In vitro growth inhibitory effect of selected 18ß-glycyrrhetinic acid esters on Theileriaannulata.

Glycyrrhetinic acid (GA) is one of the important Pentacyclic Triterpenoids (PT) found in the roots of licorice. This study aimed to evaluate the in vitro growth inhibitory effect of 18ß-GA (18ß-Glycyrrhetinic acid) and C-30 esters against Theileria annulata, the causative agent of Tropical Bovine Theileriosis. C-30 esters of 18ß-GA were synthesized and their structures were elucidated using spectroscopy. The pharmacodynamic properties of 18ß-GA and its C-30 esters were predicted using DataWarrior and Swiss ADME tools. Cattle isolates of T. annulata schizont-infected bovine lymphoblastoid cells were cultured using standard conditions and the growth inhibitory effect of GA and its esters were evaluated using MTT assay. The isopropyl ester of 18ß-GA (GI50- 1.638 µM; R2- 0.818) showed improved anti-theileriosis efficacy than other 18ß-GA derivatives. The propyl (GI50 - 5.549 µM), ethyl (GI50 - 5.638 µM), and benzyl (GI50 - 7.431 µM) esters also showed considerable inhibitory effect. The GI50 value for 18ß-GA was recorded as 6.829 µM. This study throws light on the usefulness of 18ß-GA and its esters for the treatment of Tropical Bovine Theileriosis.

First report of occurrence of Babesia gibsoni in captive Indian wolves.

Blood samples from fifteen captive Indian wolves (Canis lupus pallipes) maintained at Arignar Anna Zoological Park, Vandalur, Chennai were screened for the presence of Babesia spp., Ehrlichia canis and Trypnosoma evansi DNA by PCR. Out of 15 wolf samples, 3 samples were found positive for Babesia spp. The amplified 18S rRNA gene fragments from 3 wolves were sequenced and confirmed as Babesia gibsoni. A maximum likelihood tree was constructed using the three sequences along with other Babesia spp. sequences derived from GenBank adopting HKY nucleotide substitution model based on the Bayesian Information Criterion. The phylogenetic analysis confirmed that the three sequences were of Babesia gibsoni and highly divergent from Babesia canis, B. vogeli and B. vulpes. This might be a possible spill over event of B. gibsoni from community dogs through blood feeding dog ticks. This is the first report and molecular confirmation of B. gibsoni infection in captive Indian wolves.

A cross sectional study on molecular prevalence of Orientia tsutsugamushi in household rat population of South India.

This study aimed to assess the molecular prevalence of mite-borne zoonotic pathogen O. tsutsugamushi in household rats of South India through nested polymerase chain reaction amplification of O. tsutsugamushi 47-kDa htrA gene and to determine the most suitable sample type for screening of O. tsutsugamushi in rats. Out of 85 rats trapped in Tamil Nadu, Karnataka, and Puducherry regions, 47 rats were found positive for the O. tsutsugamushi genome with prevalence of 55.29 %. Among different sample types screened, faecal samples exhibited the highest positivity rate, followed by liver, spleen, kidney, and blood samples. Agreement between faecal and spleen samples of rats for the presence of O. tsutsugamushi was the highest. Principal component analysis revealed a positive correlation between the spleen, liver, and faeces and a negative correlation between blood and faeces for the presence of O. tsutsugamushi genome. These findings underscore the varied distribution of O. tsutsugamushi among different samples and indicate that the faecal and liver samples of rats are an ideal choice of samples for epidemiological studies. This is the first study to report a high level of presence of O. tsutsugamushi in faecal samples of rats.

Comparative efficacy of rHaa86 and rBm86 against Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus.

Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus are the most economically important tick species in India and other tropical and subtropical regions of the world and transmit pathogens causing animal and human diseases. We demonstrated that vaccination of animal by rHaa86 could be used for the control of both H. a. anatolicum and R. (B.) microplus infestations. By comparing the efficacy of rHaa86 and rBm86, it was observed that vaccine based on rHaa86 will be more effective in controlling homologous challenge infestations (68·7% against larvae and 45·8% against adults). The results of this trial demonstrated that species-specific antigens are the better choice for vaccine development and could serve as an effective tool for the integrated control of H. a. anatolicum.

Screening for the "Achilles Heel" of Hyalomma anatolicum Ticks by RNA Interference Technology and an Update on Anti-Tick Vaccine Design.

