Nirbhay Kumar Singh’s research while affiliated with Guru Angad Dev Veterinary and Animal Sciences University and other places

Tick-transmitted parasites as Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis are major health concern for dogs. Owing to prevalence and infection severity, there is need of sensitive, specific, and affordable test for their simultaneous detection. Prevalence of B. gibsoni, B. vogeli, E. canis, and H. canis infections was assessed on 719 blood samples by microscopy and multiplex PCR assay targeting 18S rRNA (B. gibsoni & H. canis), ITS1 & 5.8S rRNA (B. vogeli) and VirB9 gene (E. canis). An internal control (canine-actin) was also included to increase the accuracy of assay and effect of associated risk factors with disease prevalence was also studied. Microscopic prevalence of B. gibsoni, B. vogeli, E. canis and H. canis was 5.0%, 0.1%, 1.4% and 1.0%, respectively, whereas with multiplex PCR assay, the corresponding values were 8.9%, 1.1%, 2.6% and 5.1% besides concurrent infections of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). Analytical sensitivity of developed assay was 0.1pg (B. gibsoni & H. canis), 0.01pg (B. vogeli), and 1.0pg (E. canis). A ″fair″ (B. vogeli & H. canis) to ″substantial″ (B. gibsoni & E. canis) agreement between two tests was observed with data as statistically significant. Breed, sex and location were significantly associated with B. gibsoni infection. The developed multiplex PCR assay offers a potential solution to detect these pathogens simultaneously, aiding in timely diagnosis and effective disease management in suspected dogs.

Rhipicephalus microplus (Acari: Ixodidae) is a highly invasive tick responsible for significant economic losses to cattle industry worldwide. The increasing global reports on acaricide resistant tick populations warrant development of alternate eco-friendly approaches for suppressing the tick populations and vectored pathogens. The present study aimed to evaluate the acaricidal activity of essential oils (EOs): Cedar oil (CO), Garlic oil (GO), Peppermint oil (PO) and their combinations against R. microplus by larval packet test. Six concentrations each of individual EOs and their combinations (10, 5, 2.5, 1.25, 0.625 and 0.31%) were evaluated. The lethal concentrations (LC) of individual EOs and their combinations along with values of Combination Index (CI) and Dose Reduction Index (DRI) were estimated to assess the effects of interactions (synergistic, additive or antagonistic) of EO mixtures. Among the individual oils, GO showed the highest efficacy with lowest LC50, LC90 and LC95 (95% CL) values at 2.19% (1.96–2.43%), 4.00% (3.48–4.98%) and 4.74% (4.01–6.23%), respectively. Among combinations, highest acaricidal property was exhibited by GO + PO (LC50, LC90 and LC95 values of 2.63, 4.87 and 5.81%) with CI value of 0.598 indicating moderate synergism, while the CO + GO + PO combination (LC50, LC90 and LC95 values of 1.67, 9.97 and 16.54%) exhibited slight synergism with CI value at 0.872 and favourable DRI of 8.24, 1.85 and 4.76 for PO, GO and CO, respectively. The present study seems to be pioneer to assess the combination effect of these EOs against R. microplus and could help in development of an effective and eco-friendly product for tick control.

