Guardians of the Herd: Molecular Surveillance of Tick Vectors Uncovers Theileriosis Perils in Large Ruminants

Theileria sp. (Piroplasmida: Theileriidae) is one of the most widely known infections transmitted by hard ticks (Acari: Ixodidae) and has been linked to significant economic losses across the globe. The study’s main emphasis was theileriosis, a disease that is common in Pakistan and has an incidence ranging from 0.6% to 33%. Through DNA screening of the vector ticks and host blood, this study sought to determine the risk of tick-borne theileriosis in populations of buffalos (Bubalus bubalis) and cattle (Bos indicus) in Toba Tek Singh district of Punjab, Pakistan. Identified tick species include Hyalomma anatolicum (35.4%), Rhipicephalus (Boophilus) microplus (30.2%), and R. sanguineus (25%). Tick specimens were collected from animals and their respective microenvironments. PCR assays targeting Theileria annulata were used to investigate the infection in the DNA extracted from the collected blood samples from large ruminants and salivary glands (SGs) of the Hyalomma ticks. The 18S rRNA of T. annulata was amplified using specific primers. Positive T. annulata amplicons were sequenced and verified using BLAST analysis. Overall, 50% of SGs contained T. annulate DNA. Female ticks, and those collected from cattle and from riverine environments had significantly higher (p < 0.05) rates of Theileria infection in their acini. Overall prevalence of Theileria infection was 35.9% in blood collected from large ruminants. Cattle had a substantially greater frequency of bovine theileriosis (43.2%) than buffalos (28.7%). Age and sex of large ruminants were significantly positively associated (p < 0.05) with Theileria infection. Furthermore, compared to non-riverine cattle (35%) and buffalo (19.5%), riverine cattle (52.2%) and buffalo (36.2%) showed a considerably higher prevalence. The results of this study, which is the first in Pakistan to examine the blood of large ruminants and vectorial function of Ixodid ticks in the transmission of T. annulata along with associated risk factors, offer an important insight for risk assessment of Theileria infection in livestock using vectorial infectivity.


Introduction
In addition to restlessness and anemia in animals, ticks (Acari: Ixodidae) also act as carriers of viruses, bacteria, protozoa, and rickettsia, among other pathogens.Tick infestation poses serious danger to the livestock in many parts of the world (Asmaa et al., 2014 [1]).Compared to other tick species, Hyalomma spp.(Ixodidae) have a remarkable capacity to survive in severe and difficult environments (Hasan et al., 2012 [2]).The majority of Hyalomma spp., including Hyalomma anatolicum, H. scupense, and H. lusitanicum, are known to transmit tropical theileriosis due to Theileria annulata (Piroplasmida: Theileriidae) (Yamchi and Tavassoli, 2016 [3]).Unfortunately, many factors have led to dangerously high levels of H. anatolicum in Pakistan (Rehman et al., 2017 [4]).Pakistan's livestock industry is seriously threatened by Hyalomma and Rhipicephalus tick spp.Babesia bovis, B. bigemina, and Anaplasma marginale are among the major tick-borne diseases in Pakistan, and they are prevalent throughout the world too (Perveen, 2011;Karim et al., 2017 [5,6]).In three temporal zones of KPK (Khyber Pakhtunkhwa), R. annulatus is the dominant species, followed by R. microplus and Haemaphysalis aciculifer (Farooqi et al., 2017 [7]).A critical step in risk assessment of Theileria species in domestic animals is through the examination of infesting tick populations.A simple way to predict the danger of theileriosis in livestock and the spread of the disease is to screen tick salivary glands (SGs) for Theileria sporozoites (Amiri et al., 2021;Zeb et al., 2022 [8,9]).
Theileriosis, a serious protozoal parasitic disease of ruminants, is caused by various species of the genus Theileria (Jabbar et al., 2015 [10]).Depending on the infecting species, the infected ruminants experience a condition known as tropical theileriosis or East Coast fever (Jabbar et al., 2015;Fry et al., 2016 [10,11]) and may display clinical signs such a high temperature, anemia, anorexia, and decreased milk output.The infections may be fatal in extreme circumstances (Islam et al., 2011 [12]).As a result of decreased productivity, higher veterinary expenses, and potential death, the financial effects on herds can be overwhelming to livestock operations.According to Brown (1997 [13]), this condition estimates significant economic losses of up to US $800 million per year in India, which are attributable to decreased production, morbidity, mortality, the use of anti-theilerial medications and tick control techniques such acaricides, and cost of other management strategies.
On a global scale, efforts have been undertaken to identify theilerial sporozoites by staining tick SGs with methyl green pyronin (MGP); however, the precise tick species vector remains unknown (De La Fuente et al., 2008 [14]).It is typically impossible to morphologically distinguish T. annulata from other Theileria spp. that are not harmful.Thus, Theileria sporozoites in vectors have been identified using polymerase chain reaction (PCR) (Amiri et al., 2021 [8]).The purpose of this work was to use PCR to determine the magnitude of Theileria infection in the SGs of questing (off-host) and infesting (on-host) tick populations collected from riverine (habitats located close to or along the banks of rivers) and non-riverine (habitats located at a distance from rivers) areas and in blood samples of large ruminants and their association with various factors in Toba Tek (T.T.) Singh district of Punjab, Pakistan.

