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Open Access
Molecular characterization of Fasciola species in sheep and goats of Andhra Pradesh, India
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-11) JYOTHI SREE, Ch; MALAKONDAIAH, P(MAJOR); CHENGALVA RAYULU, V; Rama Devi, V; RAMANI PUSHPA, R.N
ABSTRACT: The present investigation has been undertaken to study the “Molecular characterization of Fasciola species in sheep and goats of Andhra Pradesh, India”. Slaughter/ necropsy examination of 5192 sheep and 2070 goats livers revealed 124 (2.38%) and 29 (1.40%) positive for Fasciola spp. with an overall prevalence rate of 2.10%. Higher prevalence was recorded in female sheep (3.50%) and goats (1.70%) than males of sheep (2.10%) and goats (1.24%), respectively. Highest prevalence was revealed in youngs (2.61% and 1.62%) than in adults (1.86% and 1.25% in 1-3 yrs and 2.11% and 1.22% in more than 3 years age) in sheep and goats, respectively. Highest infection rate was recorded in summer (2.89% & 1.53%) followed by winter (2.35% & 1.51%) and rainy seasons (1.19% & 0.91%) in sheep and goats, respectively. Present investigation also revealed that the prevalence of Fasciola spp. was the highest in April (6.05%) in sheep and in March (4.13%) for goats. Morphometric analysis was displayed large leaf like appearance of flukes with length and width ranged between 27.1 - Name of the author 36.5mm and 4.8 - 6.3mm in sheep and 27.5 - 38.1mm and 4.5 - 7.4mm in goats, respectively. Allometric values of Ventral Sucker to Posterior end (VS-P) and ratio of body length and width of adult flukes were 24.35-29.8mm and 4.71-6.29mm in sheep whereas 24.6-34.8mm and 4.89-6.73 mm in goats, respectively. Allometric values of the flukes collected in the present study were within the standard range (VS-P=26.8- 50.09mm, BL/BW=3.4-6.78mm) of F. gigantica and confirmed that the flukes from different parts of Andhra Pradesh were morphologically F. gigantica. Amplification of 275 genomic DNA samples (175 sheep and 100 goats) with their respective primers yielded a 433bp (ITS 1) and 550bp (ITS 2) products, respectively and confirmed them as Fasciola spp. Upon digestion with Rsa1 and BspHI restriction enzymes, the PCR-RFLP revealed a fragment of 233bp and 200bp in ITS 1 and 377bp and 173bp products in ITS 2 from all isolates of sheep (50) and goats (50), respectively. Sequencing analysis of randomly selected ITS 2 PCR products (3 sheep, 2 goats and 1 buffalo) revealed 100% homozygous within the species and a very few variations with the other geographical isolates. Pair wise distance analysis revealed 0% divergence in between them and 1-25% with the other geographical isolates of F. gigantica whereas 6% divergence with the F. hepatica. The phylogenetic analysis of ITS 2 sequences (6) revealed a close relationship with F. gigantica isolates of Asia (India, Iran, China, Thailand, South Korea, Vietnam, Indonesia, Pakistan) and Africa (Egypt, Zambia, Kenya, Mauritania). While the F. hepatica from Iran, Egypt, Australia and France were also exhibited relationship with the AP isolates and observed as a separate sub branch. The phylogeny of Andhra Pradesh isolates revealed that they were clustered in a same sub branch with regardless of their host, geographical origin and maternally inherited from F. gigantica.
