Srikanta Ghosh

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.

Molecular characterization of Theileria orientalis causing fatal infection in crossbred adult bovines of South India.

The disease condition attributed to have been caused by Theileria orientalis is generally benign. However, it is also thought that the parasite, at least some strains of it, can cause fatal disease. The present communication deals with the clinical signs, postmortem lesions and diagnosis of a fatal disease due to T. orientalis which caused mortality in crossbred adult bovines of South India. High body temperature, lacrimation, nasal discharge, swollen lymph nodes and haemoglobinuria were the symptoms observed. The postmortem lesions observed were punched out ulcers in abomasum, enlargement of spleen, massive pulmonary oedema, frothy exudates in trachea, epicardial and endocardial haemorrhage and haemorrhagic duodenitis. Peripheral blood smear examination revealed rod shaped Theileria sp. organisms. Polymerase chain reaction that amplify the T. orientalis specific P(32/33) gene, followed by cloning and sequencing, revealed maximum homology with Narathiwat (Thailand) and Jingole -1 (Indonesia) isolates which were positioned as isolate type 7 of T. orientalis.

Survey of pyrethroids resistance in Indian isolates of Rhipicephalus (Boophilus) microplus: identification of C190A mutation in the domain II of the para-sodium channel gene.

Monitoring acaricide resistance and understanding the underlying mechanisms are critically important in developing strategies for resistance management and tick control. Eighteen isolates of Rhipicephalus (Boophilus) microplus collected from four agro-climatic regions of India were characterized and the resistant data were correlated with bioassay results, esterase enzyme activities and with the presence/absence of point mutation in the para-sodium channel gene. The adult immersion test was standardized to assess the level of resistance and resistant factors (RF) in the range of 1.2-95.7 were detected. Out of eighteen isolates, three were categorized as susceptible (RF<1.4), five isolates at level I (RF=1.5-<5), eight at level II (RF=5.1-<25), and one isolate each at level III (RF=26-<40) and level IV (RF=>41). The esterase enzyme ratio and survival% of tick isolates was observed significantly (p<0.001) correlated with correlation coefficient (r) in α- and β-esterase activity. The correlation of determination (R(2)) for α- and β-esterase activity indicated that 73.3% and 55.3% data points of field isolates were very close to the correlation lines. For detection of point mutation, three sites (mutation in domain IIS6, T2134A mutation in domain IIIS6 and C190A mutation in domain IIS4-5 linker) of sodium channel gene were amplified and sequenced. Comparative sequence analysis identified a cytosine (C) to adenine (A) nucleotide substitution (CTC to ATC) at position 190 in domain II S4-5 linker region of para-sodium channel gene in six isolates and in reference deltamethrin resistant IVRI-IV line. The occurrence of mutation in the tick isolates having high resistance factor suggested that target site insensitivity and enhanced esterase activity is the possible mechanism of resistance to deltamethrin in the Indian isolates of R. (B.) microplus. These results also concluded that the mutation site in Indian tick isolates is similar to Australian and Brazilian tick isolates while it is different in tick isolates from Mexico and North America. This is the first report of occurrence of mutation in para-sodium channel gene of deltamethrin resistant Indian isolates of R. (B.) microplus.

Jatropha curcas (Linn) leaf extract –a possible alternative for population control of Rhipicephalus(Boophilus) annulatus

Abstract Objective To study the effect of ethanolic extract of leaves of the plant Jatropha curcas as a step towards developing a safe and ecofriendly therapeutic agent to combat the problems of tick and tick-borne diseases. Methods Pulverised leaves of J. curcas were subjected to soxhlet extraction using ethanol. The ethanolic extract of J. curcas at different dilutions such as, 50, 60, 70, 80, 90 and 100 mg / ml were tested against ticks using adult immersion test. The per cent adult mortality, inhibition of fecundity and hatching of laid ova were studied. Results The extract caused significant blocking of hatching of the laid ova by the treated ticks. Conclusions Eclosion blocking effect of J. curcas extract is a promising property of the plant that can be utilized for controlling the population of ticks.