Study Area and Collection of Ticks
Three tehsils make up the T.T. Singh district (30.98 • N, 72.48 • E): T.T. Singh, Kamalia, and Gojara.In the research area, adult and partially fed Ixodid ticks were collected from buffalos (Bubalus bubalis) and cattle (Bos indicus) that appeared to be in good health.For additional processing, ticks were brought to the Department of Parasitology at the University of Agriculture in Faisalabad, Pakistan.The study was conducted for a period of six months, from March 2022 to August 2022.

Collection of Blood
Blood samples were taken from both tick-infested and non-infested animals.Using a sterile syringe and ethylene-diamine tetraacetic acid (EDTA)-filled test tube, a blood sample (10 mL) was drawn aseptically from the jugular vein.Samples were transported to the laboratory and stored in a deep freezer at 4 • C for DNA extraction.Each animal's information (tag number, breed, etc.) was recorded in a separate file.Theileria DNA was detected through PCR analysis of the blood, as described below.
This study was approved by the Research Ethics Committee, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan, and University of Veterinary and Animal Sciences, Lahore, Pakistan.The standard guidelines for institutional animal care and use (IACU), University of Agriculture, Faisalabad, Pakistan, were followed.

Identification and Dissections of Ticks
The keys developed by Walker et al. (2003 [15]) were used to identify ticks.SGs were obtained by stereoscopic dissections of H. anatolicum ticks as described by Purnell et al. (1968 [16]).Dissections were carried out in ice-cold phosphate-buffered saline (PBS), pH 7. Individual ticks with at least 8 or more acini were analyzed for Theileria infection to determine the intensity of infection.

DNA Extraction and Amplification
Following the manufacturer's instructions, blood from host and SGs from ticks were used for genomic DNA extraction using a DNeasy kit (Qiagen, Hilden, Germany).Quantification of extracted DNA was based on the ratio of absorbance at 260/280 nm using a nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), then it was stored at −20 • C until use.Based on host species, tick gender, infestation status of ticks (infesting or questing), and ecological variables, pools were made.Using the oligonucleotide primer sets for Theileria genus, (Ta F) 5 AGT TTC TGA CCT ATC AG3 and (Ta R) 5 TTG CCT TAA ACT TCC TTG3 (Allsopp et al., 1993 [17]), and for T. annulate, (Ta F) 5 GTA ACC TTT AAA AAC GT3 and (Ta R) 5 GTT ACG AAC ATG GGT TT3 (d 'Oliveira et al., 1995 [18]), PCR experiments amplified a segment of the 18S rRNA gene.A 25 µL reaction volume consisted of 12.5 µL PCR supermix (Catalog number: 10572014) with 2 mM MgCl 2 , 0.05 u/µL Taq DNA polymerase, 0.2 mM of each deoxynucleotide triphosphate (Catalog number: PR-C1141), 0.5 µL forward and reverse primers, 4.5 µL nucleasefree water (Catalog number: AM9932), and 7 µL extracted DNA.DNA amplification began with an initial denaturation step at 94 • C for 5 min.Subsequently, 40 amplification cycles were performed, each consisting of denaturation at 94 • C for 1 min, annealing at 58 • C for 1 min, and extension at 72 • C for 1 min.This amplification process was carried out in a thermal cycler (C1000 Thermal Cycler, Bio Rad, Hercules, CA, USA).Ten minutes at 72 • C were spent on the last extension, then it was ended by cooling at 4 • C. PCR products were electrophoresed through a 1.8% agarose gel with an ethidium bromide (1 µg/mL) indicator in a running buffer containing Tris Acetate EDTA at 90 V for 45-60 min.Molecular Imager ® Gel DocTM XR+ with Image LabTM software version 6.1 was used to image gels after transfer to a gel documentation system (Bio Rad, Hercules, CA, USA).Purified PCR products were sequenced for validation, then a homology search was conducted on the NCBI website.

