S. S. Rath

Detection of amitraz resistance in Rhipicephalus (Boophilus) microplus from SBS Nagar, Punjab, India.

The resistance status of Rhipicephalus (Boophilus) microplus collected from SBS Nagar, Punjab, was evaluated against amitraz by Adult Immersion Test (AIT). The regression graph of probit mortality of ticks plotted against log values of progressively increasing concentrations of amitraz revealed the slope of mortality (95% CI) as 2.36 ± 0.61 (0.38 to 4.33). The LC50 (95% CI) and LC95 (95% CI) values were recorded as 332.52 ppm (305.06–362.44) and 1646.93 ppm (1383.97–1959.84), respectively, and the resistance factor (RF) was 13.17 which indicated level II resistance status. The dose response curves for egg mass weight, reproductive index, and percentage inhibition of oviposition were also validated by AIT and the slopes (95% CI) were −7.17 ± 2.41 (−14.85 to 0.50), −0.009 ± 0.02 (−0.16 to −0.031), and 19.99 ± 4.77 (4.81 to 35.17), respectively. The current study reports the development of resistance in R. (B.) microplus to amitraz from Punjab state and the data generated would be useful in formulation of effective control strategies against ticks of this region.

Prevalence of Theileria annulata infection in Hyalomma anatolicum anatolicum in Punjab state, India

The study was conducted to assess the prevalence of Theileria infection in tick vectors collected from healthy animals to record the natural infection level of theilerial parasite in the field condition. A total of 156 male and 110 semi-engorged female of Hyalomma anatolicum anatolicum were collected from cattle and buffaloes of two different agro climatic zones of Punjab, dissected and their salivary glands were stained with Methyl Green Pyronin (MGP) stain. The prevalence, intensity and abundance of Theileria annulata infection were higher in female ticks (15.45%, 5.08 and 32.88, respectively) than male ticks (8.97%, 1.69 and 18.86, respectively). The ticks collected from cattle had a higher prevalence, abundance and intensity (15.15%, 5.38 and 35.53, respectively) of T. annulata infection than the ticks collected from buffaloes (9.58%, 1.74 and 18.13, respectively). Hot and dry climate favored the development of T. annulata sporozoites in H. a. anatolicum ticks as the prevalence, abundance and intensity of T. annulata infection were higher in ticks collected from Western semi arid zone of Punjab as compared to those collected from the central plain zone with hot and humid climate.

Effect of various acaricides on hatchability of eggs of Rhipicephalus (Boophilus) microplus.

The effect of commonly used acaricides (amitraz, cypermethrin, deltamethrin, fenvalerate, and flumethrin) on the eggs of Rhipicephalus (Boophilus) microplus collected from Ludhiana, Punjab, was evaluated by egg hatch assay (EHA). The regression graph of probit hatchability and per cent inhibition of hatching (IH%) of eggs was plotted against log values of concentration of various acaricides. All concentrations of flumethrin and amitraz caused complete inhibition of hatching, whereas a hatchability of 31.0 ± 6.1, 40.0 ± 5.2 and 19.3 ± 1.7% was only recorded at the highest concentration of cypermethrin, deltamethrin, and fenvalerate, respectively. An increase in the concentration of the acaricide showed a significant effect on the IH% of eggs for cypermethrin (P < 0.01) and deltamethrin (P < 0.05) but was nonsignificant for fenvalerate. The slope of the regression curve of IH% was utilized for the calculation of the dose of various acaricides causing inhibition of hatching for 95% eggs (LC95) and the discriminating dose (DD). Results indicated that maximum DD was recorded for fenvalerate (2.136%), followed by cypermethrin (0.214%) and deltamethrin (0.118%). The results of the current study will be helpful in formulating effective control strategies against ticks.

