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ThesisItem Open Access IN VITRO EVALUATION OF ANTICOCCIDIAL PROPERTIES OF SELECTED EDIBLE NUT BY-PRODUCTS ON EIMERIA OOCYSTS OF POULTRY(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-06) SUDHA RANI, MOGILI; Jyothi Sree, Ch.(MAJOR); Sreedevi, C; Bharavi, KThe present study was conducted to evaluate the effect of methanolic extracts from cashew (Anacardium occidentale) nut peel, green coconut (Cocos nucifera) endocarp, almond (Terminalia catappa) hulls and partially developed groundnut (Arachis hypogea) kernel on sporulation and morphology of oocysts of Eimeria species of poultry. Sporulation inhibition bioassay was used to evaluate the activity of four extracts on the sporulation of coccidian oocysts. In this assay, unsporulated oocysts were exposed to five (300, 100, 30, 10, and 3 mg/ml) concentrations of each extract in distilled water in Petri dishes and cell-culture plates while salinomycin, ethopabate, formalin and potassium dichromate solution served as control groups. The Petri dishes and cell-culture plates were partially covered to allow the passage of oxygen and incubated at 25-29°C for 48 h, providing 80% humidity. The sporulation of the oocyst was confirmed by examining sporocysts under microscope at 40x at the end of incubation. The methalolic extracts of A. occidentale peel and T. catappa hulls showed significant (P<0.01) inhibitory effect (88.58±0.39 and 79.24±0.83%, respectively) at highest concentration of 300 mg/ml on sporulation of Eimeria oocysts in comparison with negative control, with reference drugs i.e., salinomycin, ethopabate and formalin (10%) (92.91±1.80, 84.64±2.73 and 98.03±1.04%, respectively) being more effective than the plant extracts. The inhibition of sporulation was not dose dependent for A. occidentale nut peel and T. catappa hulls extracts. At highest concentration (300 mg/ml) the rate of sporulation inhibition by C. nucifera endocarp and partially developed A. hypogea kernels was 8.07±2.34 and 1.43±0.75 per cent respectively, which was imperceptible in comparison with the reference drugs. Extracts of A. occidentale peel induced 50% inhibition at lower concentration (1.099 μg/ml) compared to T. catappa extract (7.627 μg/ml). All four extracts also damaged the normal morphology and shape of the Eimeria oocysts. Thin Layer Chromatography analysis and qualitative phytochemical screening of extracts revealed presence of flavonoids, tannins, saponins, carbohydrates, triterpenoids and alkaloids that might be responsible for the anticoccidial effects noticed. In conclusion, this in vitro study suggests that A. occidentale peel and T. catappa hulls have promising anticoccidial effects.ThesisItem Open Access CROSS SECTIONAL STUDY TO DETERMINE APPARENT OCCURRENCE OF FAMILY Anaplasmataceae IN CANINES IN AND AROUND KRISHNA DISTRICT, ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-05) SREE DIVYA DURGA, G; JEYABAL, L(MAJOR); SREEDEVI, C; SUDHAKAR, KVector-borne diseases (VBDs) are indeed a major health concern for dogs around the world, with the potential to spread zoonoses. The VBDs represent a varied group of diseases, including anaplasmosis and ehrlichiosis, which can cause serious, even life-threatening clinical conditions in dogs. In the present study, prevalence of blood parasites of canines belonging to the family Anaplasmataceae was carried out by blood smear examination in correlation with suitable haemogram and genomic DNA amplification by PCR. A total of 54 blood samples were collected at Teaching Veterinary Clinical Complex, NTR College of Veterinary Science, Gannavaram and nearby private veterinary clinics over a period of nine months (March to November, 2021) from dogs presented with tick infestation and clinical symptoms suggestive of diseases caused by Anaplasmataceae pathogens. The blood samples were initially screened for the presence of Anaplasmataceae species by microscopic examination, which revealed 14.81 (8/54) and 1.85 (1/54) per cent prevalence of Anaplamataceae species (Ehrlichia canis) by Leishman and acridine orange staining technique. None of the samples were positive for E. ewingii, Anaplasma platys, and