Open Access
EVALUATION OF IN VITRO ANTHELMINTIC ACTIVITY OF CHITOSAN ENCAPSULATED ALBENDAZOLE AGAINST GASTROINTESTINAL NEMATODES OF SHEEP
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-02) RAMYA, V; CHENGALVA RAYULU, V(MAJOR); VENU, R; ALPHA RAJ, M
ABSTRACT: Present investigation was carried out to evaluate the anthelmintic activity of chitosan encapsulated albendazole with phytochemical combinations against GI nematodes of sheep. Various microspheres including chitosan alginate (CS-ALG), chitosan encapsulated albendazole (CS-ABZ), chitosan encapsulated albendazole in combination with five phytochemicals viz., caffeic acid, cinnamic acid, curcumin, piperine and syringic acid were synthesized and characterized. Phytochemical loaded CS-ABZ microspheres were designated as chitosan encapsulated albendazole with caffeic acid (CS-ABZ-CAA), chitosan encapsulated albendazole with cinnamic acid (CS-ABZ-CIA), chitosan encapsulated albendazole with curcumin (CS-ABZ-CUR), chitosan encapsulated albendazole with piperine (CS-ABZ-PIP) and chitosan encapsulated albendazole with syringic acid (CS-ABZ-SYN). Synthesized microspheres were spherical with a diameter ranging from 0.470 to 0.634 mm. The mean diameter of CS-ALG and CS-ABZ were significantly (P<0.05) lower than CSABZ- CIA. Majority of unloaded CS-ALG microspheres had a diameter of 0.3 mm. Loading of CS-ALG with ABZ and phytochemicals resulted in microspheres with 0.3 to 0.8 mm diameter. Maximum concentration of ABZ was achieved in CS-ABZ microspheres (55.56mg/100mg). Addition of phytochemicals decreased the loading of ABZ in the microspheres. The highest concentration of ABZ was observed in CSABZ- CUR (19.53 mg/100mg) and the lowest in CS-ABZ-CIA (11.63 mg/100mg). Phytochemical concentration (mg/100mg) of microspheres ranged from 0.78 (CS-ABZ-CAA) to 1.18 (CS-ABZ-SYA). Loading efficacy of CS-ABZ, CS-ABZCAA, CS-ABZ-CIA, CS-ABZ-CUR, CS-ABZ-PIP and CS-ABZ-SYN estimated in this study was 69.45, 23.63, 14.54, 23.42, 23.23 and 19.28 percent, respectively. The highest albendazole loading efficacy (69.45%) achieved in CS-ABZ decreased with the addition of phytochemicals. The release of ABZ from the microspheres under simulated gastric conditions was minimum at pH 1.2 (1.93 to 6.65%) and maximum at pH 6.8 (10.13 to 51.73%). Adding phytochemicals to the microspheres improved the release of ABZ in basic pH with the highest release from PIP microspheres (51.73%) followed by CAA (39.04%) and SYA (38.10%) while CUR and CIA had no effect. Release of ABZ from all the microspheres followed Krosmeyer-Peppas model (Q=ktn) with R2 ranging from 0.845 to 0.915. The release from ABZ, CAA, CIA, PIP and SYA was predominantly through anomalous diffusion while release from CUR microspheres followed zero order. Examination of sheep faecal samples collected during the investigation revealed the presence of eggs of Haemonchus contortus, Strogyloides spp., Bunostomum spp. and Trichuris spp. Egg hatch test (EHT) with LC50 (μg mL-1) values of ABZ (0.51), CS-ABZ (0.40) and phytochemicals (0.57 to 1.30) indicated the development of anthelmintic resistance of GI nematodes. Whereas the LC50 (μg mL-1) of CS-ABZ-CUR (0.09) and CS-ABZ-PIP (0.08) were significantly (P<0.05) lower than ABZ and CS-ABZ. The LC50 (μg mL-1) values of CS-ABZ-CAA (0.27), CS-ABZ-CIA (0.24), CS-ABZ-CUR (0.25), CS-ABZ-PIP (0.17) and CSABZ- SYA (0.26) obtained with larval development test (LDT) were significantly (P<0.05) lower than CS-ABZ (0.41) and pure ABZ (0.46). There was no significant (P>0.05) difference between the LC50 values of various phytochemical loaded microspheres. Coproculture of positive faecal samples for GI nematodes revealed the presence of third stage larvae of H. contortus and Strongyloides spp. No third stage larvae were observed from the wells after LDT with CS-ABZ-CAA, CS-ABZ-CIA, CS-ABZ-CUR, CS-ABZ-PIP and CS-ABZ-SYA. However, live third stage larvae of H. contortus were recovered from the wells cultured with ABZ, CS-ABZ and pure phytochemicals. Pure ABZ, CS-ABZ and pure phytochemicals were found least effective against GI nematodes of sheep as evidenced by EHT and LDT. CS-ABZ-PIP and CSABZ- CUR microspheres synthesized in the present study were found to be better alternatives to routine chemical anthelmintics for treatment of GI nematodes. These microspheres can overcome ABZ anthelmintic resistance due to synergistic combination with phytochemicals. Chitosan encapsulated albendazole with phytochemical combination is an effective novel formulation with improved anthelmintic activity and release kinetics.