Canine Babesiosis in Northwestern India: Molecular Detection and Assessment of Risk Factors

In the current study, a total of 214 blood samples from dogs in and around Ludhiana, Punjab (India), suspected for canine babesiosis were examined with conventional and molecular assays. Examination of Giemsa-stained peripheral thin blood smears revealed an overall prevalence of 7.47% (16/214) for canine babesiosis encompassing 0.93% (2/214) of large Babesia and 6.54% (14/214) of Babesia gibsoni. However, molecular diagnosis revealed 15.42% (33/214) samples positive for B. gibsoni infection as evident by the presence of 671 bp amplicon. The results of multivariate analysis showed that the prevalence of B. gibsoni was associated with various risk factors, namely, age (P < 0.001; OR: 0.398; CI 95%: 0.080–1.799), sex (P = 0.022; OR: 0.849; CI 95%: 0.403–1.791), breed of host (P = 0.371; OR: 3.345; CI 95%: 1.045–10.710), and season (P = 0.230; OR: 2.143; CI 95%: 0.788–5.830). The prevalence of B. gibsoni was higher in summer as compared to winter season and in younger dogs, while breed and sex of the host were not significantly associated with the occurrence of the disease.

Molecular prevalence of Babesia bigemina in Rhipicephalus microplus ticks infesting cross-bred cattle of Punjab, India

Babesiosis is an economically important tick-borne apicomplexan protozoan disease of cattle in tropical and subtropical regions. In the present study, Rhipicephalus microplus engorged female ticks were collected from 135 apparently healthy cattle from different agro-climatic zones of Punjab, India, to investigate the carrier status of Babesia bigemina infection in vector tick by using microscopy and PCR based assays. PCR when applied on DNA extracted from the egg masses harvested from ticks showed 1.48% (2/135) samples as positive, whereas 4.44% (6/135) samples were positive when product of primary PCR was used as template in nPCR. Further, among the DNA samples isolated from the unfed larval stages that emerged from egg masses laid by ticks, only 1.48% (2/135) samples were detected as positive for B. bigemina in PCR, while 7.41% (10/135) samples were detected positive in nPCR assay. Statistically, non-significant (p > 0.05) difference in prevalence rates was observed across different agro-climatic zones and between different age groups of cattle from which engorged ticks were collected. It can, thus, be concluded that prevalence of B. bigemina in the vector tick, R. microplus in Punjab state of India indicates an endemic status of the organism and a further study is needed for the management and control of the bovine babesiosis.

Molecular prevalence, risk factors assessment and haemato-biochemical alterations in hepatozoonosis in dogs from Punjab, India

Hepatozoonosis caused by Hepatozoon canis is an important tick-borne disease of dogs in tropical and sub-tropical regions throughout the world. In the present study evaluation of blood samples collected from 225 dogs presented at Small Animal Clinics, GADVASU, Ludhiana, Punjab (India) was done for the presence of H. canis by PCR based assay targeting a portion of 18S rRNA gene. Of the total samples subjected to PCR, an amplicon of 666bp was detected in 13.78% samples whereas, routine blood smear examination revealed gamonts in 5.78% samples. Furthermore, prevalence of H. canis infection was found to be significantly associated with season, being highest in summer and lowest in winter while other risk factors e.g. age, sex and breed showed non-significant association. In terms of various clinico-pathological parameters, significant drop in haemoglobin, total red blood cell count, packed cell volume and lymphocytes were recorded in positive cases whereas the total white blood cell count was non-significantly increased. The haematological alterations in the positive cases were lymphopenia, anaemia, thrombocytopenia, relative neutrophilia, neutrophilic leucocytosis, eosinophilia, monocytosis and lymphocytosis while the biochemical profile revealed hypoproteinemia and increased levels of blood urea nitrogen and creatinine (in positive cases) pointing towards renal failure.

Detection of malathion resistance in Hyalomma anatolicum anatolicum from Bathinda District, Punjab