Acaricidal properties of Ricinus communis leaf extracts against organophosphate and pyrethroids resistant Rhipicephalus (Boophilus) microplus

Indian cattle ticks have developed resistance to commonly used acaricides and an attempt has been made to formulate an ecofriendly herbal preparation for the control of acaricide resistant ticks. A 95% ethanolic extract of Ricinus communis was used to test the efficacy against reference acaricide resistant lines by in vitro assay. In in vitro assay, the extract significantly affects the mortality rate of ticks in dose-dependent manner ranging from 35.0 ± 5.0 to 95.0 ± 5.0% with an additional effect on reproductive physiology of ticks by inhibiting 36.4-63.1% of oviposition. The leaf extract was found effective in killing 48.0, 56.7 and 60.0% diazinon, deltamethrin and multi-acaricide resistant ticks, respectively. However, the cidal and oviposition limiting properties of the extract were separated when the extract was fractionated with hexane, chloroform, n-butanol and water. The HPTLC finger printing profile of R. communis leaf extract under λ(max.) - 254 showed presence of quercetin, gallic acid, flavone and kaempferol which seemed to have synergistic acaricidal action. In vivo experiment resulted in 59.9% efficacy on Ist challenge, however, following 2nd challenge the efficacy was reduced to 48.5%. The results indicated that the 95% ethanolic leaf extract of R. communis can be used effectively in integrated format for the control of acaricide resistant ticks.

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.

Eclosion blocking effect of ethanolic extract of Leucas aspera (Lamiaceae) on Rhipicephalus (Boophilus) annulatus.

The crude ethanolic extract of aerial parts of Leucas aspera was tested for its acaricidal properties against Rhipicephalus (Boophilus) annulatus. The per cent adult mortality, inhibition of fecundity and hatching of laid ova were studied at concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50 and 100mg/ml. Adult tick mortality was significant at the highest concentration tested. Inhibition of fecundity of treated groups differed significantly from control and was concentration dependent. L. aspera extract also produced complete failure of eclosion of eggs from the treated ticks even at lower dilutions of the extract.

Immunoprotective Efficacy of a Purified 39 kDa Nymphal Antigen of Hyalomma anatolicum anatolicum

Soluble nymphal antigens (HNAg) were purified by immunoaffinity chromatography using CNBr-activated Sepharose 4B coupled with immunoglobulin ligands from animals immunized with HNAg and 69–71% protected against challenge infestations, and 8% recovery of the purified protein (Aff-HNAg) was obtained. Following immunization of crossbred calves (Bos indicus×Bos taurus) with 1600 μg of Aff-HNAg in three divided doses, significant rejections of larvae (p<0.001, 84.2%), nymphs (p<0.05, 61.4%) and adults (p<0.05, 58.7%) were recorded. No significant changes were recorded in the engorgement weights of the larvae and nymphs, but there was a significant (p<0.05) reduction in the weight of the engorged adults. Immunization conferred a significant decrease in the numbers of resultant nymphs (p<0.001) and adults (p<0.001) that had fed on the immunized animals. SDS-PAGE analysis identified a 39 kDa protein, previously isolated from larvae of Hyalomma anatolicum anatolicum, as the antigen responsible for the induction of resistance against all the stages of the tick.

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.

Experimental immunisation of crossbred cattle with glycoproteins isolated from the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus.

In India, control of tick infestations is a major problem facing the owners of highly productive crossbred cattle. To develop suitable immunoprotective measures against the multitick infestations, specific problem encountered by the Indian farmers, an attempt has been made to identify candidate protein molecules in the larvae of Hyalomma anatolicumanatolicum and Boophilus microplus. Employing two steps affinity chromatography method, glycoproteins of 34 and 29 kDa were isolated from H. a. anatolicum and B. microplus, respectively. The additive effect of the isolated glycoproteins was tested by the immunisation of crossbred male calves at a dose of 200 µg/animal of each glycoprotein, three divided dosages. Following challenge infestations by the stages of two species of ticks, a significant rejection of (73.6%; P < 0.01) of larvae and adults (75.0%; P < 0.01) of H. a. anatolicum and adults (89.8%; P < 0.01) of B. microplus were noted. The direct effect of immunisation on the stages of the challenged ticks (%DT) was calculated as 69%, 52% against larvae and adults of H. a. anatolicum and 60% against adults of B. microplus. A direct corelation between protection and anti-glycoprotein antibody titre was detected. The effect of the isolated glycoproteins on limiting the tick infestation is discussed in detail.