Open Access
PREVALENCE, HAEMATOLOGICAL, BIOCHEMICAL AND HISTOPATHOLOGICAL OBSERVATIONS OF PARAMPHISTOMOSIS OF DOMESTIC RUMINANTS IN TIRUPATI
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) PREETHI, M; VENU, R(MAJOR); SRINIVASA RAO, K; SRILATHA, Ch; VINOD KUMAR, N
ABSTRACT: The present study on ‘prevalence, haematological, biochemical and histopathological observations of paramphistomosis of domestic ruminants in Tirupati’ was conducted in cattle, sheep and goats. A total of 2133 samples (dung, blood, serum, tissues and amphistome specimens) from slaughtered domestic ruminants was examined. In direct faecal smear examination, an overall prevalence of 24.29 percent of paramphistomosis infection was recorded, whereas in faecal sedimentation method, 32.51 percent was observed. In cattle, the prevalence of infection by direct faecal smear and faecal sedimentation methods were 17.43 and 31.19 percent, respectively. In sheep, the prevalence rates were recorded higher than cattle. In goats, the prevalence of paramphistomosis by direct faecal smear examination was 20.66 percent, whereas by faecal sedimentation method, it was 30.52 percent. Out of 109 cattle carcasses, in 47 cases (43.12) amphistomes were found in rumen, reticulum and bileduct during slaughterhouse examination. In sheep and goat, the prevalence rates were 42.15 and 40.85 percent, respectively by slaughterhouse examination. Age-wise, higher prevalence was recorded in cattle of 2-4 years followed by older animals of above 4 years and young animals of <2 years. Slightly higher prevalence (26.79%) was noticed in >1- 2 years old sheep than <1year age group. In goats, the prevalence of infection was lower in the age group of <1year, when compared to their counterparts in sheep. Based on slaughterhouse study, the sheep of <1 year old were more infected (56.28%) than >1-2 years old sheep (36.85%). In contrast, the higher prevalence was noticed in >1-2 years (63.27%) than < 1 year old goats (21.74%). Sex-wise, the prevalence of infection in male cattle was slightly higher than females by direct faecal smear and slaughterhouse examinations, whereas in sedimentation method, female animals showed higher infection than male. The prevalence of infection in female sheep was higher than male sheep. Overall, statistically no significance difference was observed between male and females, in respect to their diagnostic method. Blood samples were screened for haematological observations such as PCV, Hb and RBC counts. Statistically, significant difference was noted between immature amphistomosis and uninfected groups of cattle, sheep and goats. No significant difference was noted between infected and uninfected animals of cattle and sheep in relation to PCV, Hb and RBC values, but in goats significant difference was observed. In DLC, statistically, no significant difference was observed in immature, infected and uninfected amphistomosis cattle. In immature and uninfected sheep, neutrophil and monocyte values showed significant alterations, but in goats it was in neutrophil and lymphocytes. Serum sample screening revealed, low levels of total proteins and albumins in immature amphistomosis infected cattle, sheep and goats. Gross and histopathological changes of amphistome infected rumen, reticulum and bile duct were observed. The adult amphistome parasites invading the rumen mucosa by sucker and plugging of mucosa of bile duct was noticed in cattle. In the bile duct of cattle, massive and extensive infiltration of plasma cells, lymphocytes and monocytes in between acinar structures was recorded. Hyperplasia of bile duct, degenerative changes and necrotic changes in acinar mucosal lining epithelial cells were appreciated prominently. In immature amphistomosis, duodenum of sheep revealed thickened and edematous; mucosal surface was severely congested, petechial haemorrhages and necrosis were also noticed. Histopathological studies of infected duodenum of sheep revealed, plugging of the mucosa with sucker and extensive infiltration of macrophages and plasma cells deep into the mucosa. Based on morphology, 5 amphistomes were recognized viz., Cotylophoron cotylophorum, Paramphistomum cervi, Gastrothylax crumenifer, Fischoederius elongatus and Gigantocotyle explanatum. Mixed infections were noticed higher at 50, 73.6 and 62 percent, in cattle, sheep and goat, respectively. In immature amphistomosis infected ruminants, haematological and biochemical parameters were estimated and showed a great variation and microscopic examination of duodenal wall scrapings revealed immature amphistomes.
