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Open Access
IN VITRO EVALUATION OF ANTHELMINTIC PROPERTIES OF THE HYDRO-ALCOHOLIC EXTRACTS OF SELECTED MEDICINAL PLANTS ON Haemonchus contortus
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) TULASI, DAVULURI; SREEDEVI, C(MAJOR); MALAKONDAIAH, P; SRINIVASA RAO, G
In recent years, there has been growing interest in alternative therapies and the therapeutic use of natural products especially of medicinal plants for control of parasites. In the present study in vitro assays such as egg hatch assay (EHA), larval paralysis assay (LPA) and adult worm motility inhibition assay (WMIA) were conducted to determine the anthelmintic efficiency of hydro-alcoholic extracts of Anacardium occidentale shell, Illicium verum fruit and Artocarpus heterophyllus seed on eggs, infective larvae (L3) and adult worms of Haemonchu contortus, in comparison to albendazole. Among three extracts, A. occidentale shell showed significant (P<0.01) inhibitory effect on egg hatching and larval motility followed by A. heterophyllus seed and I. verum fruit extracts. Extracts of A. occidentale shell induced 50% inhibition at lower concentration (0.0255 mg/mL) compared to I. verum fruit extract (0.0612 mg/mL) and A. heterophyllus seed (0.0372 mg/mL) extracts. The LD50 value of reference drug albendazole (positive control) was 0.237 µg/mL. Extracts of A. occidentale shell required maximum of 0.5 mg/mL, whereas extracts of I. verum fruit and A. heterophyllus seed required maximum concentration of 4 and 2 mg/mL respectively, to induce 100 per cent egg hatch inhibition. Similarly A. occidentale shell showed maximum activity on motility of L3 larvae (LD50 = 0.196 mg/mL) with 100 per cent paralysis while A. heterophyllus seed (LD50 = 0.666 mg/mL) and I. verum fruit (LD50 = 1.418 mg/mL) exhibited 84.67±1.76 and 72.66±1.76 per cent paralysis respectively, at higher tested concentration of 6 mg/mL. In WMIA, three extracts induced significant (P<0.001) mortality of adult worms; however the activity of A. occidentale shell was higher (100%) than I. verum fruit (36.6±3.3%) and A. heterophyllus seed (70.00±5.7%) at a concentration of 6 mg/mL within 1 h post exposure. Anacardium occidentale extract revealed better LD50 (1.0365 mg/mL) values in comparison with I. verum fruit (LD50 = 3.848 mg/mL) and A. heterophyllus seed (LD50 = 2.398 mg/mL) in the WMIA. Three plant extracts exhibited significant (P<0.001) dose dependant anthelmintic responses by inhibiting egg hatching and causing paralysis of larvae and mortality of worms. In vitro effect of these extracts on lactate dehydrogenase activity of H. contortus was also studied. All extracts significantly (P<0.01) inhibited the activity of LDH catalysing the oxidation of lactate. Maximum level of inhibition of LDH activity was noticed in A. occidentale shell extract treated worms. Thin Layer Chromatography analysis and qualitative phytochemical screening of three extracts revealed presence of alkaloids, flavonoids, tannins, saponins, carbohydrates and proteins which might be responsible for the anthelmintic effects noticed. Overall, this in vitro study suggests that these three plants have promising anthelmintic effects.
