D.D. Ray

Status of tick distribution in Bangladesh, India and Pakistan

On a global basis, ticks transmit a greater variety of pathogenic microorganisms, protozoa, rickettsiae, spirochaets, and viruses than any other arthropods and are among the most important vectors of diseases affecting livestock, humans, and companion animals. Ticks and tick-borne diseases (TTBDs) affect 80% of the world cattle population and are widely distributed throughout the world, particularly in tropical and subtropical countries including India, Pakistan, and Bangladesh. Ticks and tick-transmitted infections have coevolved with various wild animal hosts, which constitute the reservoir hosts for ticks and tick-borne pathogens of livestock, pets, and humans. In this region, the livestock sector is suffering from a number of disease problems caused by bacteria, viruses, fungi, and parasites. Among the parasitological problems, the damage caused by TTBDs is considered very high, and the control of TTBDs has been given priority.

Deltamethrin and cypermethrin resistance status of Rhipicephalus (Boophilus) microplus collected from six agro-climatic regions of India

A cross sectional study was conducted to assess the prevalence of synthetic pyrethroids (SP) resistance in Rhipicephalus (Boophilus) microplus in India. Twenty-seven areas located in six agro-climatic regions were selected for the collection of engorged ticks using two stage stratified sampling procedure. Adult immersion test (AIT) and larval packet test (LPT) were optimized using laboratory reared susceptible line of R.(B.) microplus (IVRI-I) for determination of 95% lethal concentration (LC(95)) of deltamethrin (29.6 ppm in AIT and 35.5 ppm in LPT) and cypermethrin (349.1 ppm in AIT and 350.7 ppm in LPT). The AIT with a discriminating dose (2 × LC(95)) was used to detect deltamethrin and cypermethrin resistance in the field isolates of R.(B.) microplus. On the basis of the data generated on three variables viz., mortality, egg masses and reproductive index, the resistance level was categorized as I, II, III and IV. The overall prevalence of SP-resistant R.(B.) microplus among the sampled farms was 66.6% (18/27). Out of these 18 areas, resistance to deltamethrin at level I was detected in 08 areas (resistance factor=2.0-4.9), at level II in 09 areas (RF=5.2-11.8), at level III in 01 area (RF=34.9) and at level IV in 01 area (RF=95.7). The resistance to cypermethrin was detected in 16 areas and level of resistance was detected at level I in 10 areas (RF=2.06-4.64) and at level II in 06 areas (RF=5.13-9.88). The middle-gangetic and trans-gangetic plains revealed higher density of resistant ticks where intensive cross bred cattle population are reared and the SP compounds are commonly used. The data generated on acaricide resistant status in ticks will help in formulating tick control strategy for the country.

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.

Prevalence and molecular characterization of bovine Cryptosporidium isolates in India.

A survey based on PCR assay of 18S SSU rRNA gene revealed a 30.2% infection with Cryptosporidium spp., out of 457 faecal samples collected from neonatal bovine calves across three different regions of India. The PCR-RFLP pattern of the gene in all the positive cases established the species as Cryptosporidium parvum. Highest prevalence was recorded in the monsoon months (37.3%) and in the calves showing acute diarrhoea (32.3%). The calves below 15 days of age were mostly affected (45.1%). The infection was more prevalent in the northern parts (35.4%) of the country than in the eastern or southern parts. Results indicated that C. parvum was the only species of Cryptosporidium prevalent in bovine calves in three different geographical regions of India.

A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata

A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.

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.

Prevalence of Cryptosporidium andersoni: A molecular epidemiological survey among cattle in India

Cryptosporidiosis is an important and established cause of calfhood morbidity in bovines. The present communication reports the prevalence of Cryptosporidium infection among juvenile and adult cattle (6-24 months old) in India based on examination of faecal samples collected from 350 animals across three different agro-climatic regions of the country and further confirmation by a two-step nested PCR assay targeting 18S ssu rRNA gene. A total of 45 samples were positive for Cryptosoridium species by nested PCR assay. The PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using SspI and VspI restriction enzymes for species differentiation. The results showed that the species involved in all the samples found positive was Cryptosporidium andersoni. The overall prevalence rate was 12.85%, with highest occurrence in the northern states (14.37%) of the country. The animals between age group of 6-12 months were mostly affected (21.67%) and the season wise prevalence of infection was more during the hot and humid monsoon season (20.16%). The results clearly demonstrated that C. andersoni is the major Cryptosporidium species affecting juvenile and adult cattle in three agro-climatically different geographical regions of India. This is the first report on prevalence of C. andersoni in bovines from India the confirmation of which is based on application of nested PCR and PCR-RFLP based molecular tools.

Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines.

The role of Cryptosporidium spp. as a major cause of diarrhoea and gastrointestinal illness of protozoan origin in neonatal calves has been established. Many coprological and serological techniques have been described for detection of the parasites with the limitations of sensitivity and specificity. Polymerase chain reaction (PCR) technique offers a useful alternative to conventional diagnosis of Cryptosporidium spp. in bovines from both clinical and environmental samples. We compared four conventional coprological techniques, viz., direct faecal smear staining (DFSS), normal saline sedimentation staining (NSSS), Sheathers flotation (SF) and Sheathers flotation sedimentation staining (SFSS) with PCR directed against the 18S SSU rRNA gene as standard reference test for the diagnosis of cryptosporidiosis in bovines. Out of 457 faecal samples collected from neonatal bovine calves, specific PCR amplification was achieved in 138 samples, whereas, 65 samples turned positive by DFSS. Normal saline sedimentation staining, SF and SFSS could detect 92, 82 and 109 samples as positive, respectively. Sheathers flotation sedimentation staining was found to be the most sensitive (82.6%) and specific (98.76%) among the coprological techniques. On per sample processing based cost analysis, DFSS was found to be the most economical method (15 cents) followed by NSSS (19.6 cents), SF (23.6 cents) and SFSS (33.9 cents). The time taken for complete processing and diagnosis varied between 70 and 100 min. PCR based diagnosis of a sample took about 7.5-8h for completion and cost of diagnosis was estimated as approximately 7.604 US

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

per sample. Among the conventional coprological methods, SFSS provided the required sensitivity and specificity along with nominal cost for diagnosis on per sample basis, and may be considered as a viable diagnostic alternative when PCR is not an option for a particular laboratory setting, especially in developing countries. This is the first comparative study describing the sensitivity and specificities of four conventional coprological techniques altogether with respect to PCR along with the economic assessment and per sample diagnosis time of all the techniques for the diagnosis of cryptosporidiosis in bovines.

Cross-reaction among four isolates of Theileria annulata from India

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.