Statistical Analyses
The Chi-square test (categorical variables) was used to examine differences between independent factors (host, sex, infestation status, and ecological variable) with respect to prevalence of Theileria infection.The software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data.By dividing the number of positive samples by the total number of samples and then multiplying the result by 100, the relative prevalence of Theileria infection was determined.

Results
Among the collected ticks, the highest (p < 0.05) prevalence was of H. anatolic (35.4%), followed in order by R. microplus (30.2%) and R. sanguineus (25%) (Table 1).D to our focused hypothesis of Hyalomma infectivity for Theileria spp., we only dissected S of Hyalomma spp.for PCR.In total, 126 of the 252 pools (50%) of Hyalomma spp.SGs w positive for T. annulate (Figure 1).After DNA sequencing of representative positive sa ples, 100% homology with T. annulata was found through BLAST analysis.The frequency distribution of Theileria DNA in the SGs of H. anatolicum ticks is sho in Table 2. Compared to ticks obtained from buffalos, ticks infesting cattle showed a hig prevalence (p < 0.05) of Theileria infection.Additionally, for both infesting and quest ticks, the infection was significantly higher (p < 0.05) in female tick acini than males.S ilarly, infesting (on-host) ticks were more likely to be infected with Theileria than quest ticks (p < 0.05).The prevalence of Theileria infection was significantly higher (p < 0.005 ticks collected from riverine compared to non-riverine areas (Table 2, Figure 2).The frequency distribution of Theileria DNA in the SGs of H. anatolicum ticks is shown in Table 2. Compared to ticks obtained from buffalos, ticks infesting cattle showed a higher prevalence (p < 0.05) of Theileria infection.Additionally, for both infesting and questing ticks, the infection was significantly higher (p < 0.05) in female tick acini than males.Similarly, infesting (on-host) ticks were more likely to be infected with Theileria than questing ticks (p < 0.05).The prevalence of Theileria infection was significantly higher (p < 0.005) in ticks collected from riverine compared to non-riverine areas (Table 2, Figure 2).Bovine tropical theileriosis was found in 35.9% of cases.In total, 84 of the 136 anim whose ticks were screened were positive for Theileria.Out of these, all 84 came from a mals whose blood samples also tested positive for Theileria, demonstrating a direct li between tick and blood infection.Cattle and buffalos were both Theileria-infected.Catt (43.23%) had a significantly higher frequency of theileriosis than buffalos (28.7%).The eriosis was most prevalent in Friesian cattle (57.9%), followed in order by Jersey (44.9 and Sahiwal (35.3%) breeds.Theileriosis was more common in Kundi buffalo (44.9%) th Nili Ravi breed (17.5%).Bovine tropical theileriosis was more prevalent in female catt and buffalos (prevalence rates of 47.9% and 31.6%,respectively) than in males (prevalen rates of 28.3% and 16.2%, respectively).

