Molecular studies on Cryptosporidium and Giardia species in cattle calves and therapeutic evaluation of Curcuma longa against experimental cryptosporidiosis in mice
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Date
2019-10-15
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Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K)
Abstract
The prevalence studies were based on identification of Cryptosporidium oocyst and Giardia cyst in cattle calves of Jammu region by examining 614 faecal samples subjected to Diethyl ether sedimentation technique (DEES) followed by modified Ziehl-Neelsen staining technique and lugols iodine wet mount technique for identification of Cryptosporidiumspp.andGiardia spp. infection. The study revealed an overall positivity of 144 animals (23.45%) for cryptosporidiosis and of 43 animals (7.00%) for Giardia infection. Highest prevalence of Cryptosporidium spp. was recorded in Samba (28.57%) district, whereas highest prevalence of giardiosis was recorded in Kathua (7.78%) district of Jammu region. The highest prevalence of Cryptosporidium infection was recorded in < 1 month of age animals (43.24%) than other age group whereas highest prevalence of Giardia infection (11.2%) was recorded in 1-6 month age group. Diarrhoeic animals revealed significantly higher prevalence (29.51%) of Cryptosporidium infection and (9.02%) Giardia infection as compared to non diarrhoeic animals. Highest prevalence of Cryptosporidium infection was recorded (34.65%) in winter months from December to February but in case of Giardia infection it was recorded highest (10.34%) in monsoon months from July to September. Cattle calves having mucus in the faeces showed significantly higher prevalence (37.33%) of cryptosporidiosis whereas in case of giardiosis faecal samples with no mucus/blood showed significantly higher prevalence (7.62%). Male animals showed non significantly higher incidence of Cryptosporidium infection (37.77%) and Giardia infection (12.77%) as compared to females. Genetic characterisation of 120 samples (40 samples per age group) found positive by mZN was carried using molecular techniques. Nested PCR of 18S small subunit (SSU) rRNA gene of Cryptosporidium spp. amplified a product of 830 bp band size whereas β-giardin gene of Giardia spp. amplified a product of 511 bp band size.RFLP analysis of nested PCR product of Cryptosporidium spp. by three restriction enzymes namely SspI, VspI and MboIIwas carried which resulted in higher prevalence of C. parvum(74.07%) in young animalswhereas, 16.66% were positive for C. andersoniand 9.25% were found to be positive for C. bovis.For subtyping of C. parvum,DNA of 17 positive samples GP60 gene was amplified and sequenced andyielded clear band at 850 bp. Molecular phylogenetic analysis of these sequences showed that these C. parvum isolates belonged to one subtype family, i.e., IIa. Fifteen of the C. parvum specimens belonged to the subtype IIaA15G2R1, whereas two specimens belonged to subtype IIaA14G2R1. The 511 bp nested product of Giardia spp. when digested by Hae III restriction enzymes, zoonotic Giardia intestinalis Assemblage B with 1.6% prevalence and Assemblage E with 14.16% prevalence in young cattle calves of Jammu region was recorded.Administration of curcumin, obtained from ethanolic extract of Curcuma longa as therapeutic agent at dose rate of 8 mg/ kg body weight for five days resulted in decreased oocyst production in experimentally infected mice. Although higher body weight gain was recorded in treated groups, but it was not significantly affected. The findings of the present study clearly suggest that molecular characterization and subtypes/assemblages of Cryptosporidium spp. and Giardia spp. established zoonotic potential of C. parvum and G. intestinalis infecting cattle of Jammu region. Curcuma longa showed promising anticryptosporidial effects in reduction of oocyst count in experimentally infected mice and highlights the significance of further trial in other susceptible host like cattle.
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