Prevalence and molecular characterisation of Cryptosporidium

Transcription

1 CRYPTOSPORIDIUM IN DIARRHOEIC GOAT KIDS IN TURKEY 229 Prevalence and molecular characterisation of Cryptosporidium spp. in diarrhoeic pre-weaned goat kids reared under traditional farming system in Diyarbakır, Southeastern Anatolia City, Turkey D. N. SAYIN İPEK University of Dicle, Faculty of Veterinary Medicine, Department of Parasitology, Diyarbakır, Turkey. *Corresponding author: ABSTRACT Cryptosporidium is an enteropathogenic protozoan that causes diarrhoea in humans and various domestic animals, worldwide. Faecal samples were collected for up to one month from 112 diarrhoeic goat kids on 12 traditional hair goat farms to assess the prevalence of Cryptosporidium in Diyarbakır, Southeastern Anatolia, Turkey. These samples were evaluated using direct immunofluorescent antibody technique. Cryptosporidium oocysts were found in 76 of 112 (67.9%) samples, with an average of 44,059 oocysts per gram feces (opg) for Cryptosporidium. PCR products of SSU rrna locus were isolated from all samples in which Cryptosporidium oocysts were detected. On the basis of these results, molecular analysis was conducted to identify the predominant pathogenic species. Two principle species were identified, including Cryptosporidium parvum (96.1%) and C. xiaoi (3.9%). Furthermore, four subtypes were found to have zoonotic potential, as revealed by sequence analysis of the 60 kda glycoprotein (gp60) gene obtained from C. parvum isolates. Furthermore, our findings show that goat kids are an excellent host for zoonotic C. parvum and its subtypes. Keywords: Diarrhoeic goat kids, Cryptosporidium, species, subtypes, Turkey RÉSUMÉ Prévalence et caractérisation moléculaire de Cryptosporidium spp. chez des chevreaux diarrhéiques pré-sevrés élevés dans le cadre d un système agricole traditionnel à Diyarbakir, Turquie. Le cryptosporidium est un protozoaire entéropathique qui cause la diarrhée chez l homme et divers animaux domestiques dans le monde entier. Des échantillons de matières fécales ont été prélevés pendant une période pouvant aller jusqu à un mois sur 112 chevreaux diarrhéiques dans 12 élevages traditionnels de chèvres à poils afin d évaluer la prévalence du Cryptosporidium à Diyarbakir, dans le sud-est de l Anatolie, en Turquie. Ces échantillons ont été analysés par immunofluorescense directe. Des oocystes de Cryptosporidium ont été trouvés dans 76 des 112 (67,9 %) échantillons, avec une moyenne de oocystes par gramme de fèces (opg). Des produits de PCR du locus ARNr SSU ont été isolés à partir de tous les échantillons dans lesquels des oocystes de Cryptosporidium ont été détectés. Sur la base de ces résultats, une analyse moléculaire a été effectuée pour identifier les espèces pathogènes prédominantes. Deux espèces principales ont été identifiées, dont Cryptosporidium parvum (96,1 %) et C. xiaoi (3,9 %). En outre, quatre sous-types se sont révélés avoir un potentiel zoonotique, comme le révèle l analyse séquentielle du gène de la glycoprotéine 60 kda (gp60) obtenu à partir d isolats de C. parvum. Ces résultats montrent que les chevreaux sont un excellent hôte pour C. parvum et ses sous-types à l origine de zoonose. Mots-clés: chevreaux, diarrhée, Cryptosporidium, Turquie Introduction Cryptosporidium, a common enteropathogenic protozoan that causes diarrhoea in humans and various domestic animals, is prevalent worldwide [50]. In neonatal ruminants, the most significant clinical symptom of a Cryptosporidium infection is yellow diarrhoea with or without blood [33, 34]. Cryptosporidium infections may cause death in goat kids, which leads to significant economic losses in the goat farming industry [8, 20, 31]. Additionally, faecal-derived oocysts, especially the potentially zoonotic species, may remain alive outside the body for a long period. This creates a public health risk by contaminating potable water supply in areas where hygiene is poor [4, 41, 54]. Prevalence studies of cryptosporidiosis in adult goats and goat kids are