Over the years, RNA interference (RNAi) has evolved as a valuable tool to study the tick gene function, screening and preliminary characterization of tick-protective antigens in a relatively short time, with a minimal use of laboratory animals before conducting expensive vaccine trials for the development of improved vaccine composition. In this process, a double-stranded RNA (dsRNA) of gene of interest is introduced into the tick system which specifically suppresses expression of a target gene. The results of RNAi-based gene silencing were interpreted by reduction in targeted gene transcript, changes in phenotypic data and anatomical/ biochemical changes in ticks; thereby, providing a clue to the probable role played by the gene in the tick biological system. Across the globe, various tick research groups applied RNAi technique for characterization and identification of new anti-tick vaccine targets. Herein, we used the RNAi tool in Hyalomma anatolicum ticks for identification and characterization of vaccine candidates.

Cloning, expression and immunoprotective efficacy of rHaa86, the homologue of the Bm86 tick vaccine antigen, from Hyalomma anatolicum anatolicum.

The Bm86 homologue of Hyalomma anatolicum anatolicum Izatnagar isolate was cloned and expressed in methylotropic yeast Pichia pastoris as intracellular, glycosylated and particulated form. It was named as rHaa86, the first recombinant protein of H. a. anatolicum. Seven epidermal growth factor-like domains predicted in Haa86 were structurally similar with that of its Bm86 counterpart. The identity between the corresponding EGF like domains of Bm86 and Haa86 were ranging from 51.3% to 78.3%. The molecular weight of the rHaa86 was 120-140 kDa, with possible 50-70 kDa glycosylation. The purified rHaa86 was characterized immunologically and evaluated for its immunoprotective potential against homologous challenge infestation in three groups of cross-bred calves. The immediate rejection percentage of females of H. a. anatolicum was 36 5%, 12.4% and 10.1% fed on immunized (group 1), adjuvant control (group 2) and untreated control (group 3) calves, respectively. The percent rejection of female ticks fed on immunized calves was 24.1% and 26.4% higher than for the ticks fed on control groups 2 and 3, respectively (P < 0.05). The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 58.0%, 9.0%, 5.0% and 61.6%, respectively. The mean reproductive index of females fed on group 1 calves was significantly lower (P < 0.05) than the females fed on the control groups and 44% reduction in the number of engorged larvae was recorded from the group 1 calves. The data demonstrated that rHaa86 antigen based vaccine could serve as one of the effective components in the integrated control of H. a. anatolicum.

Dirofilaria hongkongensis - A first report of potential zoonotic dirofilariosis infection in dogs from Tamil Nadu.

Dirofilariosisis, is a mosquito borne anthropo-zoonotic disease caused by the filarial nematodes of the genus Dirofilaria. The current paper reports a species of Dirofilaria, which has not been recorded in Tamil Nadu, in addition to differentiating it from the existing species of Dirofilaria spp. through morphological and molecular techniques. Micrometry of the microfilaria detected by Knott's technique as well as in Giemsa stained blood smears revealed that the length was on an average 280 µm to 310 µm whereas the width was 5 to 8 µm. The microfilariae were unsheathed with blunt heads and tapering tails. For molecular confirmation, the blood sample was subjected to genomic DNA isolation. PCR amplification using specific primers for D. hongkongensis was carried out using thermal cycler with the isolated genomic DNA as template. Molecular analyses revealed the band size of about 230 bp which confirmed the presence of D. hongkongensis. Phylogenetic analysis was done using UPGMA and MLM method. Dirofilaria is recognized as a zoonotic entity throughout the world, and dirofilariasis should be considered an emerging zoonosis in southern India. The control of ubiquitous mosquito population is essential to prevent the transmission of microfilaria from animals to arthropods and from arthropods to humans.

Effect of surface area to weight ratio of egg masses on the hatchability of Boophilus microplus eggs.

BACKGROUND & OBJECTIVES: On global basis, ticks transmit a number of pathogens than any other arthropod vector, and are among the most important vectors of diseases affecting humans, livestock and companion animals. Control of the vector has been focused on integrated management involving strategic use of insecticides, use of vaccines, use of herbal acaricides and breed resistance. It has been established that tick vaccine is working on limiting the egg laying potentiality and subsequent hatchability of the ticks fed on immunized animals. To generate entomological data following immunization of animals against ticks an experiment was conducted to establish the role of water content in egg masses for successful hatching into larvae. METHODS: Different size and shape of egg masses of Boophilus microplus, Izatnagar isolate were obtained by manipulating the egg laying process. The weight of the egg masses was measured, keeping their integrity and surface areas of respective egg masses were calculated with the aid of computer software. Larvae hatched from the respective egg masses were counted individually. RESULTS: It is clear that, with an increase in the exposed surface area of the cylindrical egg mass per unit weight, there is a reduction in the number of larvae hatched out. Also, the spherical egg masses significantly (0.026 at 95% confidence level) yielded more larvae per unit weight in comparison to the cylindrical egg masses. INTERPRETATION & CONCLUSION: It has been established that the larval count yielded from an egg mass is more or less dependent on the surface area : weight ratio of the respective egg masses rather than on either the surface area alone or weight of the egg mass alone.

