Molecular characterization of Bertiella studeri infecting a primate in South India.

Bertiella spp. is a mite-borne cestode parasite that inhabits the small intestine of wide range of mammals, including non-human primates. In the present study, the morphological and molecular analysis of Bertiella studeri recovered from the small intestine of a bonnet macaque (Macaca radiata) from Wayanad, Kerala (South India) was performed. Acetic alum carmine staining identified the cestode morphologically based on the characters like broader proglottids, which contain irregularly alternating genital pores, single set of reproductive organs, 280 testes and a tubular transverse uterus. Molecular characterization was done using 18SrRNA, ITS1-5.8S and COX1 genes. Phylogenetic trees were constructed using MEGA X based on the Maximum Likelihood (ML) method (Hasegawa-Kishino-Yano (HKY) model). Cytochrome oxidase I gene could detect the existence of genetic variation in the parasite from two different hosts viz., monkey (Kerala, Argentina, and Kenya) and human (Sri Lanka). A minimum spanning network of haplotypes was generated by the haplotype networking with the above sequences using the popARTv1.7. Haplotype analysis based on COX1 revealed that the parasite haplotype was different in each country with highest population frequency in Sri Lanka.

Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus.

The problem of ticks and tick borne diseases is a global threat and growing reports of resistance to commonly used insecticides further aggravated the condition and demands for country specific resistance monitoring tools and possible solutions of the problem. Establishment of standard reference is prerequisite for development of monitoring tools. For studying possible role of different mechanisms involved in development of resistance in Rhipicephalus (Boophilus) microplus population and to develop newer drug to manage the problem of resistance, a deltamethrin exposed and selected tick colony, referred to as IVRI-IV, was characterized using reference susceptible IVRI-I tick line as control. The RF values of IVRI-IV ticks against deltamethrin, cypermethrin and diazinon were determined as 194.0, 26.6, 2.86, respectively, against adults. The esterase enzyme ratios of 2.60 and 5.83 was observed using α-naphthyl and ß-naphthyl acetate while glutathione S-transferase (GST) ratio was 3.77. Comparative analysis of IVRI-I and IVRI-IV carboxylesterase gene sequences revealed 13 synonymous and 5 non synonymous mutations, reported for the first time. The C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to leucine to isoleucine (L64I) amino acid substitution was also detected in the IVRI-IV population. In the present study, monitorable indicators for the maintenance of the reference IVRI-IV colony, the first established deltamethrin and cypermethrin resistant tick line of India, were identified.

Esterase mediated resistance in deltamethrin resistant reference tick colony of Rhipicephalus (Boophilus) microplus.

Monitoring of acaricide resistance is considered as one of the important facets of integrated tick management. In an attempt of development of resistance monitoring indicators, in the present study two reference tick lines of Rhipicephalus (Boophilus) microplus maintained in the Entomology laboratory, Indian Veterinary Research Institute (IVRI), Izatnagar, India, were studied to determine the possible contributing factors involved in development of resistance to deltamethrin. Electrophoretic profiling of esterase enzymes detected high activities of EST-1 in reference resistant tick colony designated as IVRI-IV whereas it was not detectable in reference susceptible IVRI-I line of R. (B.) microplus. Esterases were further characterized as carboxylesterase or acetylcholinesterase based on inhibitor study using PMSF, eserine sulphate, malathion, TPP and copper sulphate. It was concluded that an acetylcholinesterase, EST-1, possibly plays an important role for development of deltamethrin resistance in IVRI-IV colony of R. (B.) microplus.

Ixodid Tick Vectors of Wild Mammals and Reptiles of Southern India.

BACKGROUND: We aimed to focus on the ixodid ticks parasitizing wild mammals and reptiles from Wayanad Wildlife Sanctuary, Western Ghat, southern India. METHODS: The taxonomic identification of ticks collected from wild mammals and reptiles was performed based on the morphology of adults. RESULTS: We revealed eight species of ticks including, Amblyomma integrum, Rhipicephalus (Boophilus) annulatus, Haemaphysalis (Kaiseriana) spinigera, H. (K.) shimoga, H. (K.) bispinosa, H. (Rhipistoma) indica, Rhipicephalus haemaphysaloides and R. sanguineus s.l. collected from nine species of wild mammals while four tick species Ablyomma kraneveldi, A. pattoni, A. gervaisi and A. javanense parasitizing on four species of reptiles. The highest host richness was shown by H. (K.) bispinosa and R. haemaphysaloides parasitizing six and five different host species, respectively. Reports of R. (B.) annulatus on sambar deer, A. javanense and A. kraneveldi on python as well as A. pattoni on Indian rat snake are the new host records from this region. CONCLUSION: Eight species of ticks parasitizing on nine species of wild mammals and four species of parasitizing on four species of reptiles were identified. The highest host richness was shown by H. (K.) bispinosa and R. haemaphysaloides. H. spinigera as the vector of KFD was also identified in this study.