limited. In these studies, it was found that the prevalence of this disease in goats and goat kids ranged from 2.5% to 76.4% [5, 14]. In Turkey, goat breeding has increasingly gained importance, but epidemiological studies of cryptosporidiosis in goats and goat kids has been limited to İzmir (46%), Konya (9.5%), and Van (10.7%) [7, 12, 43]. A few studies have identified Cryptosporidium spp. and their subtypes in goats and goat kids around the world. Previous studies have demonstrated that Cryptosporidium parvum and C. xiaoi are the dominant species infecting goats [9-11, 13, 16, 26, 30, 45, 49]. C. hominis, C. ubiquitum, C. andersoni, and Cryptosporidium rat genotypes are other species commonly found in goats [21-23, 40, 50]. Molecular studies identifying Cryptosporidium spp. in goats in Turkey were conducted in a limited region by TAYLAN-OZKAN et al. (2016), and only C. parvum was identified [48]. Various molecular subtyping tools have been developed to study the risks of zoonotic transmission of Cryptosporidium spp. Sequence analysis of the 60 kda glycoprotein (gp60) gene, which allows the identification of C. parvum subtype families, is one of the most commonly used molecular subtyping tools. At least 11 subtype families (IIa, IIb, IIc,

2 230 SAYIN IPEK (D. N.) IId, IIe, IIf, IIg, IIh, IIi, IIk, and IIl) have been identified among C. parvum isolates from humans and animals [42, 51, 52]. However, only subtypes IIa and IIb are found in goats, and these appear to be zoonotic [9, 13, 23, 38, 48, 49]. The objective of the present study was to examine the prevalence and intensity of Cryptosporidium infections on goat farms in Turkey, and to characterise isolates to evaluate potential zoonotic risks. Materials and methods STUDY AREAS AND ANIMAL SOURCES From February 2016 to March 2016, a total of 112 faecal samples were collected from the rectum of diarrhoeic goat kids less than one month of age (2-4 week), by using sterile gloves and swaps. Samples were obtained from 12 smallholding goat farms (46 72 goats per farm). These farms maintain animals using non-professional and traditional methods in four districts of Diyarbakır, which is located in southeastern Anatolia, Turkey. Three farms were located in Bismil (37.50 N, E; southeast Diyarbakır), three were in Dicle (38.22 N, E; north Diyarbakır), three were in Karacadağ (37.67 N, E; southwest Diyarbakır), and three were in the Central district (37.55 N, E). Faecal samples were brought to the laboratory and examined in a cold chain. Microscopic analyses were conducted within 24 h. MICROSCOPIC ANALYSIS To confirm the presence of Cryptosporidium oocysts, the commercially available Crypto/Giardia-Cel FITC Staining Kit (Cellabs Inc., Brookvale, Australia) was used. Nine ml phosphate buffered saline (PBS) containing of 0.1% sodium azide were added to faecal samples weighing 1 g, and the samples were vortexed and centrifuged at 2000 rpm for 10 min. Next, 20 µl of supernatant from each sample was added to a single well in 8-well Teflon lams. After the samples dried, they were fixed with acetone for 5 min, and 25 µl of monoclonal antibody was added to each well. Samples were incubated in a humid chamber at 37 C for 30 min. Subsequently, the lams were washed with PBS for 1 min, and moult solution was dripped into the samples in each well. Each sample was then sealed with a coverslip. Each well of the lam was examined under 200, 400, and 1000 magnification using a fluorescence microscope. The oocytes identified in each faecal sample were counted at 200 magnification, and the number of oocysts per gram faeces (opg) was calculated as follows: (number of cysts or oocysts/ [volume of sample examined (ml) weight of faeces (g)]). DNA EXTRACTION, GENE AMPLIFICATION AND SEQUENCING Total DNA extraction was conducted with the direction of suggestion of kit by using ZR Fecal DNA MiniPrep kit (Zymo Research, Irvine, CA). In order to species identification, all the DNA extracts were subjected to a nested PCR protocol to amplify a fragment of the SSU rrna