Cost effectiveness of colony lysis and colony PCR methods for screening of recombinant Escherichia coli colonies--a comparative study.

Economizing the research protocols by using low cost technologies is the need of laboratories of developing world. Screening of recombinant E. coli colonies is the crucial step in gene cloning and expression studies. In the present study, the cost effectiveness of colony lysis method and colony PCR method in the screening of recombinant E. coli colonies was compared. The colony lysis method was 20 two times more cost effective and less time consuming and can be used to screen the recombinant E. coli colonies in large scale instead of colony PCR method.

Upcoming and future strategies of tick control: a review.

Ticks are distributed worldwide and significantly impact human and animal health. Due to severe problems associated with the continuous use of acaricides on animals, integrated tick management is recommended. Increasing public health concern over the tick-borne diseases demands the strategic control of ticks on animals that transmit diseases to human beings. Immunological control of tick vector of Kyasanur Forest Disease (KFD) on cattle and other wild reservoir hosts is one of the possible alternative strategy for reducing the transmission of KFD to man. Chemical-vaccine synergies have been demonstrated and a combination of chemical and vaccine for tick and tick-borne disease control has been identified as a sustainable option. Studies have suggested the possibility of vaccine strategies directed towards both tick control and transmission of pathogens. Besides tick vaccine, use of endosymbionts, which are essential for the survival of arthropod hosts, for the control of tick vectors will be one of the targeted areas of research in near future. India with huge natural resources of herbs and other medicinal plants, the possibilities of developing herbal acaricides is discussed. The future of research directed towards target identification is exciting because of new and emerging technologies for gene discovery and vaccine formulation.

Occurrence of the pentastomid Porocephalus crotali (Humboldt, 1811) in an Indian rat snake (Ptyas mucosus): a case report.

The occurrence of the pentastomid Porocephalus crotali in an Indian rat snake (Ptyas mucosus) at Chennai, India is reported. The worms were found in the lungs and they were identified as females. The worms were cylindrical and annulated with a centrally located mouth surrounded by four hooks. The worms were rounded in cross section with tapering ends. Eggs were composed of two shell membranes. This is one of the new reports from rat snakes in Southern India.

Efficacy of rHaa86, an orthologue of Bm86, against challenge infestations of Hyalomma anatolicum anatolicum.

In an attempt to develop vaccine against Hyalomma anatolicum anatolicum, the protective efficacy of rHaa86 was evaluated against experimental challenge infestations of homologous tick species and lethal dose of Theileria annulata. Following challenge, a significant difference of 20.9% (P < 0.01) in the dropping per cent of ticks fed on immunized and control animals was recorded. A statistically significant reduction of 49.6 mg (P < 0.01) in the weight of ticks fed on immunized animals in comparison with control was noted. The ticks dropped from immunized animals laid fewer eggs and a reduction of 68.1 mg (P < 0.05) in comparison with the ticks fed on control animals was noted. The DT%, DO%, DR% and E% were calculated as 73.8, 31.3, 15.8 and 82.3% respectively. In all the calves fever (rectal temperature

Vaccine Efficacy of Bm86 Ortholog of H. a. anatolicum, rHaa86 Expressed in Prokaryotic Expression System.

The use of tick vaccine in controlling ticks and tick borne diseases has been proved effective in integrated tick management format. For the control of H. a. anatolicum, Bm86 ortholog of H. a. anatolicum was cloned and expressed as fusion protein in E. coli as E. coli-pETHaa86. The molecular weight of the rHaa86 was 97 kDa with a 19 kDa fusion tag of thioredoxin protein. The expressed protein was characterized immunologically and vaccine efficacy was evaluated. After 120 hours of challenge, only 26% tick could successfully fed on immunized animals. Besides significant reduction in feeding percentages, a significant reduction of 49.6 mg; P < .01 in the weight of fed females in comparison to the females fed on control animals was recorded. Following oviposition, a significant reduction of 68.1 mg; P < .05 in the egg masses of ticks fed on immunized animals in comparison to the ticks fed on control animals was noted. The reduction of number of females, mean weight of eggs, adult females and efficacy of immunogen were 73.8%, 31.3%, 15.8%, and 82.3%, respectively. The results indicated the possibility of development of rHaa86 based vaccine as a component of integrated control of tick species.

