Epidemiology, Molecular Characterization and Therapeutic Control of Hard Ticks: Tick-Borne Theileriosis and Anaplasmosis of Ruminants in Pakistan

Abstract

Ticks are important vectors which cause life-threatening problems in animals worldwide. The first part of the present study aimed to investigate morphometric epidemiology of hard ticks infesting sheep and goats in three agroecological zones of Pakistan. A total of 4488 animals (2184 sheep and 2304 goats) were examined for ticks’ infestation. Tick genera were identified based on their morphological features through conventional taxonomic keys. All ticks collected were belonged to four tick genera: Rhipicephalus, Hyalomma, Haemaphysalis, and Ixodes. The results showed slightly higher tick infestation in sheep (23%) compared to goats (20.1%). Male sheep had higher infestation of 23.4%, whereas in goats females had a higher infestation of 23.0%. However, the difference was not significant(P≥0.05). Tick prevalence was similar across the study zones (P=0.79), with sheep in Muzaffarabad showing a slightly higher prevalence (47.0%). The prevalence was higher in sheep and goats younger than 1 year (25.7% and 20.8%, respectively) and did not vary significantly (P=0.99). Shinwar- White sheep breed 34.4% (132/384), and Taddy goats 29.7% (38/128) were noticeably more infested with ticks when compared to other sheep and goat breeds; however, the difference was not significant (P=0.79). The study demonstrates higher infestation of tick suggesting their biological and therapeutic control measures. In the second part, the molecular characterization of the Ixodid ticks belonging to the genus Rhipicephalus was carried out. Prevalence of Rhipicephalus ticks was 4.5% in sheep and 3.9% in goats. Molecular characterization based on two mitochondrial and one internal nuclear spacer DNA sequences placed the sequences obtained in this study in paraphyletic clades along with sequences in GenBank. The ITS2 sequence of the R. sanguineus (OK642408) and R. microplus (OK642409) form a distinct clade with sequences from other countries. The 16S rRNA sequences of R. sanguineus (OK560870) clustered with sequences form three lineages, tropical, temperate, and south-eastern lineages. The Cox I gene identified R. turanicus (OK623472) fall in clad with Pakistan, China, Kazakhstan and Bulgaria, while R. microplus (OK623463) form separate clades with sequences from Pakistan, Iran, India and China. The study demonstrates the diversity of Rhipicephalus species infesting small ruminants in Pakistan suggesting their possible involvement in the transmission of tick-borne diseases. Further studies on tick-borne diseases are required for control purposes. xvi In the third part, tick-borne pathogens (TBPs) were investigated in sheep, goats and cattle from Khyber Pakhtunkhwa (KP). This study develops real-time PCR to target Pan-Theileria, the primers and probes were designed to target hypervariable V4 region in the 18S rRNA gene of Theileria sp., which allows detection of different Theileria sp. We also examined the utility of the 16S rRNA gene sequence for discriminating Anaplasma samples to the species level. Microscopically suspected positive blood samples for Theileria and Anaplasma were subjected to DNA extraction. A total of fifty-one blood samples for Theileria and 14 for Anaplasma were subjected to molecular characterization. In Theileria qPCR synthetic gBlock™ gene fragments and clinical specimens were used for analytical and clinical validation. PCR positive Theileria samples were then confirmed using Sanger sequencing on 18S rRNA gene. Regression coefficient (R2) of five reactions was 0.9637 showed a great linearity. Analytical sensitivity ranged from 10 to 100 copies per reaction. The coefficient of variation was less than 5%. Total 51 samples tested for the clinical validation, 47 were positive in Pan Theileria, also amplified on the speciation reaction and confirmed by sequencing. Three samples were found positive to T. orientalis during sequencing, two were confirmed by qPCR. One sample was found positive by qPCR as T. ovis. Overall assay brought us good sensitivity and specificity for our clinical test. Anaplasma was observed in the blood of 14 animals, out of these12 Anaplasma sequences were of good quality. Across the Anaplasma species for which genomes are available, sequence similarity in 16S rRNA with Anaplasma marginale was 99.25% and below this indicating a distinct species. This study adds insight into the epidemiology of TBPs around the KP, province and highlights the need for proactive surveillance of TBPs in livestock. In the last part, this study investigates the effect of commercial synthetic compounds i.e. Cypermethrine (CYM), Deltamethrin, Trichlorphon + Dimethylester, Ivermectin (IVM) and Fipronil on natural infestations of ticks in goats. The In-vivo quantitative assessment of five tick genera i.e. Hyalomma, Rhipicephalus, Ixodes, Haemaphysalis and Boophilus revealed that both CYM and IVM treated groups resulted in significantly lower (P<0.05) tick counts relative to other medicines and controls on all post-treated days. The maximum reduction in mean number of ticks in the CYM and IVM treated group was recorded from days 3 to 4, followed by complete shedding of ticks on day 5. However, Deltamethrin, Trichlorphon+ Dimethylester and Fipronil showed 100% efficacy on sixth day. In-vitro efficacy trail fipronil (0.25g/100ml) recorded 100% tick’s mortality within 18th hours in post-treated group, while Deltamethrin, Trichlorphon + Dimethylester and CYM were ranked 2nd, 3rd and 5th based of their 100% efficacy within 24-33 hrs, 33-42 hrs and 39-48 hours, respectively. The investigation has shown xvii that tested acaricides varied in their performance to reduce the tick infestation and further experimentation on different formulations of other members of major acaricidal classes need to be standardized.