gene of Cryptosporidium (830 bp). For the primary PCR, a PCR product of 1325 bp was amplified using the forward primer 5 -TTCTAGAGCTAATACATGCG-3 and the reverse primer 5 -CCCTAATCCTTCGAAACAGGA-3 [53]. PCR reactions were carried out in a total volume of 50 µl containing 10 PCR buffer, 3 mm MgCl2, 0.2 mm each dntp, 20 pmol of each primer and 2 U of Taq DNA polymerase under the following conditions: initial denaturation at 94 C for 3 min, 35 cycles at 94 C for 45s, 55 C for 45s, and 72 C for 1 min, and a final extension step at 72 C for 7 min. For the second round of PCR, reactions were carried out using primers forward r 5 -GGAAGGGTTGTATTTATTAGATAAAG-3 and 5 -AAGGAGTAAGGAACAACCTCCA-3 [53]. The nested PCR mixture and conditions was identical to the primary PCR except that a concentration of 1.5 mm MgCl 2 was used. In order to identify the subtypes of C.parvum, approximately 550bp fragment of 60 kda glycoprotein (Gp60) gene was amplified with nested PCR by using 5 -ATAGTCTCCGCT GTATTC-3 and 5 -GCAGAGGAACCAGCATC-3 primer set at the first PCR step and 5 -TCCGCTGTATTCTCAGCC-3 and 5 -GAGATATATCTTGGTGCG-3 primer set at second PCR step [37]. The PCR products were subjected to electrophoresis in a 1% agarose gel and visualized by staining with ethidium bromide. Negative and positive controls were included in all PCR sets. The secondary PCR products were sequenced in both directions on an automated sequencer (ABI PRISM 310 model; Perkin-Elmer, USA). STATISTICAL ANALYSIS Statistical analysis was used by SPSS 15.0 software for Windows. Pearson s chi-square test was performed to evaluate differences of prevalence in the study area. Differences were considered significant when P < Results MICROSCOPIC FINDINGS Microscopic analysis of 112 diarrhoeic faecal samples revealed the presence of Cryptosporidium oocysts in 76 samples (67.9%) obtained from 12 goat farms. The prevalence of Cryptosporidiosis across districts ranged from 57.1% to 75.9% (χ 2 = 2.67; P > 0.05, Table I). The average number of Cryptosporidium oocysts per gram faeces (opg) was 44,059 and ranged from 1,036 to 156,238. CRYPTOSPORIDIUM SPECIES AND SUBTYPES A fragment of the SSU rrna gene was amplified from the 76 samples in which Cryptosporidium oocysts were microscopically identified. The sequences of the PCR products were analysed, and it was determined that 73 of the 76

3 CRYPTOSPORIDIUM IN DIARRHOEIC GOAT KIDS IN TURKEY 231 samples (96.1%) harboured C. parvum oocysts, and the other three (3.9%) contained C. xiaoi oocysts (Table I). C. parvum was isolated from all farms, while C. xiaoi was only found at two farms in the central district. The C. parvum subtypes identified in this study were identical to those submitted under the GenBank numbers AH , KM , and HQ Meanwhile, the C. xiaoi subtype matched the GenBank sample submitted as KM PCR products for the gp60 locus were obtained for all 73 isolates identified as C. parvum. However, sequence analyses could not obtained for six of these samples. The sequence analyses identified the IIa and IId subtype families, and IIa was the most common subtype family (74.6%; 50/73). Two C. parvum IIa subtypes were found at the goat farms, including IIaA14G1R1and IIaA15G1R1. IIaA14G1R1 subtype was present in 25 goat kids on seven farms in the Karacadağ, Bismil, Central, and Dicle districts. Meanwhile, the IIaA15G1R1 subtype was identified in 25 animals from six farms in the Karacadağ, Bismil, Central, and Dicle districts. Two IId subtypes were found in 17 samples, including IIdA17G1 (8/17) and IIdA18G1 (9/17). Subtype IIdA17G1 was found on four farms in Karacadağ, Bismil, Central, and Dicle districts. Additionally, subtype IIdA18G1 was detected on three farms in the Central and Dicle districts (Table II). Finally, the IIaA14G1R1, IIaA15G1R1, IIdA17G1, and IIdA18G1 subtypes matched the GenBank submissions KF , AB , LT , and AY , respectively. Discussion Our study revealed that Cryptosporidium is a prevalent and widely distributed enteric pathogen in diarrhoeic preweaned goat kids reared under traditional