins were obtained from 10 snakes from the families Viperidae (three Bothrops jararaca and three Crotalus durissus), Colubridae (two Pantherophis guttatus), and Boidae (two Boa constrictor amarali), and 2.2. Clinical and parasitological evaluation these snakes were housed in the Vivarium of Venom Pro- duction of the Butantan Institute. They were negative for The clinical evaluation was conducted daily for all ani- Cryptosporidium spp. based on periodical screening of fecal mals to observe any physical or behavioral changes. Body samples using the Kinyoun’s acid-fast staining and nested weight was measured at the beginning of the experi- PCR (Xiao et al., 2000). Three milliliters of blood was col- ment and every six or 12 months, unless an animal died lected once from each snake, forming a pool of serum from (Tables 1 and 2). different species. The parasitological assessment was conducted in all The gamma globulin fraction of the snake serum pool the animals. One hundred eighty-four fecal samples were was purified by precipitation with 45% ammonium sul- collected at approximately one-month intervals for 12 fate and centrifuged at 7000 × g for 30 min. The resulting months, unless an animal died (Tables 1 and 2). The samples sediment was diluted in PBS, pH 7.2, transferred to a dial- were strained with folded gauze, centrifuged at 1500 × g ysis membrane, and submerged twice in 0.025 M Tris–HCl for 10 min, and stained using the Kinyoun’s acid-fast stain- buffer, pH 8.8 for 18 h to remove excess ammonium sul- ing, which was also used to stain smears of regurgitated fate (Hebert et al., 1973). The snake gamma globulins were material. The snakes that died during the experiment were quantified with the BCA1 kit (Sigma, Saint Louis, MO, USA) weighed and underwent necropsy. and yielded a solution of 30.26 mg/ml. The species of Cryptosporidium that were present in To produce chicken IgY anti-snake gamma globulin, fecal samples were identified at the beginning and end four commercial laying hens, from the Isa Babcock strain, of the experiment. For molecular identification, genomic were inoculated intramuscularly four times with 100 ␮g or DNA was extracted from 200 mg of fecal samples using the 500 ␮g of snake gamma globulins (500 ␮l of PBS containing QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA), the gamma globulins and 500 ␮l of Freund’s adjuvant) at according to the manufacturer’s protocol after an initial 10-day intervals. The first inoculation was conducted with step of freezing and thawing in liquid nitrogen 5 times, fol- complete Freund’s adjuvant, and the other inoculations lowed by nested PCR (Xiao et al., 2000) and sequencing of were conducted with incomplete Freund’s adjuvant.
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