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

Ticks and tick-borne diseases (TTBDis) are a major constraint to the health and production of small ruminants in Pakistan. Despite being the subject of intermittent studies over the past few decades, comprehensive information on the epidemiology and control of TTBDis is lacking. Herein, we have systematically reviewed the current knowledge on TTBDis of small ruminants in Pakistan. Critical appraisal of the selected 71 articles published between 1947 to 2020 revealed that morphological examination had been the most widely used method for the identification of TTBDis in Pakistan. Tick fauna comprise at least 40 species, mainly belonging to Haemaphysalis, Hyalomma and Rhipicephalus. The prevalence of ticks is the highest in summer (June–September) and it is also higher in goats than sheep. Anaplasma, Babesia and Theileria spp. are the major tick-borne pathogens (TBPs), and their prevalence is usually higher in sheep than goats. Spatio-temporal distribution, genetic diversity and control of ticks and TBPs of small ruminants as well as the competence of tick vectors for various TBPs remain to be explored. Therefore, coordinated and focused investigations are required to fill knowledge gaps in these areas to maximise the health, production and welfare of small ruminants and minimise economic losses associated with TTBDis in Pakistan.


Introduction
Food security is one of the challenges faced by the rapidly growing human population worldwide, particularly in developing countries [1]. For example, the livestock sector plays a crucial role in the national economies and household food security of both developed and developing countries [2], and increasing the production of livestock species (e.g., cattle, sheep and goats) could be one of the ways to address the food shortage in the near future [3]. For this purpose, small ruminants (goat-Capra hircus and sheep-Ovis aries) are promising livestock species due to their resistance to drought and climatic extremes, low-input production, multipurpose use (for milk, meat and wool) and their ability to utilise household by-products and residues efficiently [4,5]. Small ruminants constitute a major

Literature Search
A literature search was conducted for studies published from 1947 to October 2020 on TTBDis of small ruminants in Pakistan using four databases (i.e., Google Scholar, Web of Science, PubMed and CAB Direct). The keywords used for search included tick(s), tick-borne disease(s), tick borne disease(s), small ruminant(s), livestock, sheep, goat(s), haemoparasite(s), anaplasmosis, Anaplasma, babesiosis, Babesia, theileriosis, Theileria, Crimean-Congo haemorrhagic fever, CCHF, Q fever, Coxiella burnetii, coxiellosis, and Pakistan. Combinations of various keywords were used to retrieve full-text research articles, postgraduate theses and conference proceedings that reported TTBDis of sheep and goats in Pakistan. Reference lists of retrieved articles were also screened to identify relevant articles (accessed until 14 October 2020).

Quality Assessment and Selection
The literature assessment and selection criteria are illustrated in Figure 1. Following the initial identification of references searched through online databases, primary screening was performed based on the titles and abstracts to remove duplicates and irrelevant articles. Full-text articles and theses unavailable online were retrieved through inter-library loans available via the University of Melbourne as well as contacting local libraries in Pakistan. Furthermore, an additional screening step was performed to exclude those articles, theses and conference proceedings that were unavailable as full-text. Where both articles and theses were available, preference was given to published articles. A total of 96 articles related to TTBDis of small ruminants from Pakistan was finally included in this review. However, 25 studies were excluded due to the duplication or poor-quality study design and/or data. Out of 71 eligible studies, 28, 36 and 7 studies were on ticks, TBPs and TTBPs, respectively ( Figure 1). Subsequently, data were extracted about the location, study type, study period, host species, tick/pathogen species and reported prevalence. Moreover, attempts were made to extract information about risk factors and interventions, where possible.

Literature Search
A literature search was conducted for studies published from 1947 to October 2020 on TTBDis of small ruminants in Pakistan using four databases (i.e., Google Scholar, Web of Science, PubMed and CAB Direct). The keywords used for search included tick(s), tick-borne disease(s), tick borne disease(s), small ruminant(s), livestock, sheep, goat(s), haemoparasite(s), anaplasmosis, Anaplasma, babesiosis, Babesia, theileriosis, Theileria, Crimean-Congo haemorrhagic fever, CCHF, Q fever, Coxiella burnetii, coxiellosis, and Pakistan. Combinations of various keywords were used to retrieve full-text research articles, postgraduate theses and conference proceedings that reported TTBDis of sheep and goats in Pakistan. Reference lists of retrieved articles were also screened to identify relevant articles (accessed until 14 October 2020).

