Genotypic analysis of hepatitis E virus (HEV) from sporadic symptomatic cases in Pakistan.

Pakistan is the fifth most populous nation in the world and faces several challenges, including devastating floods, sub-optimal sanitary conditions, clustered accommodations, and unregulated cross-border movements. These drastic population shifts make it vulnerable to the efficient spread of the Hepatitis E virus (HEV). The current study analyzed the genotypic characteristics and variants of the Hepatitis E virus circulating in the population of Pakistan. A total of 75 ELISA-IgM positive samples were collected from three metropolitan cities: Lahore, Peshawar, and Karachi, and subjected to viral RNA extraction. The amplification of the HEV RNA-dependent RNA polymerase (RdRp) region was done using Nested PCR and degenerate primers. Out of the total, 40% of the samples were positive for HEV RNA. Sequencing and phylogenetic analysis identified the new HEV isolates as Subtype 1 g, a subtype within an existing HEV genotype 1. This shift warrants investigation into its impact on clinical manifestation and disease severity. Importantly, this study marks the first HEV subtype analysis in Pakistan, contributing valuable insights into subtype diversity and prevalence in the region.

Mechanistic insight for improving butenyl-spinosyn production through combined ARTP/UV mutagenesis and ribosome engineering in Saccharopolyspora pogona.

Butenyl-spinosyn is a highly effective, wide-spectrum and environmentally-friendly biological insecticide produced by Saccharopolyspora pogona. However, its scale-up is impeded due to its lower titer in wild-type strains. In this work, ARTP/UV mutagenesis and ribosome engineering were employed to enhance the butenyl-spinosyn production, and a stable mutant Saccharopolyspora pogona aG6 with high butenyl-spinosyn yield was successfully obtained. For the first time, the fermentation results in the 5 L bioreactor demonstrated that the butenyl-spinosyn produced by mutant Saccharopolyspora pogona aG6 reached the maximum value of 130 mg/L, almost 4-fold increase over the wild-type strain WT. Furthermore, comparative genomic, transcriptome and target metabolomic analysis revealed that the accumulation of butenyl-spinosyn was promoted by alterations in ribosomal proteins, branched-chain amino acid degradation and oxidative phosphorylation. Conclusively, the proposed model of ribosome engineering combined with ARTP/UV showed the improved biosynthesis regulation of butenyl-spinosyn in S. pogona.

Seroprevalence and Molecular Evidence of Coxiella burnetii in Dromedary Camels of Pakistan.

Coxiellosis is a zoonosis in animals caused by Coxiella burnetii. A cross-sectional study was conducted on 920 (591 female and 329 male) randomly selected camels (Camelus dromedarius) of different age groups from 13 districts representative of the three different ecological zones in the Province Punjab, Pakistan to determine the prevalence and associated risk factors of coxiellosis. The blood samples were collected and tested for anti-C. burnetti antibodies using indirect multispecies ELISA. Real-time PCR was used for the detection of C. burnetii DNA to determine the prevalence in heparinized blood pools. Out of 920 investigated camels, anti-C. burnetii antibodies were detected in 288 samples (31.3%) (95% CI: 28.3-34.4%). The highest (78.6%) and lowest (1.8%) seroprevalence were detected in Rahimyar Khan (southern Punjab) and in Jhang (central Punjab), respectively. Potential risk factors associated with seropositivity of the Q fever in camels included desert area (42.5%; OR = 2.78, 95% CI 1.12-3.21) summer season (35.7%; OR = 2.3, 95% CI: 1.31-3.2), sex (female) (39.1; OR = 2.35, 95% CI: 1.34-2.98), tick infestation (51.3%;OR = 2.81, 95% CI: 1.34-3.02), age (>10 years; 46.4%; OR = 1.56, 95% CI: 0.33-2.05) and herd size (38.5%; OR = 1.21, 95% CI: 0.76-1.54). Coxiella burnetii DNA was amplified in 12 (20%) and 1 (10%) of 60 ELISA-negative and 10 suspected camels, respectively. DNA could not be detected in ELISA positive blood pools. This study emphasizes the seroprevalence and associated risk factors of coxiellosis as well as its potential to spill over to animals and humans in contact with these camel herds.