Open Access
IDENTIFICATION OF SARCOCYSTIS SPECIES IN CATTLE (Bos taurus) BY PCR-RFLP
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-10) MOUNIKA, K; SREEDEVI, C(MAJOR); VENU, R; SRINIVASA RAO, T
ABSTRACT: The present study was carried out to determine the prevalence of bovine sarcocystosis and identify various species of Sarcocystis from cattle in different regions of Chittoor district, Andhra Pradesh by PCR-RFLP. Macroscopic examination and microscopic examination by pepsin-HCl digestion method of 150 slaughtered cattle from Tirupati, Chandragiri, Chittoor, Renigunta and Pakala regions in Chittoor district revealed an overall 91.33 per cent of prevalence of sarcocystosis. The infection was highly prevalent in Tirupati (97.14 %) compared to that of Chandragiri (92.5 %), Renigunta (90.0 %),Chittoor (80.0 %), and Pakala (70.0 %) regions of Chittoor district. The prevalence of macroscopic and microscopic sarcocysts was 6.57 and 93.43 per cent respectively. Macroscopic cysts were exclusively observed in oesophagus. The prevalence of infection increased with advance in age and there was a significant relationship between the prevalence of infection and age groups of cattle (P<0.001). There was no significant difference (P>0.001) between the prevalence of Sarcocystis infection in male (91.76 %) and female (90.76 %) cattle. Genomic DNA was extracted separately from bradyzoites of all 137 cattle that were positive for sarcocystosis and amplified 18S rRNA gene of Sarcocystis that yielded PCR product of 900 bp. Digestion of 18S rRNA gene PCR products was performed with restriction endonuclease BseLI to detect different species of Sarcocystis. On digestion with restriction endonuclease three different patterns were observed on agarose gel electrophoresis, one with 513 bp and 343 bp and other with 525 bp, 241 bp and 141 bp which were referred to Sarcocystis cruzi and S. hirsuta respectively. While another with 532 bp and 335 bp was referred to S. fusiformis. S. cruzi (93.43 %) was significantly more prevalent in Chittoor district in comparison with the S. hirsuta (4.38 %) and S. fusiformis (2.19 %). Infection of cattle with S. hominis was not observed in the study area. These findings provide evidence that Sarcocystis species of cattle and water buffaloes are not strictly intermediate host specific but might occasionally infect water buffaloes and cattle, respectively where both hosts occur and natural cross transmission through dogs are possible. S. fusiformis is able to use the cattle as an intermediate host and is not restricted to buffalo. PCR-RFLP was helpful in studying the molecular epidemiology of sarcocystosis in cattle and was a good method in discriminating between species of Sarcocystis.