Objective: The resistance status against malathion in Hyalomma anatolicum anatolicum ticks collected from Bathinda district, Punjab, was evaluated by adult immersion test (AIT). Materials and Methods: Technical grade malathion was used for conduction of AIT with 2 min immersion time protocol. The regression graph of probit mortality of ticks was plotted against log values of increasing concentrations of malathion and was utilized for the determination of slope of mortality, LC50, LC95 (95% confidence interval [CI]) and resistance factor (RF). The reproductive parameters of treated ticks viz. egg mass weight, reproductive index (RI), and percentage inhibition of oviposition (% IO) were also studied. Results: The increasing concentration of malathion showed an upward trend in per cent tick mortality. The slope of mortality (95% CI) was 2.489 ± 0.719 (2.489 ± 0.719) and value of goodness of fit (R2) was 0.799. The LC50 (95% CI) and LC95 (95% CI) values were recorded as 9099.2 (8378.6–9881.7) and 41,511.3 (35,060.2–49,149.4) ppm, respectively, with RF as 16.60 indicating level II resistance status. A negative dose-dependent slope of egg mass weight (−91.79 ± 25.15 [−171.8 to −11.76]) was recorded as the survived ticks laid significantly (P = 0.0355) fewer eggs. The mean RI of treated ticks decreased with increasing concentrations of drug and the slope (95% CI) was −0.293 ± 0.059 (−0.482 to −0.105). Further, a dose-dependent significant increase (P = 0.0157) in the mean % IO was recorded in treated ticks. Conclusions: The results of the current study would be useful in the formulation and implementation of effective tick strategies in the region.

Effect of synergists on ivermectin resistance in field populations of Rhipicephalus (Boophilus) microplus from Punjab districts, India

The status of ivermectin resistance in Rhipicephalus (Boophilus) microplus collected from various districts of the Punjab state, India was determined using larval immersion test (LIT). Regression graphs of probit mortality of larval ticks of various field isolates were plotted against log values of increasing concentrations of technical grade ivermectin for determination of slopes of mortality, lethal concentrations for 50% (LC50) and resistance factors (RF). Values for the coefficient of determination (R2) in LIT assay ranged from 0.82 to 0.98 indicating the model to be a good fit. The RF values against ivermectin ranged from 1.65 to 9.07 revealing resistance status in all the field isolates. Pre-exposure to a single pre-determined sub-lethal concentration of ATP-binding cassette transporter inhibitors (cyclosporin-A, MK571) and p-glycoprotein inhibitor (verapamil) lead to reduction in LC50 values of ivermectin in different field tick isolates. Among the various field isolates, the highest synergistic factor for MK571 and verapamil was recorded in the Moga isolate as 4.97 and 3.21, respectively whereas for cyclosporin-A, the highest value was recorded in the Mansa isolate as 2.81. Among the three synergists used in the current study, MK571 caused the highest increase in toxicity against ivermectin in the field ticks. Therefore, combination products of ivermectin with the above synergists could prolong the useful life of this drug for effective control of ticks.

Effect of Malathion on Reproductive Parameters of Engorged Female Rhipicephalus (Boophilus) microplus Ticks of Punjab Districts, India

The present study was aimed at evaluating effects of malathion on the various reproductive parameters, namely, egg mass weight (EMW), reproductive index (RI), percentage inhibition of oviposition (%IO), and hatchability percentage of eggs of Rhipicephalus (Boophilus) microplus (Canestrini 1887) females from 19 districts of Punjab, India. The effect on various parameters was found to be dose dependent and more discernible upon exposure to higher concentrations. Complete cessation of egg laying was recorded in tick isolates on exposure to 5000 ppm and above. The values of %IO ranged in 4.4–68.6, 25.2–76.2, 35.6–100.0, 45.7–100.0, and 71.4–100.0 in groups treated with 1250, 2500, 5000, 10000, and 20000 ppm of malathion, respectively. A low hatching % was recorded in eggs of all treated female ticks in comparison to control treated with distilled water and complete inhibition of hatching was recorded at 10000 ppm and above. However, the survival of the hatched larvae was not affected and was similar to control group. The results of the current study can be of immense help in formulation and implementation of effective tick control measures.

Molecular prevalence of Babesia bigemina infection in cattle in Western semi-arid and Foothill regions of Punjab (India)

A nested PCR based prevalence study was done from June, 2011 to May, 2013 to determine the carrier status of Babesia bigemina infection in apparently healthy crossbred cattle from Western semi-arid and Foothill regions of Punjab (India). Of the 278 blood samples collected, an amplicon of 170 bp size was detected in 16.19% animals by nested PCR aa compared to the routine blood smear examination which showed parasitic piroplasms in only 1.80% samples. Regarding various risk factors, statistically nonsignificant variation was observed with respect to zone-wise, sex-wise and age-wise prevalence whereas season-wise disease prevalence showed significant variation (p<0.05).