Prevalence and Molecular Analysis of Cryptosporidium Spp. Isolated From Wild and Domestic Birds

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1 ISSN IDOSI Publications, 2015 DOI: /idosi.apg Prevalence and Molecular Analysis of Cryptosporidium Spp. Isolated From Wild and Domestic Birds 1 2 Ghaidaa A. Jasim and Ikhlas A. Marhoon 1 Branch of Microbiology, College of Veterinary Medicine, Al-Qadisiya University, Iraq 2 Department of Biology, College of Education, Al-Qadisiya University, Iraq Abstract: The purpose of this studywas: Initially to revealed the distribution of Cryptosporidium spp. in wild and domestic birds in Al- Qadisiya province in Iraq, secondly to determinate genotypic characterization and phylogenetic analyze based on 18S rrnasequences of Cryptosporidium which obtained from these birds. A total of 236 fecal specimens from six types of birds were screened microscopically to look for Cryptosporidium oocysts using modified Ziehl Neelsen stain. Cryptosporidium spp. in 32 microscopy-positive specimens were analyzed genetically through DNA extraction after that DNA was amplified and processed with the small-subunit 18S rrna gene by using ordinary PCR and then produceddna wasanalyze to determinate their sequences. Results showed Cryptosporidium oocysts were observed in 137(58.1%) specimens of birds' feces. They was 54.5% of the turkey, 57.5% of the domestic chickens, 53.8% of the broiler chickens, 62.5% of the common ducks, 76.7% of the quails and 26.7% of the feral pigeon. Sequencing andfurther phylogenetic analyses identified Cryptosporidiumparvum ()in all birds in our study, Cryptosporidium.baileyiobserved in domestic and broiler chickens, quail and feral pigeon ducks, Cryptosporidium. meleagridisisolated from turkey and quail, finally the species Cryptosporidium.gallionly recorded from domestic chickens.because of a high infection percentages with cryptosporidiosis in domestic and wild birds species. So that we suggesting that our birdsin the present study would be considered as biological transporters of Cryptosporidium spp. contributing to environmentaldissemination with pathogens which infect human and other animals. Key words: Cryptosporidium Sp. Domestic Birds 18srrna of Cryptosporidium INTRODUCTION budgerigars, cockatiels, quails and ostriches [6], finaly, C.galli, which discovered recently, infects several hosts Cryptosporidium spp. are one of the most prevalent such as finches, domestic chickens and pine grosbeaks protozoan pathogen which have a wide range of [9]. In 1929, Tyzzerwas recordedthe first description of varietyhosts like: mammalian, reptilian, amphibian and Cryptosporidium infection in the ceca of chickens [4]. fish, beside of all species of birds [1]. Cryptosporidiosis, Later, a report in 1955 described structurally similar a disease caused by Cryptosporidium spp., has been parasites in turkeys and these parasites were named reported in over 170 host species [2, 3] and in more than C. meleagridis [9]. In 1986, an organism from chickens 30 avian species worldwide [4]. was isolated, described and gave the name C. baileyi [10]. In birds there are three main Cryptosporidium Currently, there are 16 species of Cryptosporidium that species that have been described namely C. baileyi, have been identified which have a different morphologies C. galli and C. meleagridis [5, 6]. Only C. meleagridis, and hosts [11, 12]. Several researches refers to infection which infects turkeys and parrots, is a known threat to with another species of Cryptosporidium in birds in humans [7, 8]. While, C. baileyiis likely the most common addition tothethree species mentioned previously. The avian Cryptosporidium species because its ability to most important of these species is C. parvumwhich have infect many birds like: domestic and caged chickens, foundnatural infection with it in different wild and pet turkeys, geese, ducks, feral pigeon, lovebirds, birds [4, 13]. Corresponding Author: Ikhlas A. Marhoon, Department of Biology, College of Education, Al-Qadisiya University, Iraq. 65

2 Avian cryptosporidiosis can manifest as respiratory extracted from the purified oocysts using a Stool Genomic form Dhillon et al. [14] and intestinal form Adejinmi and DNA extraction Kit (Bioneer Company, South Korea) in Oke [15]. In some cases, cryptosporidiosis might even accordance with the manufacturer s instructions and kept manifest as a renal form, which can be fatal [16], also at 20 C until detected by the PCR method. Trampel et al. [17] were described cryptosporidiosis in Molecular Analysis: A total of 32 Cryptosporidiumlaying hens in tubule urinary and ureterand they named positive samples from studied birds were characterized as"urinary tract cryptosporidiosis" the stages of genetically. Cryptosporidium spp. were genotyped by evolutionary parasite seen on the apical surface of amplifying an 830bp of the small subunit 18S rrna gene epithelial cells of the collected tubules urinary and ureters. by direct PCR [9]. these samples from different birds were The traditional methods of diagnosis of analyzed to identified DNA sequencingby using AB DNA cryptosporidium spp. was confirmed through sequencing