MOLECULAR DETECTION AND TYPING OF CRYPTOSPORIDIUM IN CAPTIVE WILDLIFE
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Date
2014-10
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT :
The present research work has been undertaken with an objective to investigate the Cryptosporidium infection in captive wildlife in Andhra Pradesh. Conventional microscopy (mZN staining method) was adopted for preliminary screening of faecal samples for detection of Cryptosporidium infection. Subsequently, the mZN staining positive isolates were confirmed by nested PCR. Further, the selected nested PCR amplicons were sequenced to determine the Cryptosporidium species. Seven hundred and eighty eight faecal samples were collected from 127 captive wildlife species of all the three zoological parks viz., Sri Venkateswara Zoological Park (S. V. Zoological Park), Tirupati (n=242); Indira Gandhi Zoological Park (I. G. Zoological Park), Visakhapatnam (n=218); Nehru Zoological Park, Hyderabad (n=328), in Andhra Pradesh and screened for the presence of the Cryptosporidium infection. In mZN staining method, a total of 57 (7.23%) out of 788 faecal samples screened were found positive for Cryptosporidium oocysts. The highest prevalence of Cryptosporidium infection was detected by mZN staining method in the faecal samples of wildlife at Nehru Zoological Park, Hyderabad (8.23%), followed by S. V. Zoological Park, Tirupati (7.44%) and I. G. Zoological Park, Visakhapatnam (5.50%). Among the Cryptosporidium positives (n=57) by mZN staining method from the wildlife of all the three zoological parks, the highest prevalence (47.37%) was recorded in the wildlife of Nehru Zoological Park, Hyderabad, followed by S. V. Zoological Park, Tirupati (31.58%) and I. G. Zoological Park, Visakhapatnam (21.10%). Regarding, the kind of wildlife the highest prevalence of Cryptosporidium was observed in rodents (18.18%) followed by reptiles (11.54%), primates (11.11%), herbivores (9.29%), birds (7.79%) and the lowest was recorded in carnivores (1.54%).
All the 57 Cryptosporidium positive faecal samples by mZN staining method were subjected to nested PCR targeting 18S rRNA gene for confirmation of Cryptosporidium DNA and were successful for 51 (89.47%) samples. Eighteen mZN Cryptosporidium positive isolates from wildlife of S. V. Zoological Park, Tirupati were subjected to nPCR for confirmation of Cryptosporidium DNA. Except two samples, remaining amplicons were detected the Cryptosporidium DNA with 88.89% confirmation. All the twelve mZN Cryptosporidium positive isolates from the wildlife of I. G. Zoological Park, Visakhapatnam were confirmed the Cryptosporidium DNA by nPCR. Out of 27 mZN Cryptosporidium isolates from Nehru Zoological Park, Hyderabad screened by nPCR, 23 samples were confirmed the Cryptosporidium DNA at 85.19 % level. Fourteen nPCR Cryptosporidium positive amplicons were subjected to sequencing and only 12 were successful in speciation of Cryptosporidium. Species discrimination using BLAST determined the Cryptosporidium speciation and the percent nucleotide identity of the five species ranged between 98-99% with their respective available sequences in the GenBankTM. Five species of Cryptosporidium were characterized in the wildlife of all the three zoological parks in Andhra Pradesh. (Cryptosporidium parvum; C. ryanae, C. suis, C. muris and Cryptosporidium avian genotype III). The observations in the current study conclude that, Cryptosporidium infection was prevalent in the captive wildlife from the zoological parks of Andhra Pradesh. For the first time, the zoonotic species, C. parvum was recorded in one rescued elephant calf of S. V. Zoological Park, Tirupati apart from other four Cryptosporidium species in different kind of wildlife. As per the available published literature, this study is the first of its kind on the prevalence of Cryptosporidium in the captive wildlife in India.
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