Quality Assessment and Selection
The literature assessment and selection criteria are illustrated in Figure 1. Following the initial identification of references searched through online databases, primary screening was performed based on the titles and abstracts to remove duplicates and irrelevant articles. Full-text articles and theses unavailable online were retrieved through inter-library loans available via the University of Melbourne as well as contacting local libraries in Pakistan. Furthermore, an additional screening step was performed to exclude those articles, theses and conference proceedings that were unavailable as full-text. Where both articles and theses were available, preference was given to published articles. A total of 96 articles related to TTBDis of small ruminants from Pakistan was finally included in this review. However, 25 studies were excluded due to the duplication or poor-quality study design and/or data. Out of 71 eligible studies, 28, 36 and 7 studies were on ticks, TBPs and TTBPs, respectively ( Figure 1). Subsequently, data were extracted about the location, study type, study period, host species, tick/pathogen species and reported prevalence. Moreover, attempts were made to extract information about risk factors and interventions, where possible.

Estimation of Prevalence
Prevalence was estimated as the number of hosts infested/infected with at least one individual of a particular parasite divided by the total number of hosts examined for the parasite [37]. We collated the prevalence data on TTBDis from all studies where possible, then estimated the overall prevalence and 95% confidence interval (CI) of TTBPs in different hosts and locations using cumulative population data in Excel spreadsheet (Microsoft 365 ® ) and R using the package "binom" [38] following the Clopper-Pearson interval method [39].

Studies on Ticks
To date, 28 studies have investigated ticks of sheep and goats in Pakistan whereas only one and six studies focused separately on ticks of goats and sheep, respectively. Details of tick species, host, estimated prevalence, region and identification methods are given in Table 1 and Figure 2.

Estimation of Prevalence
Prevalence was estimated as the number of hosts infested/infected with at least one individual of a particular parasite divided by the total number of hosts examined for the parasite [37]. We collated the prevalence data on TTBDis from all studies where possible, then estimated the overall prevalence and 95% confidence interval (CI) of TTBPs in different hosts and locations using cumulative population data in Excel spreadsheet (Microsoft 365 ® ) and R using the package "binom" [38] following the Clopper-Pearson interval method [39].

Studies on Ticks
To date, 28 studies have investigated ticks of sheep and goats in Pakistan whereas only one and six studies focused separately on ticks of goats and sheep, respectively. Details of tick species, host, estimated prevalence, region and identification methods are given in Table 1 and Figure 2.

Tick Species
A total of 40 species belonging to six ixodid (Amblyomma, Dermacentor, Ixodes, Hyalomma, Haemaphysalis and Rhipicephalus) and three argasid (Argas, Otobius and Ornithodoros) ticks have been reported in sheep and goats from Pakistan ( Table 2). The majority of tick species belonged to three genera, i.e., Hyalomma (13 species), Haemaphysalis (11 species) and Rhipicephalus (8 species) ( ). However, we could not reliably compare the prevalence of ticks in sheep and goats due to the differences in various parameters (such as climate, sample size and target population) of various studies from Pakistan. Although not supported through scientific evidence, sheep wool could provide a barrier against tick infestation on some parts of the body [19,24] whereas agile, restless and grooming characteristics of goat behaviour can make
Data analyses of the reported prevalence estimates [number of studies (n) = 21] of ticks in sheep and goats revealed that 27.85% (8032/28,840; range: 0-86.5%; 95% confidence interval (CI): 27.3-28.4%) of the studied population of small ruminants were infested. Tick infestation was apparently higher in goats (30.67%; 5592/18,229; range: 6.7-86.5%; 95% CI: 30.0-31.3%) than sheep (23%; 2440/10,611; range: 0-81.5%; 95% CI: 22.2-23.8%). However, we could not reliably compare the prevalence of ticks in sheep and goats due to the differences in various parameters (such as climate, sample size and target population) of various studies from Pakistan. Although not supported through scientific evidence, sheep wool could provide a barrier against tick infestation on some parts of the body [19,24] whereas agile, restless and grooming characteristics of goat behaviour can make them relatively resistant to ectoparasites such as ticks [40]. Therefore, both sheep and goats could be equally susceptible to tick infestation in the subtropical conditions of Pakistan.