A. phagocytophilum. DNA from whole blood was isolated to amplify E. canis virB9, E. ewingii p28, A. phagocytophilum 16SrRNA and A. platys 16SrRNA genes. Out of fifty four, 46.29 per cent of dogs were found positive for at least one pathogen of Anaplamataceae by PCR with E. canis (40.74%) being the most prevalent species followed by A. platys (5.56%). Sequences of E. canis and A. platys obtained from dogs demonstrated 95.75% and 99.86% homology with the published sequences of E. canis and A. platys, respectively. There was close similarity between clinical and haematological signs of E. canis and A. platys infections. Pyrexia and lymphadenopathy were frequent signs noticed in infected dogs. Based on clinical signs and haematological findings, dogs found positive for ehrlichiosis were graded as acute, chronic and sub clinical cases. In acute case, fever, anorexia, reduction in all the erythrogram values along with relative monocytosis, neutropenia, lymphocytosis, leucopenia and mild thrombocytopenia was observed. In chronic cases, the erythrogram values were found in the normal range with marginal leucopenia, relative neutrophilia, mild monocytosis and thrombocytopenia. In subclinical cases, the leucogram was found in normal range with severe reduction of erythrogram values. Because of low prevalence of A. platys we could not reveal any correlation between infected dogs and hematological changes but platelets count in infected dogs was found normal. Ehrlichia canis infection was more prevalent (P>0.05) in females and German shepherds dogs. The study reported the prevalence of A. platys for first time in Andhra Pradesh. Further study involving larger number of dogs from other districts of Andhra Pradesh may be beneficial for the effective characterization of Ehrlichia and Anaplasma species in Andhra Pradesh.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF SARCOCYSTIS spp. ISOLATED FROM PIGS IN COASTAL DISTRICTS OF ANDHRA PRADESH, INDIA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-01) MADHURI, GATTA; Jyothi Sree, Ch (MAJOR); Sreedevi, C; Sudhakar, KThe present study was carried out to determine the prevalence, morphological and molecular characteristics of Sarcocystis species in pigs in coastal Andhra Pradesh by microscopy and polymerase chain reaction using tissue samples collected from 163 slaughtered pigs for a period of one year from January 2021 to December 2021. Data regarding age, sex, breed and area of the animals were noted. Microscopic examination of tissue samples collected from different predilection sites such as heart, tongue, oesophagus, intercostals and diaphragmatic pillar muscles from each of 163 slaughtered pigs by intact cyst isolation and pepsin digestion method revealed sarcocysts in 6 (3.68%) and 20 (12.26%) pigs samples, respectively. Tissue samples positive for sarcocysts were fixed in 10% formalin for histopathological examination. Sarcocysts were microscopic with a smooth and thin cyst wall and were filled with banana-shaped bradyzoites. No thick walled cysts were identified. The morphological features of the cystic stage were suggestive of S. suihominis. Histopathological examination of affected muscle tissues revealed mild leucocytic infiltration, myositis and hyaline degeneration along with fatty degeneration. DNA was isolated from each pig tissue sample separately and was separately used to amplify partial segment of 18S rRNA gene of Sarcocystis spp. with the estimated amplicon size of 915 bp. Out of 163 samples, 34 (20.85 %) samples were found to be positive for Sarcocystis species. Subsequent digestion of PCR amplicons with SspI, two different patterns were visualized on electrophoresis, one with 915 bp (S. suihominis) and other with 650 bp and 265 bp (S. meischeriana). Of the 34 positive pigs, 31 (91.2%) were found to be positive for S. suihominis infection, one (2.9%) was found to be positive for S. meischeriana and two pigs (5.9%) were positive for mixed species infection with S. suihominis and S. meischeriana. Sequences of S. suihominis obtained from pigs demonstrated 90-100% identity with the Indian isolates of S. suihominis as single monophyletic group along with Italian isolate of