Discussion
A significant component of the livestock industry, which is essential to Pakistan's agricultural economy, is large ruminants.Livestock production is at risk due to tick-borne infections such as T. annulata, which are indigenous to tropical and subtropical regions of the world, including Pakistan.Tropical theileriosis causes the livestock industry in underdeveloped nations significant financial losses (Rehman et al., 2019;Zeb et al., 2020 [19,20]).
The acini of female tick SGs were more frequently T. annulata-infected than those of male ticks.This is consistent with the finding that female ticks have greater frequencies of Theileria infection (Yamchi and Tavassoli, 2016 [3]).The different feeding habits of male and female ticks may account for variation in infection rates.Male ticks are sporadic feeders; they frequently attach to the host, feed early, and then detach to mate with females.If females are not present, male ticks may either stay connected to the host or drop off.While this is not directly related to the infection rate of females, it does imply that males may feed earlier and more erratically, which could facilitate the sporogony (Kocan et al., 1988 [30]).Males may then transmit sporozoites to the host during the first few days of attachment before mating with females.Due to early sporozoite transfer to the host, SGs of male ticks may have lower observed infection rates.To comprehend the mechanisms underlying the different infectivity rates of males and females, however, more research is required (Woods et al., 2021 [31]).The differential tick infection rates across the sexes raise interesting issues regarding the biology of these ticks.It is possible that hormonal or physiological variations between male and female ticks account for the increased infection rates seen in female tick acini.Investigating the biological differences between male and female ticks may provide vital information about the dynamics of the transmission of pathogens carried by ticks.
An interesting finding was that cattle had a considerably higher rate of Theileria infection than buffalos, which may indicate host susceptibility or vector preference differences for the parasite.In line with earlier studies (Kolte et al., 2017 [32]), we found that Theileria sporoblast prevalence was higher in ticks collected from cattle than from buffalos in the same region.It is crucial to remember that this does not suggest that the host is the cause of the higher Theileria infectivity in H. anatolicum ticks, but rather that the higher infestation rate of H. anatolicum on cattle compared to buffalos may be responsible.Additionally, because their nymphal stage may feed on calves showing high parasitemia, Hyalomma ticks that infest cattle may be more frequently infected (Kumar et al., 2020 [33]).This result confirms that crossbred cattle are more susceptible to Theileria spp.than buffalos, perhaps as a result of higher vector susceptibility of the former (Sajid et al., 2009 [26]).To estimate the risk posed to vertebrate hosts, it is essential to evaluate the prevalence of Theileria spp. in infesting and questing tick populations.To quantify the risk of tropical theileriosis in cattle and buffalos in the research area, our determination of Theileria spp.distribution in tick vectors is extremely helpful.It is possible that this variation results from a variety of variables, such as variations in these animal species' tick feeding habits or host immune responses.Understanding these elements may shed light on the mechanisms underlying the host's vulnerability to Theileria infections and open the door to specialized treatment plans.
Theileria annulata, which causes tropical theileriosis and is mostly transmitted by H. anatolicum ticks in Pakistan, was most prevalent in tick pools (6.7%), followed by T. orientalis (3.5%) and T. ovis (0.2%), according to Rehman et al. (2019 [20]).The most virulent of these species, T. annulata, has a large number of strains that are widely dispersed over the globe.In Africa and Asia, the dairy industry suffers significant financial losses from T. annulata, which causes a serious and potentially deadly disease in calves (Bishop et al., 2009;Haque et al., 2010 [34,35]).According to Jabbar et al. (2015 [10]), the disease is more severe in exotic and cross-bred cattle, in which the case-fatality rate can reach up to 80%, vs. 20% in native breeds.
Analysis by age revealed that young ruminants were more susceptible to Theileria infection than adults.Age as a risk factor yielded notable outcomes as well.These results are in line with previous studies (Qayyum et al., 2010, Farooqi et al., 2017, Zeb et al., 2020, Ullah et al., 2021 [19,36-38]) that revealed a similar profile of Theileria infection from semi-arid and arid agro-climatic zones of the country.The owner of the herd may have taken better care of adult animals, especially dairy animals, while neglecting young ones, which may be the cause of the higher incidence in young ruminants.Young animals' immune systems are not fully developed to resist or limit infection with T. annulata (Kabir et al., 2011 [39]).On the other hand, older animals may have a lower prevalence of Theileria because of a lifetime history of immune development and recurring infections (Ilhan et al., 1998;Gharbi and Darghouth, 2014 [40,41]).Bovine tropical theileriosis, with breed-and age-related variations, was common in both cattle and buffalos.These insights are essential for the creation of focused strategies for the control of ticks and tick-borne diseases in the area.
There were also gender-related differences, with female ticks and female ruminants having greater infection rates.These results call for more research into Theileria-specific interactions between ticks and gender of vector and hosts.Valente et al. (2023 [42]) found a greater proportion of females with T. annulata infection, similar to our study.The findings of Valente et al. (2023 [42]) contradicted our findings, which showed no statistically significant correlation between T. annulata positivity and sex.This could be explained by the fact that we studied a significantly higher proportion of females than males.This may be the outcome of immunosuppression in female animals brought on by ongoing stress and hormonal changes during nursing and pregnancy.Additionally, this is consistent with the findings of Kamani et al. (2010 [43]) and Parveen et al. (2021 [44]), which point to a higher prevalence in females due to their prolonged retention for various functions, such as reproduction and milk production, as well as the possibility that they may not receive sufficient feed to satisfy the high nutritional demands associated with their reproductive function.
Due to the complex relationship of ecological conditions, riverine ticks have a higher prevalence of Theileria infection than non-riverine ticks.Both tick vectors and Theileria parasites are prevalent in riverine habitats.Ticks have more possibilities to feed in these places since there are more compatible hosts available, which increases their likelihood of contracting and spreading the parasite (Salih et al., 2003;Magzoub et al., 2021 [45,46]).Additionally, ticks found in riverine areas may have greater Theileria vector competency, increasing the incidence of disease transmission.Theileria infectivity in ticks within riverine habitats may also be more prevalent due to regional livestock management practices and the existence of wildlife reservoirs (Iweriebor et al., 2022 [47]).The need for focused surveillance and control strategies to handle tick-borne diseases in such areas is successfully highlighted by these complicated ecological dynamics.