Epidemiology of Ticks
To date, the majority of studies (24/35) aimed at the epidemiology of ticks have been conducted in Punjab and Khyber Pakhtunkhwa, whereas only 1-4 studies were available from other regions which inhabit more than half of the population of small ruminants in Pakistan [11]. To the best of our knowledge, the first detailed account on the morphological characterisation of ticks in various mammalian hosts (including livestock, companion and wild animals) from Pakistan was provided by McCarthy [41]. 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].
Seasonal variation of tick infestation in a region is dependent upon the fluctuation of monthly/annual temperature and moisture [67]. To date, a few studies have assessed the seasonal variation in tick prevalence in small ruminants in Pakistan and reported a higher tick infestation in summer (June-September) [23,25,50,51,53,56,58,[64][65][66], possibly due to higher temperature-and moisture-levels suitable for the development of ticks [68,69]. A number of risk factors can favour the tick infestation in animals [19,70] and only two studies have investigated risk factors associated with tick infestation of sheep and goats in Pakistan [19,53]. These authors found that traditional housing, free grazing, the lack of acaricidal drug use and the absence of rural poultry were the main risk factors for the higher occurrence of ticks in small ruminants [19,53]. Furthermore, tick prevalence is affected by climatic conditions across different agro-ecological zones (AEZs) of a region/country [71]. Recently, we demonstrated a significant variation in the prevalence (22.2-70.5%; p < 0.0001) of bovine ticks (Hy. anatolicum, Hy. hussaini, Hy. scupense, Rh. annulatus and Rh. microplus) across five AEZs of Pakistan [72]. However, for small ruminants, only a small population of sheep [number of individuals (N = 18)] and goats (N = 80) from arid and semi-arid AEZs of Punjab province [19] was examined for tick infestation. Given that the tick infestation of animals can be influenced by various factors such host, husbandry, management and environment, it is pivotal to enhance our understanding of the epidemiology of ticks of small ruminants by assessing agro-climatic and spatio-temporal differences across various AEZs of Pakistan so that effective and sustainable control programs for TTBDis of small ruminants could be developed.

Identification of Ticks
Accurate identification of ticks is central to the understanding of the epidemiology of TTBDis and developing effective control strategies [73]. Morphological characterisation using dichotomous keys has been the most commonly used method to identify ticks to species level (22/25) followed by a combination of microscopic and molecular methods (3/25) (Table 1). However, six studies provided only genus-level identification of ticks [27,46,49,54,57,65] whereas three studies did not provide information on the identification of ticks [43,45,64]. Although the microscopic examination of ticks is simple and cheap, it has several limitations such as the requirement of entomological expertise for the identification of closely-related species (e.g., Rh. sanguineus and Rh. turanicus; Hy. anatolicum and Hy. excavatum), immature or larval stages, and engorged or damaged specimens [74][75][76][77]. Such limitations could sometimes lead to unreliable data, including reports of non-endemic tick species such as those of Rh. appendiculatus [42] and Amblyomma hebraeum [58] from Pakistan. 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]. However, due to the limitations associated with each method, no single method is ideal for accurate characterisation of ticks. Therefore, the use of a combination of morphological and molecular methods would be essential for the surveillance and control of ticks of veterinary and public health significance in Pakistan.