S. suihominis. The prevalence was high in Krishna (36.36%) followed by Guntur (22.22%), Visakhapatnam (20.0%), East Godavari (18.18%), Srikakulam (15.83%), Nellore (14.28%), West Godavari, (8.33%), Prakasam (7.69%) and Vijayanagaram (7.14%) districts of coastal Andhra Pradesh. Age and breed influenced the prevalence of Sarcocystis infection being more prevalent in Non descriptive pigs (P<0.01) and adults (P<0.05). No significant difference was observed between the prevalence of male and female pigs. Overall, the prevalence of S. suihominis (20.24%; 33/163) was more compared to that of S. miescheriana (1.84%; 3/163) in the study area possessing public health risk.ThesisItem Open Access OCCURENCE OF OVINE INTRACELLULAR HAEMOPROTOZOAN PARASITES IN ANDHRA PRADESH AND THEIR ASSOCIATION WITH DRB1 GENE POLYMORPHISM(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) SAI SUMANTH, P; SREEDEVI, C (MAJOR); JYOTHI SREE, CH; SUDHAKAR, KThe present study was carried out to detect Babesia spp. and Theileria spp. in sheep from Andhra Pradesh and to determine the relationship between ovine MHC-DRB1 gene polymorphism and genetic resistance/susceptibility to babesiosis and theileriosis. A total of 249 blood samples including healthy (n=224) and those showing clinical signs (n=25) suggestive of haemoprotozoan infection were collected from sheep from different zones in Andhra Pradesh. The blood samples were initially processed to detect Babesia and Theileria spp. by microscopic examination. Microscopic examination of Giemsa-stained thin blood smears revealed 12.9 (32/249) per cent prevalence of Theileia spp. DNA was isolated from each sample by modified high salt method and amplified by polymerase chain reaction for simultaneous detection of 18S rRNA gene of Babesia and Theileria spp. The Babesia/Theileria genus specific PCR amplified only 430 bp 18S rRNA specific to Theileria spp. in 34.9 (87/249) per cent of sheep samples. None of the samples yielded amplification specific for Babesia spp. or mixed infections. Samples positive for Theileria spp. were further subjected to semi-nested PCR for species specific differentiation. Out of 87 theileriosis sheep, 76 (87.6%) were found to be positive for T. luwenshuni infection and eleven sheep (12.4%) were positive for mixed infection with T. luwenshuni and T.ovis. Overall, the prevalence of T. luwenshuni and T. ovis in sheep was 34.9 (87/249) and 4.4 (11/249) per cent, respectively. Age was found to be significantly (P=0.05) associated with positivity for Theileria infection in sheep being high in adult animals. The prevalence of theileriosis in sheep was not significant with respect to gender, breed and region. The Ovar-DRB1 exon 2 of 249 sheep were amplified by nested PCR from DNA samples. The 296 bp PCR products were digested by REnase BstNI, which generated three genotypes ‘aa’ (86 bp, 87 bp and 123 bp), ‘ab’ (86 bp, 87 bp, 123 bp, and 210 bp) and ‘bb’ (86 bp and 210 bp) with frequencies of 72.0, 22.0 and 6.0 per cent, respectively and two alleles ‘a’ and ‘b’ with frequencies of 0.82 and 0.18, respectively. The χ2 test of independence revealed a significant association (P<0.05) between genotypes and infection status in sheep. Though the frequency of ‘aa’ genotype was more in both the healthy (66.7) and infected (81.6) group, a significantly high frequency of ‘bb’ (6.8) and ‘ab’ (26.5) genotypes was observed compared to the infected group. A proportion of 39.7 per cent of ‘aa’, 23.2 per cent of ‘ab’ and 21.4 per cent of ‘bb’ genotypes were positive for theileriosis among the respective genotype sheep. The frequencies of ‘bb’ genotypes were significantly (P<0.05) higher in the healthy group compared with the theileriosis sheep, which implied that there was an association between these genotypes and theileriosis resistance or susceptibility. Among theileriosis positive sheep the genotype frequencies of ‘aa’, ‘ab’ and ‘bb’ pattern were 81.6 (71), 14.9 (13) and 3.4 (3) per cent, respectively. The frequency of ‘a’ and ‘b’ allele among infected sheep was 0.89 and 0.11 and is not consistent with Hardy-Weinberg equilibrium (P<0.01). The corresponding allele frequencies in the healthy sheep were 0.80 and 0.20.