system [22]. The acquired sequences were findingoocysts in the feces [18]. Recently, molecular submitted to a BLAST search to initially define the diagnosis was followed to investigate a new species and species/genotypes and to confirm the high similarity and genotypes of cryptosporidiumin different host [19, 20]. homology with other known sequences of Because of the local studieson Cryptosporidiosis in Cryptosporidium spp. in NCBI-GenBank. Sequences of birds in Iraq were rarely and its risk on human health, the partial 18S rrna gene of C.parvum, C. meleagridis economically as caused loss of livestock and therole of C. baileyi and C. galli which isolated from studied birds birds in the dissemination of infection with this parasite, were compared with analogous species which deposited our study aimed to investigate the prevalence of in NCBI-GenBank under accession numbers L1699.1, cryptosporidiosis in fecal samples of wild and domestic eu , AY and HM respectively. birds in different regions in Al-Qadisiya province, Iraq and molecular analysis ofcryptosporidium species which Cryptosporidium Phylogeneticanalysis: All sequences isolated from birds and definethe relationship among were multiple-aligned and analyzedby using MEGA6 species genetically according to 18S rrna gene. p h y l o g e n e t i c t r e e p r o g r a m ( And then a neighbor- MATERIALS AND METHODS joining or phylogenetic tree was built. Collection of Samples and Examination: A total of 236 Statistical Analysis: Data generated were analysed on birds were collected from different regions of Al-Qadisiya the computerstatistical package SSPSusing Chi-square province between May 2013 to June 2014, Birds included test. Differences expressed as significant at P < 0.05 [23]. six species which are: 22 Turkey (Meleagrisgallopova), 60 Quail (Coturnixcoturnix ), 40domestic chicken RESULTS (Gallus gallusdomesticus), 52broiler chicken (Gallus gallus), 32 commenduck (Anasplatyrhynchos) and Results revealed through examination of 236 samples 30 Feral pigeon (Columba livia). Fresh fecal samples from wild and domestic birds belonging to six species of which took from birds were examined by used hot birds under study that the percentage of the overall modified Zeihl-Nelseen stain [18]. infection was 58.1%.The results in Table 1. Showed the The samples were examined for Cryptosporidium prevalence of Cryptosporidiosis according to species of oocysts by light microscopy at x400 magnification. birds which revealed the highest infection percentage was Then the oocysts had been concentrated by Sheather s in quails(coturnixcoturnix) which reached to 76.7% and sugar flotation technique as described bywebster et al. the least in feralpigeon (Columba livia) which reached to [21]. Cryptosporidium-positive samples were stored in 26.7%. 2.5% potassium dichromate and kept at 4 C until DNA Our study recorded four species belong to extraction. Cryptosporidium be responsible for intestinal cryptosporidiosis in birds which are: C. parvum, DNA Extraction: Cryptosporidiumoocysts were isolated C. meleagridis, C. baileyi and C. galli. In addition to their from positive fecal samples using discontinuous density high occurrence, oocysts were also observed in large sucrose gradient centrifugation. Genomic DNA was numbers in fecal samples collected from the same bird. 66

3 Cryptospordium spp.(s13) Cryptospordium spp.(s2) Cryptospordium spp.(s14) Cryptosporidium parvum (L ) Cryptospordium spp.(s32) Cryptospordium spp.(s9) Cryptospordium spp.(s18) Cryptospordium spp.(s19) Cryptospordium spp.(s25) Cryptospordium spp.(s8) Cryptospordium spp.(s26) Cryptospordium spp.(s30) Cryptospordium spp.(s11) Cryptospordium spp.(s1) Cryptospordium spp.(s7) Cryptospordium spp.(s27) Cryptosporidium meleagridis (EU ) Cryptospordium spp.(s15) Cryptospordium spp.(s17) Cryptospordium spp.(s29) Cryptospordium spp.(s6) Cryptospordium spp.(s10) Cryptospordium spp.(s20) Cryptospordium spp.(s16) Cryptospordium spp.(s22) Cryptospordium spp.(s23) Cryptospordium spp.(s4) 100 Cryptospordium spp.(s12) Cryptospordium spp.(s24) Cryptospordium spp.(s28) Cryptospordium spp.(s3) Cryptospordium spp.(s5) Cryptosporidium baileyi (AY ) Cryptospordium spp.(s21) Cryptospordium spp.(s31) 100 Cryptosporidium galli (HM ) Fig. 1: Phylogenetic tree of Cryptosporidium spp. isolated from wild and domestic birds. The numbers on tree branches represent bootstrap values for neighbor-joining and the numbers near specimens represent the obtained samples from birds under study(1, 7, 15, 17 from Turkey; 2, 9, 11, 14, from Common duck; 3, 4, 5, 6, 16, 19, 21, 30, 31, 32 from Domestic chicken; 8, 13, 22, 27, 29 from Quail; 10, 23, 24, 25 from Broiler chicken; 18, 20, 26, 28 from Feral pigeon) Table 1: The prevalence of Cryptosporidiosis according to species of birds Species of birds No. examined No. positive Positive % Meleagrisgallopova Gallus gallusdomesticus Gallus gallus Anasplatyrhynchos Coturnixcoturnix Columba livia Total No The molecular analysis of Cryptosporidium species was done through extracting DNA amplified with 18SrRNA by used PCR technique. Results of electrophoresis revealed that DNA bands were 830 bp. and their