Control of Ticks in Small Ruminants
There is a scarcity of data on method(s) used for the control of ticks in small ruminants from Pakistan, partly because farmers pay less attention to the husbandry and management of sheep and goats than those of bovines due to the higher economic value of latter (Ghafar et al. unpublished data). Despite the serious environmental and health implications associated with acaricides (such as macrocyclic lactones, trichlorfon and cypermethrin), their periodic application is the main tick control method used in small and large ruminants in Pakistan [14,84,85]. Additionally, grooming, i.e., manual picking of ticks by the farm workers, is also commonly practised for tick control in Pakistan [19]. To date, only two studies have assessed the in vivo efficacy of acaricidal drugs, including coumaphos, cypermethrin, diazinon and ivermectin [48,53], and these authors concluded that cypermethrin was the most effective drug against ticks in both sheep and goats (Table 3).  [32,34,35] Owing to the limited understanding of spatio-temporal epidemiology of ticks of small ruminants in Pakistan, no regular tick control program is followed [14]. During the last decade, due to increasing cases of CCHF infections in humans, event-based tick control campaigns (known as anti-Congo campaign) were launched every year by the provincial governments just before Eid-ul-Adha-a religious festival of Muslims when they slaughter animals at their homes [90][91][92]. Additionally, sporadic campaigns are also common during the summer season. These campaigns involved the repetitive use of same acaricidal drugs (mostly injectable ivermectin and/or cypermethrin spray) over the years which could possibly have contributed to the development of acaricidal resistance in ticks as reported from elsewhere [53,[93][94][95]. There is a need to test the efficacy of alternative acaricidal drugs and other prophylactic measures such as tick vaccines [96]. Furthermore, future studies investigating the status of acaricidal resistance in tick populations of small ruminants would guide integrated control of ticks in this country. Such integrated tick control strategies, consisting in the systematic combination of at least two control technologies, including anti-tick recombinant vaccines, aiming to reduce selection pressure in favour of acaricide-resistant individuals, while maintaining adequate levels of animal production, have been implemented in some countries such as Cuba with promising results [97].

Tick-Borne Pathogens in Pakistani Small Ruminants and Their Ticks
To date, bacterial (anaplasmosis and Q fever) protozoal (babesiosis and theileriosis) and viral (CCHF) TBDs of veterinary and public health significance have been reported in small ruminants as well as their ticks from Pakistan (Figure 3, Tables 2 and 4-6). The following sections provide an overview of the key TBDs of ruminants in Pakistan.

Tick-Borne Pathogens in Pakistani Small Ruminants and Their Ticks
To date, bacterial (anaplasmosis and Q fever) protozoal (babesiosis and theileriosis) and viral (CCHF) TBDs of veterinary and public health significance have been reported in small ruminants as well as their ticks from Pakistan (Figure 3, Tables 2 and 4-6). The following sections provide an overview of the key TBDs of ruminants in Pakistan.

Anaplasmosis
Anaplasmosis is one of the most important TBDs of livestock in Pakistan [14] and, in small ruminants, it is caused by members of an intracellular, Gram-negative bacteria, Anaplasma (Rickettsiales: Anaplasmataceae; A. ovis, A. phagocytophilum and A. marginale) [101,102]. It is transmitted by various genera of ticks, including Dermacentor, Hyalomma, Haemaphysalis and Rhipicephalus [103][104][105]. 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.

Anaplasmosis
Anaplasmosis is one of the most important TBDs of livestock in Pakistan [14] and, in small ruminants, it is caused by members of an intracellular, Gram-negative bacteria, Anaplasma (Rickettsiales: Anaplasmataceae; A. ovis, A. phagocytophilum and A. marginale) [101,102]. It is transmitted by various genera of ticks, including Dermacentor, Hyalomma, Haemaphysalis and Rhipicephalus [103][104][105]. 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, a total of 12 studies (Punjab = 3; Khyber Pakhtunkhwa = 8; Sindh = 1) has reported the occurrence of three Anaplasma species (A. ovis, A. marginale and A. centrale) in small ruminants (n = 10) [29,87,98,[106][107][108][109][110][111][112] and ticks (n = 2) [22,86] from Pakistan (see Table 4). Based on these studies, the estimated overall prevalence of anaplasmosis in Pakistani small ruminants are 1.7-55.3% and 25.3-47.2% using microscopic and molecular methods, respectively. However, slightly higher prevalences were reported in sheep (13.9-55.3%; 23.9-36.8%; and 28-47.2%) than goats (1.7-30.7%; 20.6-32.8%; and 25.3-34.8%) using microscopic, serological (enzyme-linked immunosorbent assay [ELISA]) and molecular methods, respectively. This higher prevalence in sheep could possibly be due to a higher susceptibility of sheep to clinical anaplasmosis than goats [105]. The prevalence of Anaplasma spp. in both sheep and goats was the highest (25.3-55.3%) in Punjab province followed by Khyber Pakhtunkhwa (1.7-47.2%) and Sindh (13.3%). To date, only one study has estimated the occurrence of Anaplasma spp. in ticks of small ruminants from Khyber Pakhtunkhwa province which reported a higher occurrence of the bacteria in ticks from sheep (39.1%) than those collected from goats (35.5%) [22]. Despite a widespread occurrence and the reported higher prevalence of A. ovis, the epidemiology of anaplasmosis in Pakistani small ruminants is poorly-understood, probably due to mild and/or asymptomatic infections in sheep and goats as well as the lack of record keeping by small-holder farmers [113,114]. Given the recently identified zoonotic potential of A. ovis and its ability to cause severe clinical disease particularly when present as a co-infection [113,115], future research should focus on understanding the disease epidemiology and vector competence of potential ticks known to infest small ruminants in different AEZs of the country.