A multiplex PCR assay was standardized and evaluated to detect the DNA of Babesia gibsoni, B. vogeli, Ehrlichia canis and Hepatozoon canis in dogs along with internal control in a single tube reaction. The self-designed primers revealed amplicons of 182 bp, 131 bp, 407 bp and 268 bp for the DNA of B. gibsoni (18S rRNA gene), B. vogeli (18S rRNA gene), H. canis (18S rRNA gene), and E. canis (VirB9 gene) respectively, without any non-specific amplification. A product size of 88 bp was obtained for the internal control targeting the canine actin gene. The results of multiplex PCR assay were further compared with the microscopy. Blood samples from 719 dogs collected from different agro-climatic zones of Punjab, when screened revealed the prevalence of B. gibsoni, B. vogeli, E. canis and H. canis as 5.0%, 0.1%, 1.4% and 1.0%, respectively whereas, with multiplex PCR assay the values were 8.9%, 1.1%, 2.6% and 5.1% along with concurrent infection of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). The comparative result of microscopy with multiplex PCR assay revealed the sensitivity values at 95% CI as 100% with range of 2.50% to 100% while the specificity was 99.03% with range of 94.04% to 99.61%. The strength of agreement between the two tests was found to be fair (B. vogeli & H. canis) to substantial (B. gibsoni & E. canis) by kappa value statistics and the data was statistically significant. The analytical sensitivity of multiplex PCR assay in the detection of B. gibsoni, B. vogeli, E. canis and H. canis was 0.1 pg, 0.01 pg, 1 pg and 0.1 pg, respectively. Among various risk factors, a significant association of breed, sex and location was recorded with the prevalence of B. gibsoni infection.

In this study, molecular characterisation and comparative phylogenetic analysis of Ehrlichia canis (Ludhiana isolate) targeting the partial 16S ribosomal RNA gene was carried out. The results of study revealed a very high identity (99-100%) with the gene sequences from Nagpur, Mizoram, and Bareilly isolates from India as well as from those in other countries viz.,

As per 20th livestock census, India possessing 193.46 million cattle and 109.85 million buffaloes in organized and unorganized sectors. These animals are suffering from tick infestation almost throughout the year causing both direct and indirect losses. Repeated use of chemical acaricides for tick management resulted in the establishment of acaricide resistant tick populations, insecticide residues in livestock products and environmental pollution. In the present study, analysis of the data generated during 1990–2021 revealed pooled prevalence of infestations in cattle and buffaloes was 53% [95% CI = 47–60%] and 38% [95% CI = 28–49%], respectively. Based on peripheral blood smear examination the prevalence of theileriosis, babesiosis and anaplasmosis in cattle is estimated as 17% [95% CI = 12–24%], 4% [95% CI = 3–6%] and 5% [95% CI = 3–7%], respectively, while in buffalo the prevalence of infection is estimated as 7% [95% CI = 2–21%], 2% [95% CI = 0–5%] and 8% [95% CI = 2–36%],respectively.To estimate economic impact, both direct (reduction in milk production, cost of treatment, leather depreciation) and indirect (milk loss and treatment cost) losses were taken into consideration. Loss of milk production was predicted as 13.91, 56.91 and 85.34L/cross-bred cow/lactation in low, moderate and high tick infestation conditions, respectively. Whereas, 20.10, 7.01L milk/buffalo/lactation in Hyalomma spp. and Rhipicephalus spp. infestation was estimated. Similarly,the estimated loss of milk production due to clinical theileriosis, babesiosis and anaplasmosis was 57.96, 30.96 and 59.22L, respectively. The cumulative (milk loss, treatment cost and leather loss) loss due to tick infestation was calculated as 46199.31 million INR (USD595.07 million) while due to TBDs 14877.15 million INR (USD191.15 million) = 61076.46 million INR (USD787.63 million). The data provided base line information for the policy maker to develop strategies at government level so that the significantly high cumulative loss of 787.63 million USD due to ticks and tick borne diseases (TTBDs) can be minimized.

The cattle tick, Rhipicephalus microplus Canestrini (Acari: Ixodidae) is one of the most important tick species severely affecting health and causes huge losses to dairy industry. Chemical acaricides are mainly applied for tick control but development of resistance, environmental pollution and contamination of milk and meat products with residues has led to exploration alternative eco-friendly tick control strategies. The dried fruits Piper longum L. (Indian long pepper, Thippali or Pippali) generally used as flavoring agent have also been shown to have insecticidal property. Different concentrations (0.625%–10%) of alcoholic and aqueous extracts of Piper longum L. were prepared and evaluated for acaricidal activity against amitraz resistant R. microplus adult and larval stages. Against larval stages a dose-dependent mortality response was recorded for both extracts and higher acaricidal property was exhibited by the alcoholic extract with LC50 and LC95 (95% CL) values of 0.488% (0.48–0.49) and 1.39% (1.35–1.44), respectively. Similarly, against adult engorged females, ethanolic extract showed higher acaricidal property with LC50 and LC95 (95% CL) values of 4.67% (4.61–4.74) and 12.38% (12.05–12.73), respectively. Significant (p < 0.05) reduction was recorded in reproductive index of ticks treated and but no effect on hatchability of eggs was recorded in treated groups. The present study establishes acaricidal activity of P. longum fruit extracts against both larval and adult stages of amitraz resistant population of cattle tick.