Open Access
ASSESMENT OF ACARICIDAL ACTIVITY OF NANOSCALE ZnO ENCAPSULATED PIPERINE FORMULATION AGAINST RHIPICEPHALUS MICROPLUS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2018-03) SNIGDHA, KANCHARANA; CHENGALVA RAYULU, V(MAJOR); SRINIVASA RAO, K; SREEDEVI, B
ABSTRACT: The present study was undertaken with an aim to synthesize and evaluate the acaricidal activity of nanoscale zinc oxide piperine formulation against Rhipicephalus microplus ticks. Nanoscale zinc oxide piperine formulation (NZPF) was prepared by using 0.1% zinc oxide nanoparticles (ZnONPs) solution and 20% piperine solution employing encapsulation technique. The synthesised NZPF was characterized by employing UV–Vis spectroscopy, Fourier Transformed Infrared (FT-IR) analysis, X-ray Diffraction (XRD), Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM) and Energy Dispersion Spectroscopy (EDS) analysis. The maximum absorbance peak of the Localized Surface Plasma Resonance (LSPR) of ZnONPs and NZPF was observed at 240 nm and 345 nm, respectively by using UV-Vis Spectrophotometer. The FT-IR spectra peaks of ZnONPs and NZPF were found to be 3844, 3739, 3619, 2351, 1694 and 1526 cm-1 and1633, 1583, 1490, 1448 and1436 cm-1, respectively. The XRD analysis showed strong peaks of 31.854°, 34.497°, 36.327°, 47.64°, 56.70°, 63.060°and 68.10° corresponds to Bragg’s reflections at (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0) and (1 0 3) planes conforming the wurtzite crystalline structure of NZPF. Hydrodynamic diameter and zeta potential of the hydrosol of ZnONPs and NZPF were found to be 38.6nm and - 28.8 mV and 75nm and -36.4 mV, respectively with DLS technique. The EDS micrograph of ZnONPs showed the peaks of zinc and oxygen elements confirming the chemical composition and presence of chemical constituents (Zn77.51 % and O 22.49%).The SEM images revealed that the synthesized ZnONPs were spherical in shape with mean size of 30nm. Whereas synthesized NZPF were in rectangular rod shape with occasional agglomeration, highly poly dispersed and measured 1-2 μm. Acaricidal activity of deltamethrin, piperine, ZnONPs and NZPF on Rhipicephalus microplus was evaluated by two bioassays viz., Larval Packet Test (LPT) and Adult Immersion Test (AIT). LPT with a discriminating dose of deltamethrin (75 ppm) showed 59% mortality of R. microplus larvae. Total mortality (100%) was seen against R. microplus larvae at concentrations of 9ppm, 8ppm and 7 ppm with piperine, ZnONPs and NZPF, respectively. AIT with a discriminating dose of deltamethrin (75ppm) against adult R. microplus showed a mortality of 40%, oviposition inhibition of 78.309% and the lowest egg mass weight with17.8±1.31 mg. Mortality rate and oviposition inhibition of R. microplus were 100% whereas egg mass and reproductive index were completely nil with both piperine and ZnONPs at a concentration of 20ppm and NZPF at a concentration of 15 ppm. The egg mass values and reproductive indices were inversely proportional to the piperine, ZnONPs and NZPF concentrations whereas oviposition inhibition percent was directly proportional to the piperine, ZnONPs and NZPF concentrations. LC50 values of LPT and AIT were at the lowest concentration of 1.312 ppm and 3.505 ppm for NZPF whereas LC99 values were seen at the lowest concentration of 12.690 ppm and 33.741 ppm for ZnONPs. NZPF showed a potent ovulation inhibitory activity with significantly (P<0.05) lower IC50 and IC99 values compared to ZnONPs and piperine. Both LPT and AIT results clearly indicate the development of resistance in R. microplus ticks against deltamethrin. Synthesised NZPF, ZnONPs and piperine were found to have significantly (P<0.05) higher acaricidal activity. However, NZPF had high acaricidal efficacy at lower concentrations than pure phytochemical piperine, ZnONPs and deltamethrin. NZPF could be potential alternative to routine chemical acaricides for control of tick infestation of cattle in the wake of development of acaricidal resistance, residual effect and environmental pollution. Synthesis of NZPF and evaluation for its acaricidal activity in vitro is probably the first kind of report and further detailed study must be carried out before their application in vivo.
Open Access
SYNTHESIS OF MYCOGENIC SILVER NANOPARTICLES AND EVALUATION OF THEIR ACARICIDAL ACTIVITY AGAINST RHIPICEPHALUS MICROPLUS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2018-01) PRIYADARSHINI, A.T; SRINIVASA RAO, K(MAJOR); CHENGALVA RAYULU, V; PRASAD, T.N.V.K.V
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).

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