sequences were determinate by sent DNA product to Bioneer company in South Korea. Results of DNA sequences by used NCBI-BLAST showed that locally species of parasite, which isolate in this study, give a high identical ratio that was between 99%-100% compared with the global analogous species that recorded in NCBI Genbank for the same gene. The phylogenic tree of parasite species was drown by used MEGA6 program, the results of neighboring tree revealed two major branches: First branch include specimens of C. galli while the second branch include the other three species (C. parvu, C. meleagridis, C. baileyi). Also genetic tree showed presence of two difference strains of C. parvum can infected the birds with neighboring ratio between them reached to 83% and the same thing was found for C. meleagridis but the neighboring ratio was 65% (Fig. 1). Sequencing and phylogenetic analyses identified C. parvumin all birds in 67

4 our study, C. baileyiobserved in domestic and broiler On the other hand, C. parvum was isolated from all chickens, quail and feral pigeon ducks, C. meleagridis sex species of studied birds feces, so this refer to ability isolated from turkey and quail, finally the species C. galli to infect awide range of hosts [4, 12]. The presence of only recorded from domestic chickens. C. baileyi was observed in domestic and broiler chickens, quail and feral pigeon, this species is usually found as a DISCUSSION parasite in many variety of birds hosts. C. baileyi is probably the most common avian Cryptosporidium spp. The local birds in our study showed a higher it reported in more than 17 other avian hosts [4, 11, 30]. prevalencerate of Cryptosporidiumoocysts (58.1%) as While C. meleagridis just isolated from turkey and quail compared withother concerned studies, the present study and C. galli diagnosed from domestic chickens only, agree with previous researches [5, 15, 24, 25]. Statistical these may be belong to its weak host specificity, exposure analysis showed no significant differences in infection to infection sources, age of birdsand immunity of birds ratios among turkeys, domestic and broiler chicken and [26]. common duck while a significant difference found in quail Molecular analysis of Cryptosporidium spp. isolated and feral pigeon compared with the other bird species from birds depending on 18SrRNA revealed distinguish under study. This variation in the percentages attributed among species clearly, this gene used as a distinction to the different areas and environments which samples gene, many studies confirmedthis fact [6, 12, 31]. were collected from it, as well as the different among According to molecular characterization and phenotypic studied birds in their sensitivity and resistance to infect differences the genusc.galliconsiderable a distinct with oocysts of parasite, its age [13, 26] and bird species compared with other speciesbased on three management may also contribute to high infection rates genetic sites which are 18Sr RNA, HSP70 and actin locus such as the following methods of feeding, it may be [20, 32]. Among the species/genotypes, phylogenetic tree opening breeding type (Free in the fields) as: domestic showed a big genetic similarity between C. parvum and chickens, turkeys, common duck in agricultural areas C. meleagridis with neighbor-joining reached to [15, 27], or follow the closed breeding method (Caged 99%-100%. Many researches agreed with our results inside poultry home), as in the case of broiler chicken and [28, 33]. C. meleagridiswas the third most common sometimes the presence of small rodents in the pet shops, Cryptosporidium parasite in human [12], C. meleagridis which could be infected with C. parvum, probably could have a possible to transfer from infected birds to human have caused the spread of oocysts in their home [27]. [24, 29]. Through phylogenetic tree we observed two We know that birds play very important role as genotypes belong to C. parvum and C. meleagridis can disseminators of many pathogens [28], Cryptosporidium infect birds with neighbor-joining reached to 83% and are one of them, our study recorded two important species 65% respectively. C. parvum and C. meleagridis which have ability to infect Finally, our study established the presence of four human and variation mammals [8, 29], so that the species belong to genus Cryptosporidium caused detection of Cryptosporidium oocysts inwild and cryptosporidiosis in birds and these birds represent as a domestic birds is very significant because of biological transporters for Cryptosporidium oocysts to theirmovement from one source to another and contact human, livestock, poultry and other birds. withsporulatedoocysts by man and livestock lead to disseminate cryptosporidiosis infection beside of REFERENCES environmental contamination with viableoocysts [4, 28]. A huge outbreaksof cryptosporidiosis have been 1. Fayer, R., Taxonomy and species delimitation in associated withdrinking water in Milwaukee in1994 in Cryptosporidium. Exp. Parasitol., 124: which about 403, 000 people wereinfected [26]. Therefore 2. Nagano, Y., M. Finn, C. Lowery, J. 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