Conclusions
In conclusion, ecological investigations coupled with molecular screening tools used in the current investigations offered significant information about factors that influence tick prevalence and Theileria infection, such as species, breed, age, and sex of host, sex and infestation status of ticks, and ecological variables, as well as new insights into the PCR-based vectorial capacity for risk assessment in the community.The results of this comprehensive study shed light on the prevalence, patterns of distribution, and host connections of the complex web of tick-borne diseases.Hyalomma anatolicum is the most common species of tick, highlighting the wide habitat that ticks survive in.This diversity emphasizes the necessity for sophisticated approaches that take into account the unique ecological niches occupied by various vectors in order to combat diseases carried by ticks.Our data on the abundance of H. anatolicum and T. annulata in the host blood and vector ticks in the area emphasize how crucial it is to institute specific tick and theileriosis control methods.A stunning 50% positivity rate, verified by PCR, for the presence of T. annulata in tick salivary glands illustrates the importance of this point.The accuracy of these results is further supported by the 100% homology revealed by BLAST analysis.This confirms that H. anatolicum ticks are T. annulata carriers and highlights the risk they pose to large ruminants in the research area.
Compared to those found on buffalos, ticks infesting cattle had a greater infection rate.On-host ticks were more likely to be infected than questing ticks, and female tick acini were noticeably more contaminated than male ones.Furthermore, compared to ticks from non-riverine locations, those collected from riverine areas had higher infection rates.Bovine tropical theileriosis was found in 35.9% of cases when it was investigated.A total of 84 out of 136 animals had positive results for both tick and blood samples, proving the causal relationship between tick and blood infection.Geographically, ticks from riverine areas had greater infectivity rates, highlighting the significance of taking environment into account when analyzing disease dynamics.This study provides important information about the prevalence and variables affecting Theileria infection in ticks and large ruminants.These results can help to develop more practical methods for preventing and controlling bovine tropical theileriosis, which will ultimately improve the welfare of animals and the standard of living for farmers.

Figure 2 .
Figure 2. Prevalence of Theileria infection in tick populations in Toba Tek Singh district, Punj Pakistan.

Figure 2 .
Figure 2. Prevalence of Theileria infection in tick populations in Toba Tek Singh district, Punjab, Pakistan.

Table 1 .
Frequency distribution of hard ticks (Acari: Ixodidae) in large ruminants of Toba Tek Singh district, Punjab, Pakistan.
n = 384 (total number of animals examined).roorganisms 2023, 11, x FOR PEER REVIEW 4 o

Table 1 .
Frequency distribution of hard ticks (Acari: Ixodidae) in large ruminants of Toba Tek Si district, Punjab, Pakistan.
n = 384 (total number of animals examined).

Table 3 .
Association of various risk factors with Theileria infection in blood collected from large ruminants of Toba Tek Singh, Punjab, Pakistan.