Babesiosis
Babesiosis, caused by intraerythrocytic protozoa of the genus Babesia (Piroplasmida: Babesiidae), is one of the most common and economically important TBD of domestic and wild ruminants worldwide [116]. The disease is mainly transmitted by Dermacentor, Hyalomma, Haemaphysalis and Rhipicephalus ticks [17,105]. In sheep and goats, clinical babesiosis is caused by Babesia ovis, B. motasi and yet unidentified Babesia sp. (China) whereas B. crassa is usually associated with mild infections [105]. Acute babesiosis is characterised by high fever, anaemia, tachycardia, jaundice, haemoglobinuria, abdominal pain and death [104]. Higher infection rates and severe clinical manifestations are more common in sheep than goats [17,105,117]. To date, only B. ovis has been reported in small ruminants in Pakistan [28,88,118]. Similarly, B. ovis has been detected using the microscopic examination of the haemolymph of Rh. sanguineus collected from sheep and goats [48] as well as from a tick collected from bovines, Hy. anatolicum using a microfluidic-based real-time PCR [119].
Among all major TBDs of small ruminants in Pakistan, babesiosis is the least-studied disease (Punjab = 5; Khyber Pakhtunkhwa = 4) (see Table 5). Based on the available data on the occurrence of babesiosis in small ruminants, 7-41.7% and 23.9-55% of the studied population of goats and sheep was positive using microscopic and molecular methods, respectively. The prevalence of babesiosis was variable in sheep (7-29% and 50-55%) and goats (13.5-41.7% and 23.9%) using microscopic and molecular methods, respectively. Like anaplasmosis, the higher prevalence of babesiosis (using molecular methods) in sheep could be due to the natural resistance of goats to TBDs [17,105,120].
To date, only one study has reported the occurrence of B. ovis in ticks (microscopic examination) from sheep (1.5%) and goats (1%) [48] whereas the molecular screening of a small number of ticks (N = 54) from small ruminants in Khyber Pakhtunkhwa did not detect B. ovis [22]. Without the large-scale epidemiological investigation of caprine and ovine Babesia species using high-throughput techniques, it is not possible to assess the level of risks associated with babesiosis in small ruminants from Pakistan.