Open Access
CLONING, EXPRESSION AND CHARACTERIZATION OF PARAFLAGELLAR ROD GENE OF TRYPANOSOMA EVANSI
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-01) SIVAJOTHI, S; CHENGALVA RAYULU, V(MAJOR); MALA KONDAIAH, P; SREENIVASULU, D; SRILATHA, Ch
ABSTRACT : Present study was undertaken with an objective to isolate, clone, express and characterize the paraflagellar rod gene of Trypanosoma evansi. Local isolate of Trypanosoma evansi collected from naturally infected cow was multiplied in Wistar rats. Total RNA was extracted from DEAE-cellulose column chromatography purified trypanosomes by using Trizol LS reagent. Complementary DNA (cDNA) was synthesized from the RNA of host cell free T. evansi parasites by reverse transcription using oligo dT primers. RT-PCR was standardized to amplify the cDNA by targeting 1800 bp unique for PFR 2 gene of T. evansi. Amplification of cDNA was confirmed on agarose gel electrophoresis. The concentration of PCR amplicon was found to be 40 ng/μl after extracting from the gel. The gel purified PCR product (PFR 2 gene of T. evansi) was cloned into pTZ57R/T vector system. Transformation of competent Escherichia coli DH5α cells with ligated PFR 2 gene T. evansi was successfully carried out in LB agar with X-Gal and IPTG. Developed recombinants were observed as white colonies and non-recombinants as blue colonies. Presence of inserts was confirmed initially by Colony-PCR and then by Plasmid-PCR. Nucleotide sequence of the PFR 2 gene of T. evansi S.V.V.U. isolate (Accession No. KT277497) of the present study revealed 100 % homology with T. evansi China isolate and 99% homology with T. evansi Izatnagar and Bikaner isolates. Variation in nucleotide mutations at 4 positions with T. evansi Izatnagar and 3 positions with T. evansi Bikaner isolates were observed. The amino acid mutations in the PFR 2 gene of T. evansi S.V.V.U. isolate displayed regularity at 4 positions when compared to T. evansi China, Izatnagar and Bikaner isolates. Tree topology based on the Neighbor-joining (NJ) method of phylogenetic analysis has showed a close homology with other Trypanosomatidae species sequences with 100% bootstrap values. Restriction digestion of insert DNA of PFR 2 gene as well as pET 32a vector was carried out with EcoR I and Hind III enzymes and subjected for ligation by using T4 DNA ligase. The recombinant protein was sub-cloned into pET 32a and expressed in the BL21 (DE3) pLysS expression system. A high level of expression of recombinant protein of PFR 2 gene of T. evansi was noted following four hours of induction with 1 mM IPTG. Molecular weight of the Ni-NTA column chromatography purified recombinant protein of PFR 2 gene of T. evansi was found to be approximately 90 kDa after resolving by SDS-PAGE. PFR 2 gene of T. evansi S.V.V.U. isolate was further characterized by determination of its protein profile with SDS-PAGE analysis and western blotting against hyper immune serum. Indirect ELISA was optimized for detection of specific antibody titre against recombinant protein of PFR 2 gene of T. evansi. Based on the ELISA result, it is evident that PFR 2 gene products are eliciting very good immune response. However, further study is required to know the protective effect of the antibodies in laboratory animal models and to explore the PFR 2 gene of T. evansi as potential candidate for diagnostic and vaccine target against surra. Findings of the present study confirmed the existence of PFR 2 gene in Indian cattle isolate of T. evansi. Cloning, expression and characterization of PFR 2 gene of T. evansi of cattle isolate carried out in the present investigation is probably the first report in India.
Open Access
DIAGNOSIS OF BOVINE SARCOCYSTOSIS BY IMMUNOFLUORESCENT ANTIBODY TECHNIQUE
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-03) DASMA BAI, BANOTHU; UDAYA KUMAR, M(MAJOR); CHENGALVA RAYULU, V; NARASIMHA REDDY, Y; ANAND KUMAR, A
ABSTRACT: Sarcocystis species are widely prevalent in man and animals, causing significant impact on animal and public health throughout the world. A laboratory standardized immunofluorescent antibody technique was used to study the seroprevalence of bovine sarcocystosis and the efficacy of same was compared with the traditional diagnostic methods like macroscopic, microscopic (squash and pepsin HCl acid muscle digestion). Histopathological changes and viability of bradyzoites in the affected esophageal muscles were also studied in the present investigation. The gross examination of oesophagi revealed creamy white colored thin walled macrocysts of Sarcocystis spp appearing in different shapes (fusiform, elliptical, ovoidal