Efficacy of Azadirachta indica extracts against Boophilus microplus.

Extracts were prepared from leaf, bark, and seed of Azadirachta indica, leaf and seed of Prunus persica, bark of Mangifera indica, and leaf of Psidium guajava and were evaluated against Boophilus microplus. Of the eight extracts screened, the extracts prepared from the A. indica seed showed very high level of efficacy (80%) after 5 h of treatment. Besides the immediate effect on adult ticks, the egg-laying properties of the survived ticks was also assessed, and a significant reduction (P < 0.01) in the reproductive index of ticks fed on animals treated with A. indica seed extracts was noted in comparison to control. The efficacy of the neem seed extracts was compared with the commonly used synthetic pyrethroids, and comparable efficacy against B. microplus fed on animals treated with neem seed extracts and acaricide treated was noted. The possibility of using the extracts in IPM format for the management of ticks is discussed.

Progress in development of vaccine against Hyalomma anatolicum anatolicum-Indian scenario.

Hyalomma anatolicum anatolicum, a three host tick vector transmitting the causative agent of bovine tropical theileriosis, is widely distributed throughout India. As a component of integrated control measures against the tick vector, attempts have been made to identify candidate protein molecules for development of an anti-tick vaccine in the different stages of this tick species. By strategic methods of isolation of the targeted molecules using affinity purification of proteins showing reactivity with immunoglobulins of animals previously immunized with different sources of tick antigen, six proteins were isolated in a significantly pure form. The recovery percentage of the candidate proteins was very low in the range of 1.8-8.0%. The protective potentiality of the antigens was tested in immunization and challenge trials and maximum potential was observed in the proteins isolated from total larval extracts, nymphal extracts and in larval glycoprotein. One of the antigens with a molecular weight of 37kDa isolated from larvae of H. a. anatolicum was found to have some adverse effect on development of Theileria annulata in the vector tick. The progress in the development of immunoprophylactic measures against H. a. anatolicum is discussed.

Laboratory rearing of Theileria annulata-free Hyalomma anatolicum anatolicum ticks.

Hyalomma anatolicum anatolicum is a three-host tick which transmits Theileria annulata infection in Indian cattle. Laboratory rearing of ixodid ticks is an essential requirement of any laboratory engaged with research on ticks and tick borne diseases. The Entomology laboratory of Indian Veterinary Research Institute is fully equipped with all the facilities and skilled manpower to maintain a homogenous H. a. anatolicum population throughout the year. The continuous supply of eggs, larvae and adults of H. a. anatolicum is maintained to meet out the demand of different experiments viz., preparation of tick antigens for immunization of animals, experimental challenge, isolation of genomic DNA and RNA. Maintenance of a H. a. anatolicum colony free of T. annulata infection is imperative for the experimental challenge infestation on cross-bred (Bos indicus x B. taurus) calves, in order to prevent the transmission of T. annulata infection to the experimental animals. A system has been developed in the laboratory in which the larvae of H. a. anatolicum were fed on New Zealand white rabbits and the dropped fed nymphs molted to adults are fed on cross-bred calves free of T. annulata infection. This synthetic cycle prevents the transstadial transmission of T. annulata as the rabbits are unsusceptible to T. annulata infection and only the adults were fed on cross-bred animals. Moreover, absence of transovarial transmission of T. annulata prevents the chance of carry over infection to experimental animals in the next cycle.

Control of ticks of ruminants, with special emphasis on livestock farming systems in India: present and future possibilities for integrated control--a review.

India is predominantly an agricultural country with about 70% of her population dependent on income from agriculture. Although India accounts for a significant share of world's livestock resources, livestock production is greatly affected by ticks and tick-borne diseases (TTBDs). Therefore, India represents a particularly interesting scenario for the study of TTBDs. Herein, we review the problems and opportunities for the integrated control of ticks of ruminants with special emphasis on livestock farming systems in India. Developments discussed in the review in the area of tick vaccines and other tick control measures should have an impact on the future of Indian livestock production.