Theileriosis
Theileriosis is caused by members of genus Theileria (Piroplasmida: Theileridae) and three pathogenic (Theileria lestoquardi, T. luwenshuni and T. uilenbergi) and three non-pathogenic (T. ovis, T. separate and T. recondite) species are known to infect small ruminants [121]. Transmission occurs via tick species belonging to three main genera, Haemaphysalis, Hyalomma and Rhipicephalus [122]. In most parts of the world, malignant theileriosis in sheep and goats is caused by T. lestoquardi and is transmitted by Hy. anatolicum [121], and the main clinical signs include anorexia, anaemia, naso-lacrimal discharge, fever, emaciation, enlarged prescapular lymph nodes, haemoglobinuria, cardiac dysfunction and even death [123,124].
Another major tick-borne zoonotic disease in Pakistan is CCHF and only a few studies have detected this virus in blood (1%, 8/800) and ticks (3.8%, 20/525) collected from sheep [61], and specific antibodies in sheep (18.6-32.5%) and goats (4.6-18.9%) [32,35]. In Pakistan, the incidence of CCHF is usually higher in urban areas before Eid-ul-Adha when people slaughter animals as a religious ritual [90,92]. Moreover, it is believed that the last two decades of the Afghan war also resulted in a large influx of refugees along with their livestock, leading to an increase in CCHF cases [92]. In small ruminants, CCHF is usually asymptomatic, but it can be life-threatening in humans [138], who usually become infected upon exposure to a vector (Hyalomma) or body fluids of the infected animals [138]. In Pakistan, farmers and veterinarians are at a higher risk of CCHF due to the limited knowledge of the disease and its transmission, high tick prevalence-particularly ticks of the genus Hyalomma which is the principal vector for CCHFV-on small-scale farms, poor diagnostic facilities and the lack of control and preventive measures for both ticks and the virus [72,92].

Diagnosis and Control of TBPs in Pakistan
Microscopic examination of Giemsa-stained blood smears is considered the gold standard for the diagnosis of haemoparasitic infections worldwide [139]. Several serological assays (such as ELISA, indirect fluorescence assay and CFT) are also available for the detection of antigens or antibodies against TBPs [106,[140][141][142][143]. However, microscopic and serological methods are of limited value due to several limitations, including lower sensitivity and specificity, cross-reactivity, inability to detect carrier infections, and the requirement of expertise and time [140,144,145]. These limitations have been overcome through the use of highly sensitive molecular methods, including conventional PCR (cPCR), quantitative PCR (qPCR), nested PCR (nPCR), reverse line blotting (RLB), loop-mediated isothermal amplification (LAMP), high-resolution melting (HRM) assays, high-throughput microfluidics-based real-time PCR and the next-generation sequencing (NGS) [105,[146][147][148][149][150].
In Pakistan, microscopy is the most commonly used method for the detection of TBPs in scientific studies. However, field diagnosis is usually made based on clinical signs and the history of tick exposure, mainly due to the unavailability of well-equipped veterinary diagnostic laboratories in the country [14,114,151]. To date, a few studies have used serological (ELISA, indirect fluorescence assay (IFA) and CFT) and molecular methods (cPCR, qPCR, nPCR and microfluidic real-time PCR); however, the sequencing of PCR amplicons has rarely been performed, thereby no detailed information is available on the genetic diversity of TBPs in small ruminants from Pakistan. Moreover, a number of studies also investigated changes in the haematological profiles of animals infected with TBPs and reported a decrease in haemoglobin and packed cell volume associated with anaplasmosis, babesiosis and theileriosis [30,31,87,106,133].
Control of TBDs in small ruminants mainly relies on the use of acaricides (listed under the Section 3.1.4), antibiotics (oxytetracycline) and antiprotozoal drugs (imidocarb dipropionate, diminazene aceturate and buparvaquone) [139,152]. In Pakistan, data on the control of TBDs in small ruminants is scarce and acaricidal drugs are used to control ticks [53], whereas clinical cases of anaplasmosis, babesiosis and theileriosis are usually treated with a combination of babesicidal and theilericidal drugs and/or antibiotics. A few studies have tested the efficacy of various drugs against TBDs of small ruminants in Pakistan and reported that imidocarb dipropionate and oxytetracycline/diminazene aceturate were effective against babesiosis [99,100] and anaplasmosis [53,87], respectively.

