An Assessment of the Molecular Diversity of Ticks and Tick-Borne Microorganisms of Small Ruminants in Pakistan

Abstract

This study investigated ticks and tick-borne microorganisms of small ruminants from five districts of the Federally Administered Tribal Area (FATA) of Pakistan. Morphological (n = 104) and molecular (n = 54) characterization of the ticks revealed the presence of six ixodid ticks: Rhipicephalus (Rh.) haemaphysaloides, Rh. microplus, Rh. turanicus, Haemaphysalis (Hs.) punctata, Hs. sulcata and Hyalomma anatolicum. Phylogenetic analyses of nucleotide sequence data for two mitochondrial (16S and cytochrome c oxidase 1) and one nuclear (second internal transcribed spacer) DNA regions provided strong support for the grouping of the six tick species identified in this study. Microfluidic real-time PCR, employing multiple pre-validated nuclear and mitochondrial genetic markers, detected 11 potential pathogens and endosymbionts in 72.2% of the ticks (n = 54) tested. Rickettsia (R.) massiliae was the most common pathogen found (42.6% of ticks) followed by Theileria spp. (33.3%), Anaplasma (A.) ovis and R. slovaca (25.9% each). Anaplasma centrale, A. marginale, Ehrlichia spp., R. aeschlimannii, R. conorii and endosymbionts (Francisella- and Coxiella-like) were detected at much lower rates (1.9–22.2%) in ticks. Ticks from goats (83.9%) carried significantly higher microorganisms than those from sheep (56.5%). This study demonstrates that ticks of small ruminants from the FATA are carrying multiple microorganisms of veterinary and medical health significance and provides the basis for future investigations of ticks and tick-borne diseases of animals and humans in this and neighboring regions.

Systematic Review of Ticks and Tick-Borne Pathogens of Small Ruminants in Pakistan

“…However, this study did not provide information on the prevalence and burden of ticks from various host species. Subsequently, the majority of studies conducted adopted a convenience or opportunistic sampling strategy and/or covered smaller geographical (mostly peri-urban or near veterinary institutions in major metropolitan areas) zones targeting smaller sample sizes to investigate ticks in small ruminants [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 ].…”

“…Molecular characterisation and analyses of short regions of genetic material (known as DNA barcode) can provide an alternative approach to species-level identification [ 76 , 78 ]. For this purpose, several nuclear (second internal transcribed spacer) and mitochondrial (cytochrome c oxidase subunit I, 12S and 16S ribosomal RNA) markers have been utilised successfully worldwide [ 22 , 79 , 80 , 81 , 82 ]. During the last decade, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has also been successfully used for the identification of ticks [ 73 , 83 ].…”

“…Infections with A. ovis in sheep and goats are characterised by haemolytic anaemia and a low-grade fever, respectively [ 96 ]. In Pakistan, only two studies have reported the detection of A. ovis DNA (using microfluidic real-time polymerase chain reaction (PCR) and reverse line blot assay) in Rhipicephalus , Hyalomma and Haemaphysalis ticks of small ruminants [ 22 , 86 ]. However, there is no experimental evidence for the transmission of A. ovis by these or any other tick species from Pakistan.…”

“…To date, several studies have reported the prevalence of ticks ( Hyalomma , Rhipicephalus and Haemaphysalis spp.) [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ] and tick-borne pathogens (TBPs) ( Anaplasma , Babesia and Theileria spp.) in small ruminants from various parts of Pakistan [ 27 , 28 , 29 , 30 , 31 ].…”

“…in small ruminants from various parts of Pakistan [ 27 , 28 , 29 , 30 , 31 ]. Recently, using a high-throughput microfluidic technique, a broad spectrum of microorganisms in ticks collected from sheep and goats in the Federally Administered Tribal Areas of Pakistan were reported [ 22 ]. Moreover, tick-borne zoonotic diseases such as Crimean-Congo haemorrhagic fever (CCHF) and Q fever have also been reported from different parts of Pakistan [ 32 , 33 , 34 , 35 ].…”