and globular etc) and sizes ranging from 2.0-18.0 x 1.0-5.0 mm with an average size of 10.47 + 0.295 x 3.08 + 0.089 mm. None of the organs showed any kind of gross lesions around the macrocysts embedded in the muscles. Microscopic examination of esophagus and diaphragmatic muscles by squash technique revealed the presence of microcysts arranged horizontally in between the muscle fibers of esophagus where as the pepsin HCl acid digestion of muscle samples of esophagi and diaphragm showed live bradyzoites in gliding motion. Histopathological studies suggested two possible etiologic agents of bovine sarcocystosis namely: S. cruzi, characterized by having elongate and septate cysts and S. hirsuta or S. hominis, characterized by having spherical or rounded cysts with thick radially striated cyst wall. The muscle degeneration with focal or diffused mononuclear cells viz: leukocytic infiltration, eosinophils, lymphocytes and macrophages observed in the tissues under study were attributed to the pathogenic effects of S. cruzi. The ruptured or degenerated state of some of the mature sarcocysts surrounded by eosinophils indicated the advanced age of the cyst. Immunofluorescent antibody technique was standardized in the laboratory for the diagnosis of Sarcocyst infection in bovines. Purified, host cell free bradyzoites collected from macrocysts of Sarcocystis spp, in aliquots of 6-8 applied to glass slides and fixed in chilled acetone over night followed by preservation at -200C worked well with 1:16 dilution of positive and negative control sera and 1:40 dilution of rabbit anti-bovine FITC conjugate. The positive sera did not show any cross reaction with T. gondii RH strain and non specific reactions were absent with negative sera. The serosurveillance of bovine sarcocystosis by laboratory standardized IFAT showed 80.14% (323) of cattle and 78.59% (246) of buffaloes positive for anti sarcocystis antibodies out of 403 cattle and 313 buffalo sera tested, respectively showing an overall prevalence of 79.46% out of 716 animals screened. The antibody titers of 6 randomly selected positive samples from different age groups of <2 years, 2-5 years, 5-10 years and >10 years old bovines ranged from 16-64, 32-256, 32-128 and 16-64 with an average titer of 32 + 2.92, 106.6 + 34, 74.6 + 17 and 34.6 + 9, respectively. The age wise prevalence of sarcocystosis in cattle indicated low rate of infection in the age group below 2 years (60%) and an ascending rate of infection in the age groups of 2-5 years (81.33%), 5-10 years (80.52%) and above 10 years (90.9%). Similarly, the incidence was significantly low in the buffaloes of below 2 years (64%) and high percentage of infection (86.51%) in 5-10 years followed by 78.94% in 2-5 years and 77.27% in above 10 years of age groups. No significant difference of infection was observed between male (81.87%) and female (75.19%) animals as well as between non-descriptive (79.63%) and cross bred (77.58%) animals. Esophageal and diaphragmatic muscle samples collected from 100 animals slaughtered at Chengicherla slaughter house, Hyderabad were subjected to visual examination, squash and pepsin HCl acid muscle digestion techniques which revealed the presence of macrocysts in 16% and 0%, microscopic sarcocysts in 8% and 0% and bradyzoites in 76% and 52% esophageal and diaphragmatic muscles, respectively. The sera collected simultaneously from corresponding animals were screened for anti Sarcocystis antibodies by laboratory standardized IFAT and the results were compared with those of visual examination, squash and pepsin HCl acid muscle digestion techniques. The IFAT was found superior in diagnosing sarcocystosis with positivity of 82%, followed by muscle digestion, gross examination and squash techniques with positive rates of 52%, 16% and 8%, respectively. The present study indicated that the visual and microscopic examination of bovine carcass is by no means a satisfactory diagnostic tool and recommends Immunoflourescent antibody technique for the antemortem diagnosis of animals waiting for slaughter at abattoirs in large scale. Experiments were also undertaken to determine whether Sarcocystis would survive storage at different refrigeration temperatures for a period of 9 days. The number of live and dead bradyzoites in one gram of pepsin HCl acid digested bovine esophageal muscle samples previously stored at room temperature, 40C, 00C, and -200C for a period of 48thhr, 8 days, 24thhr, and 24hr were 2x104 and 4 x104, 1x104 and 1 x104, nil and no bradyzoite, respectively when compared to those stored at 0th hr (10x104 and 0 x104).