Conclusions and Future Perspectives
To date, most of the previous studies on TTBDis of small ruminants in Pakistan have (i) reported point prevalences of ticks and TBPs, mainly from Punjab and Khyber Pakhtunkhwa provinces, (ii) been conducted in peri-urban areas or near veterinary institutes using convenient and random sampling strategy, and (iii) utilised morphological methods for the identification of TTBPs. Although these studies have provided the information about the prevalence of TTBPs and some seasonal variation of ticks in small ruminants from Pakistan, there are still knowledge gaps about important epidemiological aspects of TTBDis, including (i) the lack of accurate species identification of ticks and TBPs using molecular methods as the microscopic examination has been the most common method of identification which has very low sensitivity and specificity [74,75], (ii) unavailability of data on risk factors, (iii) limited knowledge of TTBPs across different AEZs and production systems, seasonal variation of TBPs, and the efficacy of drugs used against TTBDis and acaricidal resistance.
For TBPs, several studies also used molecular and serological diagnostic methods. However, in most cases, molecular methods were only applied to amplify the target DNA from the positive samples screened by microscopic examination. Moreover, PCR amplicons were only rarely sequenced, and the target pathogens were identified solely based on the visualisation of expected PCR amplicon size on the agarose gel. Additionally, a few studies tested ticks for the presence of TBPs of veterinary (n = 6) or zoonotic significance (n = 4). Finally, no attempts were made to determine the vector competence of various tick species.
Future research should be directed towards investigating the epidemiology of TTBDis across different AEZs, in different seasons and under various production systems. In developing countries like Pakistan, where resources and laboratory facilities are limited, newly developed field-oriented and low-cost diagnostic methods could be very useful for routine and large-scale surveillance of TTBPs. One such method is LAMP [153]. It is a highly specific, simple, sensitive, robust and rapid method [154] and has the capacity to detect pathogens efficiently in partially processed tick specimens [155,156]. Furthermore, recent innovations of paper-based microfluidics for malaria diagnosis [157] and image-processing based platforms using smartphones for the detection of Chikungunya, Dengue, and Zika viruses [158,159] could be adopted for field diagnosis and surveillance of major TBPs of small ruminants. Concurrently, research should also be focused on unexplored areas of population genomics of TTBPs using high-throughput techniques such as NGS and microfluidics. As discussed earlier, the current focus of research is on the control of TTBDis of large ruminants in this country [72,119,[160][161][162], however, in a predominantly mixed-species farming system where small and large ruminants are reared together, sheep and goats could serve as alternative hosts for TTBPs of large ruminants [163,164]. Therefore, for the effective control of TTBDis of livestock in Pakistan, it is imperative to design future studies including common livestock species that are kept in proximity.
Climate change is playing a preeminent role in the expansion of tick population ranges as well as enhancing the pathogen transmission to humans and animals worldwide [165][166][167]. Data-based modelling studies suggest that the changing rainfall patterns and rising environmental temperature would cause a long-term change in the dynamics of TTBPs, resulting in an increased risk of infection in humans and animals [165]. It is likely that developing countries like Pakistan could be exposed to the significant impact of climate change, mainly due to the lack of awareness about mitigation measures, a weak economy and poor institutional capacity to combat this emerging issue [168,169]. In order to design preparatory strategies to tackle climate change in the area of livestock diseases, TTBPs-specific data are required from different mammalian hosts and their habitats from various AEZs of the country.
Studies on assessing the efficacy of various drugs against TTBPs would be pivotal for the identification of potentially effective control methods of TTBDis of livestock in Pakistan. Furthermore, alternative, eco-friendly and sustainable measures (such as multivalent vaccines) should be explored for the control of TTBDis and omics-derived tick microbiome information could be useful for this purpose. Farmer awareness campaigns should also be launched about the rational use of acaricides and risks associated with TTBPs, particularly in case of zoonotic TBPs. Participatory epidemiology (PE) could be very useful for such campaigns as well as for large-scale epidemiological investigations as it has been recently utilised for investigation of bovine health and production constraints in Pakistan [114]. Mobile phone-based applications to process images [170] could also be used for investigations on incidence, prevalence, risk factors and control practices for TTBDis of small ruminants as well as assessing related knowledge within communities in Pakistan and other parts of the world.
Overall, this review has demonstrated that the prevalence of TTBDis in Pakistani small ruminants is high and emphasises the need for further intensive research on the epidemiology, ecology, population genomics and control of TTBPs. The use of advanced but 'practical' diagnostic tools will be critical in attaining an improved understanding of interactions among vectors, microbiomes, mammalian hosts and the environment, and should guide the development of integrated and sustainable control of TTBDis through the One Health perspective. It is believed that such a strategy would provide effective control of TTBDis of small ruminants and benefit the resource-poor farmers in Pakistan and elsewhere to address the challenge of food security.