Status of ivermectin resistance was studied in 16 isolates of Rhipicephalus (Boophilus) microplus ticks collected from northwestern Himalayan region of India using larval immersion test (LIT). Regression graphs of probit mortality of larval ticks plotted against log values of concentrations of ivermectin were utilized for the determination of slope of mortality, lethal concentrations (LC 50 and LC 95) and resistance ratios (RRs). Out of 16 isolates examined, 15 were found resistant against ivermectin. Level II resistance was detected in eleven isolates (RR 50 ranged from 5.72 to 7.62), whereas four isolates exhibited level I resistance (RR 50 ranged from 1.58 to 2.77). Only one isolate was found susceptible (RR 50 : 1.23). Higher RR values were observed in tick isolates from low altitude areas where intensive animal husbandry activities are practiced as compared to tick isolates of high altitudes accredit the fact that managemental practices, availability of drug and its frequency of application play an important role in development of resistance.

Background Microscopy is a routinely used technique for the diagnosis of canine tick-borne haemoparasitic diseases in various clinical laboratories worldwide. In an attempt to provide better diagnostic assay to the clients for effective management of these diseases duplex real-time PCR assays were applied. Methods and results Blood samples (n = 338) aseptically collected from suspected dogs of Central Plain Zone of Punjab state, India were subjected to SYBR Green based real-time duplex PCR assays for simultaneous detection of B. vogeli & E. canis and B. gibsoni & H. canis. Results revealed an overall prevalence rate of canine tick-borne haemoparasites as 54.1%, amongst which H. canis was the predominant (25.4%), followed by B. gibsoni (16.3%), E. canis (10.7%) and B. vogeli (1.8%). Sensitivity and specificity of the duplex assays ranged from 59.04 to 100.0% and 58.12 to 92.52%, respectively and their strength of agreement was ″fair″ with kappa value statistics. A significant (p < 0.05) association between prevalence of B. gibsoni, H. canis and E. canis infection with risk factors like sex, breed, season and location was recorded. The ancestral background of the field isolates of haemoparasites was also studied by phylogenetic analysis of their nucleotide sequences. Conclusions SYBR Green dye based duplex real-time PCR assays proved to be highly sensitive, specific, rapid and affordable diagnostic tests for use by clinicians to save the life of pets.

To combat the problem of acaricide resistance in ticks, efforts have been made to develop eco-friendly herbal acaricides which are safe for animal use with no possibilities of evoking environment and residual toxicity. In the present study, the acaricidal properties of a commonly available plant, Annona squamosa have been established to develop a novel antitick phytoformulation using leaf extract, suitable solvents and emulsifier for managing resistant tick infestations in animals. The quality control of the safe and stable phytoformulation using Rutin as a major constituent was maintained by HPTLC profiling. Several phytformulations (A-E) were prepared and formulation ‘C’ is identifried as the most effective showing 52.5-75% antitick activity against in vitro treated resistant strains of R. microplus (IVRI-IV and V) with 33.8-40.2% inhibition of oviposition while 65-85% mortality against multiacaricide resistant field populations. The phytoformulation provided 70.4% efficacy after first larval challenge in experimentally infested animals. The field efficacy of the formulation in different multilocational field trials was 68.53-77.8%. The detail antitick efficacy of the formulation along with safety and stability are explained as a component of antitck technology to manage resistant tick problem in livestock.