Open Access
A CROSS SECTIONAL SURVEY ON ENDOPARASITES OF BACKYARD POULTRY
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) ANUPAMA, B(MAJOR); MALAKONDAIAH, P(MAJOR); SREEDEVI, C; RAVI KUMAR, P; SATHEESH, K
ABSTRACT: The present study has been investigated to determine the prevalence and histopathology of endoparasites in backyard poultry in Krishna district of Andhra Pradesh. Examination of a total of 1447 birds samples viz., faeces, blood, gastrointestinal tracts (GIT) and visceral organs from Kesarapalli, Gannavaram, Bapulapadu, Srinarsannapalem, Bathulavarigudem, Unguturu, Ravicherla and Indupalli villages in Krishna district revealed an overall 74.22 per cent of prevalence of endoparasites. The infection was highly prevalent in Kesarapalli (78.50 %) village compared to that of Gannavaram (76.41 %), Bapulapadu (75.54 %), Srinarsannapalem (75.16 %), Bathulavarigudem (73.85 %), Unguturu (71.83 %), Ravicherla (70.85 %) and Indupalli (70.61 %) villages of Krishna district. Examination of GIT of 702 birds revealed presence of endoparasites in all samples (100 %). Examination of visceral organs and blood smears revealed absence of endoparasites. Mixed infection was identified in 756 (70.39 %) infected birds and cestodes were the common parasites in all infected birds. Out of 1074 infected birds, 100 per cent were found to be positive for cestodes, 65.36 per cent for nematodes and 9.49 per cent for protozoa infection. No trematode parasite was identified during the study period. The species of cestodes identified included Amoebotaenia sphenoides (23.84 %), Cotugnia digonopora (36.76 %), Davainea proglottina (23.77 %), Hymenolepis carioca (2.55 %), Raillietina cesticillus (29.44 %), R. echinobothridia (25.43 %) and R. tetragona (22.66 %) whereas nematodes viz., Ascaridia galli (30.89 %), Capillaria spp. (8.84 %), Heterakis gallinarum (24.46 %), Strongyloides avium (3.31 %), Subulura brumpti (3.24 %), Tetrameres mohtedai (1.45 %) and Dispharynx spiralis (0.96 %) and protozoa viz., Eimeria tenella (2.62 %), E. necatrix (2.21 %), E. mitis (1.93 %) and E. acervulina (0.96 %) were identified respectively. The prevalence of infection was increased with advance in age and there was no significant relationship between the prevalence of infection and age groups of birds (P>0.05). There was no significant difference (P>0.05) between the prevalence of endoparasites in male (72.66 %) and female (76.74 %) birds. Among all helminth parasites identified Cotugnia digonopora was the highest (36.76 %) prevalent parasite whereas Hymenolepis carioca was the lowest (2.55 %) in all age group of birds. Strongyloides avium and Subulura brumpti were not observed in chicks. Among Eimeria spp. viz., Eimeria tenella (1.15 %) and E. mitis (1.15 %) were identified in chicks. Dispharynx spiralis was exclusively found in chicks. Tetrameres mohtedai was identified in female birds only. The prevalence of endoparasites in rainy, winter and summer seasons was 77.07, 73.66 and 71.71 per cent respectively. No significant (P>0.05) relationship between the seasonality and prevalence of endoparasites was observed in this study. Infection of Dispharynx spiralis was observed during summer season whereas Eimeria spp. viz., Eimeria acervulina, E. tenella, E. necatrix and E. mitis were identified during rainy season. The endoparasitic infections revealed presence of few to numerous parasites in the affected part of gastrointestinal tract (proventriculus and intestine) obliterating the lumen with mild enteritis, congested and enlarged wall of the affected part. Histopathologically, congested blood vessels, presence of cut sections of respective parasites associated with infiltration of inflammatory cells especially of mononuclear cells and eosinophils were noticed.