Comparative in vitro anti-tick efficacy of commercially available products and newly developed phyto-formulations against field collected and resistant tick lines of Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is considered as one of the most widely distributed tick species ecto-parasitizing on livestock and causes fatal diseases with significant production loss. To address the problem of controlling acaricide resistant tick infestations on animals, attention has been paid to develop eco-friendly phyto-acaricides. The present study was undertaken to evaluate the comparative anti-tick activities of commercially available herbal and chemical products with chemically characterized phyto-formulations developed recently against field ticks and resistant tick lines of R. (B.) microplus. The chemical product Butox® Vet was found nearly passive against all the tested resistant tick lines. However, one of the commercial polyherbal product, Zerokeet® showed an efficacy (E%) of 41.8-75.4 % ([Formula: see text]) using recommended dilution (1:2) against field ticks and resistant tick lines. However, the other commercial product, Erina® EP has very limited efficacy against all the tested tick. In comparison, the newly developed phyto-formulation, NBA/13/B/2 and NAC-01 conferred an E% of 82.4-91.3 % ([Formula: see text]) and 62.3-94.6 % ([Formula: see text]), respectively, against tested resistant ticks. Results indicated higher marketing potentiality of newly developed formulation in the existing tick problem scenario.

Survey of acaricides resistance status of Rhipiciphalus (Boophilus) microplus collected from selected places of Bihar, an eastern state of India.

Monitoring acaricide resistance in field ticks and use of suitable managemental practices are essential for controlling tick populations infesting animals. In the present study, the acaricide resistance status in Rhipicephalus (Boophilus) microplus ticks infesting cattle and buffaloes of five districts located in the eastern Indian state, Bihar were characterized using three data sets (AIT, Biochemical assays and gene sequences). Adult immersion test (AIT) was adopted using seven field isolates and their resistance factor (RF) was determined. Six isolates (DNP, MUZ, BEG, VSH, DRB and SUL) were found resistant to both deltamethrin and diazinon and except VSH all were resistant to cypermethrin. One isolate (PTN) was susceptible with a RF below 1.5. To understand the possible mode of resistance development, targeted enzymes and gene sequences of the para sodium channel and achetylcholinesterase 2 (AChE2) were analyzed. The esterase, monooxygenase and glutathione-S-transferase (GST) activity of reference susceptible IVRI-I line was determined as 2.47 ± 0.007 nmol/min/mg protein, 0.089 ± 0.0016 nmol/mg of protein and 0.0439 ± 0.0003 nmol/mg/min respectively, which increased significantly in the resistant field isolates. However, except esterases, the fold increase of monooxygenase (1.14-2.27 times) and GST (0.82-1.53 times) activities were not very high. A cytosine (C) to adenine (A) nucleotide substitution (CTC to ATC) at position 190 in domain II S4-5 linker region was detected only in one isolate (SUL) having RF of 34.9 and in the reference deltamethrin resistant line (IVRI-IV). However, the T2134A mutation was not detected in domain IIIS6 transmembrane segment of resistant isolates and also in reference IVRI-IV line despite of varying degree of resistance. The flumethrin specific G215T and the recently identified T170C mutations were also absent in domain II sequences under study. Four novel amino acid substitutions in AChE2 gene of field isolates and in organophosphate (OP) resistant reference IVRI-III line were identified which can possibly have a role in resistance development.

Acaricidal effects of fenvalerate and cypermethrin against Rhipicephalus (Boophilus) annulatus.

The acaricidal effects of two most commonly used acaricides viz., fenvalerate and cypermethrin against Rhipicephalus (Boophilus) annulatus were studied using Adult Immersion Test (AIT). The LC50 values observed for fenvalerate and cypermethrin were 1570 ppm and 184 ppm respectively. The death of ticks was not an immediate process. Fenvalerate caused death only after 7 days while cypermethrin after 5 days of treatment. The eggs laid by treated ticks did not hatch at all concentrations tested.

In vitro evaluation of acaricidal activity of fipronil against Haemaphysalis bispinosa based on adult immersion test.

Fipronil is a phenylpyrazole family insecticide which mainly affects the nervous system of insects. In the present study, the in vitro acaricidal effects of the compound against the widely prevalent multihost tick, Haemaphysalis bispinosa was assessed. The lowest concentration at which complete adult tick mortality was observed was at 25 ppm while complete absence of egg mass observed at 10 ppm. Hundred per cent inhibition of fecundity was observed at 1 ppm while complete blocking of hatching of the laid ova was observed even at 500 ppb.

Effects of solvents and surfactants against Haemaphysalis bispinosa.

As per the report published by FAO (2004), the tick population in India has developed resistance against all the available acaricides. Hence, newer methods of control including potential herbal agents are required to reduce the problems caused by the ticks. Most of the herbal extracts or their fractions are dissolved in polar or non-polar solvents or detergents before tested for acaricidal activity and these diluents should be of little acaricidal activity. In the present study, adult immersion test (AIT) was carried out on adult engorged female Haemaphysalis bispinosa ticks to detect the acaricidal activity of different solvents viz., nbutanol, glycerol, acetone, ethanol, methanol and surfactants (at 1 per cent dilution) like dimethyl sulphoxide (DMSO), tween 20 and triton X- 100. The study revealed that methanol was the least toxic solvent while tween 20 (1 per cent) was the least toxic detergent against H. bispinosa.