farming system in Diyarbakır, southeastern Anatolia. The high prevalence (67.8%) is in line with previous studies in goat kids in Turkey and world [9, 14, 28, 48]. It was reported that the number of oocysts excreted in an animal s waste contributes to infection rates [32]. In a study conducted on goat kids, GEURDEN et al. (2008) found that oocyst excretion averaged 231,926 opg, and TZANIDAKIS et al. (2014) reported that oocyst excretion in goat kids was 47,744 opg [13, 49]. In our study, oocyst excretion averaged 44,059 opg in diarrhoeic goat kids. Thus, the correlation between high rates of cryptosporidiosis infections and a large amount of oocysts excreted in the waste of diarrhoeic goat kids indicates that oocyst load is a significant risk factor for the spread of this disease. In studies conducted in various areas of the world, C. parvum was reported to be the most common species infecting goats and goat kids [3, 15-17, 25, 30, 31, 38]. In one study conducted in Turkey, all nine goat kids that were diagnosed with Cryptosporidium infections were found to harbour C. parvum [48]. In our study, C. parvum was identified as the dominant species (96.1%, 73/76) similar to these results reported in Turkey and around the world. In recent years, C. xiaoi was found to be the dominant species in studies conducted on goat kids in countries such as Greece [49], China [26], Spain [10] and France [40]. Additionally, ADAMU et al. (2014) reported that C. xiaoi was present in two HIV/AIDS patients in Ethiopia, and they believed that this created a potential human health risk [1]. Along District Number of goat examined Crytosporidium positive C.parvum Karacadağ (65.38%) 17 Bismil (57.14%) 16 Central (75.86%) 19 3 Dicle (72.41%) 21 C.xiaoi Total (67.8%) 73 (96.1%) 3 (3.9%) Table I: Prevalence and molecular characterization of Cryptosporidium spp. in diarrhoeic goat kids in different district Subtype of C. parvum District llaa14g1r1 llaa15g1r1 llda17g1 llda18g1 Unidendifed Karacadağ Bismil Central Dicle Total 25 (34.2%) 25 (34.2%) 8 (10.5%) 9 (12.3%) 6 (8.2%) Table II: Crytosporidium parvum GP60 subtypes identified in diarrhoeic goat kids

4 232 SAYIN IPEK (D. N.) with that the number of molecular studies concerning to Cryptosporidium infections is very limited in Turkey, a publication which reported the existence of C. xiaoi was not found [48]. Thus, our study is the first to describe C. xiaoi in Turkey, even though it was only seen in 3.9% (3/76) of the infected goat kids. Previous studies of gp60 sequences have identified C. parvum isolates from goats as members of the potentially zoonotic subtype families IIa and IId [9, 13, 23, 38, 48, 49]. Similar to previous studies, the IIa and IId families were also found in Turkey in this study, and IIa (74.62% 50/73) was identified as the most common subtype family. While the IIa subtype family was reported to be dominant in studies conducted in Spain (55/58) [9], Norway (1/1) [23], Papua New Guinea (2/2) [22] and Turkey (3/8) [48], IId was found to be the predominant family in Belgium (8/11) [13], Greece (2/2,) [49], Spain (17/17) [38] and China (8/12 ) [26]. Subtypes IIaA15G1R1(25/50) and IIaA14G1R1 (25/50) belong to the IIa subtype family, which was dominant in our study and observed in equal ratios. Subtype IIaA15G1R1 was found in goats in previous studies in China [26] and Turkey [48]. It was also isolated from humans in Egypt [18], Lebanon [34] and Kuwait [45]. Subtype IIaA14G1R1 was the most common subtype in humans [19, 29, 46]. It was also found in yaks and cattle [26,39]. The IIaA14G1R1(8/17) subtype detected in this study is the first case reported in goats. Subtype IIdA17G1(8/17), which we identified in goats, was reported previously in studies of goats in Spain [9], and it was mainly found in cattle [2, 24] and humans [2, 6]. Subtype IIdA18G1 (9/17) was observed in goats in Turkey [48], and it was reported in children in Iran [29], Kuwait [47]. It was also found in yaks in China [27]. 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