Open Access
MOLECULAR DETECTION AND TYPING OF CRYPTOSPORIDIUM IN LAMBS IN ANDHRA PRADESH
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2015-01) THIRUMALESWARA RAO, J; VENU, R(MAJOR); SRINIVASA RAO, K; ASWANI KUMAR, K
ABSTRACT : The present research work has been undertaken with an objective to investigate the Cryptosporidium infection in lambs in Andhra Pradesh. Conventional microscopy (mZN staining method) was adopted for preliminary screening of faecal samples for detection of Cryptosporidium infection. Subsequently, the mZN positive isolates were confirmed by nested PCR (nPCR). Further, randomly selected nested PCR amplicons were sequenced to determine the Cryptosporidium species. In mZN staining method, 74 (9.87%) Cryptosporidium positives were detected out of 750 faecal samples screened. The highest prevalence of Cryptosporidium infection was detected in the faecal samples of unorganized sheep farms (11.08%) and the lowest in organized sheep farms (8.36%). The higher prevalence of infection was observed in the age group of 8-14 days in organized farms (12.70%), whereas in unorganized sheep farms the higher risk age group was 15-21 days (13.86%). Lowest prevalence was observed in lambs of both organized and unorganized sheep farms at 0 to 7 (1st week) days. The prevalence of Cryptosporidium infection rate in male lambs (8.70%) was recorded lower than the female lambs (10.99%). The male and female lambs that belonged to unorganized sheep farms showed a higher prevalence of Cryptosporidium infection than those of respective lambs in organized farms. Breed wise, the highest prevalence of Cryptosporidium infection was observed in Deccani (17.89%) followed by Nellore brown (11.97%), Rambouillet (10.91%), Vizianagaram local (6.96%) and Nellore jodipi (6.94%). Regarding, faecal consistency character the highest prevalence of Cryptosporidium infection was determined in diarrhoeic lambs (21.80%), followed by semi-solid (11.71%) and the lowest was in non-diarrhoeic animals (5.10%). Diarrhoeic, semi-solid and non-diarrhoeic lambs belong to organized sheep farms showed lower rate of infection than their respective lambs of unorganized farms. Region wise an overall highest prevalence of Cryptosporidium infection was recorded in the faecal samples of lambs in Telangana region (13.39%) followed by Coastal Andhra (8.64%) and Rayalaseema region (8.13%) of Adhra Pradesh. Seventy four Cryptosporidium mZN positive faecal samples were subjected to nested PCR, targeting 18S rRNA gene for confirmation of Cryptosporidium DNA and was successful. Randomly selected nPCR confirmed Cryptosporidium DNA isolates were sequenced for species determination. Molecular characterization revealed three Cryptosporidium species were observed in the sheep farms of Andhra Pradesh viz., C. ubiquitum, C. parvum and C. xiaoi. Sequence analysis revealed that, C. ubiquitum was the major species identified from the faecal samples of lambs that belonged to organized sheep farms viz., Chittoor (LRS, Palamaner), Anantapur (LRS, Siddarampuram), Vizianagaram (LRS, Garividi) and Mahaboobnagar (LRS, Mahaboobnagar). In addition to C. ubiquitum in the organized sheep farms, C. parvum was also detected in the lambs of Vizianagaram (LRS, Garividi). Cryptosporidium ubiquitum was the major species identified from the faecal samples of lambs belonging to unorganized sheep farms viz., Chittoor (Vijayapuram Mandal), Anantapur (Siddarampuram Mandal), Nellore (Venkatagiri Mandal) and Vizianagaram (Garividi Mandal). In addition to C. ubiquitum in the unorganized sheep farms, C. xiaoi was also detected in the lambs of Warangal (Wardannapet Mandal) and C. parvum was isolated from the faecal samples of lambs of Mahaboobnagar (Bandimeedipalli Mandal). Geographically, C. ubiquitum is the only species distributed among the lambs of Rayalaseema region, whereas in Coastal Andhra region both C. ubiquitum and C. parvum were identified. In Telangana region, all the three Crptosporidium species i.e., C. ubiquitum, C. xiaoi and C. parvum were isolated from the faecal samples of lambs. Overall, in all the three geographical locations, C. ubiquitum was recorded among the sheep flocks, irrespective of their rearing system. In conclusion, Cryptosporidium infection was prevalent and first time observed species variation in the sheep flocks of Andhra Pradesh.
Open Access
MOLECULAR DETECTION AND TYPING OF CRYPTOSPORIDIUM IN CAPTIVE WILDLIFE
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2014-10) RAMA DEVI, P; VENU, R(MAJOR); JEYABAL, L; VINOD KUMAR, N
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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