Multiscale engineering of microbial cell factories: A step forward towards sustainable natural products industry.

Microbial cell factories (bacteria and fungi) are the leading producers of beneficial natural products such as lycopene, carotene, herbal medicine, and biodiesel etc. These microorganisms are considered efficient due to their effective bioprocessing strategy (monoculture- and consortial-based approach) under distinct processing conditions. Meanwhile, the advancement in genetic and process optimization techniques leads to enhanced biosynthesis of natural products that are known functional ingredients with numerous applications in the food, cosmetic and medical industries. Natural consortia and monoculture thrive in nature in a small proportion, such as wastewater, food products, and soils. In similitude to natural consortia, it is possible to engineer artificial microbial consortia and program their behaviours via synthetic biology tools. Therefore, this review summarizes the optimization of genetic and physicochemical parameters of the microbial system for improved production of natural products. Also, this review presents a brief history of natural consortium and describes the functional properties of monocultures. This review focuses on synthetic biology tools that enable new approaches to design synthetic consortia; and highlights the syntropic interactions that determine the performance and stability of synthetic consortia. In particular, the effect of processing conditions and advanced genetic techniques to improve the productibility of both monoculture and consortial based systems have been greatly emphasized. In this context, possible strategies are also discussed to give an insight into microbial engineering for improved production of natural products in the future. In summary, it is concluded that the coupling of genomic modifications with optimum physicochemical factors would be promising for producing a robust microbial cell factory that shall contribute to the increased production of natural products.

Advances in sustainable approaches utilizing orange peel waste to produce highly value-added bioproducts.

Orange peel waste (OPW), a discarded part of orange fruit, is a rich source of essential constituents that can be transformed into highly value-added bioproducts. OPW is being generated in million tonnes globally and returns to the environment without complete benefit. Thus, a high volume of annually produced OPW in the industry requires effective valorization. In this regard, limited data is available that summarizes the broader spectrum for the sustainable fate of OPW to produce value-added bioproducts. The main objective of this treatise is to explore the sustainable production of bioproducts from OPW. Therefore, this review covers all the aspects of OPW, from its production to complete valorization. The review encompasses the extraction technologies employed for extracting different valuable bioactive compounds, such as: essential oil (EO), pectin, and carotenoids, from OPW. Furthermore, the suitability of bioconversion technologies (digestion/fermentation) in transforming OPW to other useful bioproducts, such as: biochemicals (lactic acid and succinic acid), biopolysaccharides (xanthan and curdlan gum), and bioenergy (biomethane and bioethanol) is discussed. Also, it includes the concept of OPW-based biorefineries and their development that shall play a definite role in future to cover demands for: food, chemicals, materials, fuels, power, and heat. Lastly, this review focuses on OPW-supplemented functional food products such as: beverages, yogurts, and extruded products. In conclusion, insights provided in this review maximize the potential of OPW for commercial purposes, leading to a safe, and waste-free environment.

Kinetically modelled approach of xanthan production using different carbon sources: A study on molecular weight and rheological properties of xanthan.

The present study emphasizes improving the overall yield, productivity and quality of xanthan by Xanthomonas campestris using different carbon sources via optimizing the fermentation media and kinetic modelling work. After optimization, six carbon sources and one nitrogen source were selected for xanthan production in 5 L bioreactor. Kinetic modelling was applied to assess the experimental fermentation data and to check its influence on scale-up production. In this work, xanthan production reached 40.65 g/L with a growth-associated rate constant (α) of 2.831, and highest specific growth rate (µm) of 0.37/h while using maltose as the sole carbon source. Furthermore, rheological properties were determined, and Herschel-Bulkley model was employed to assess the experimental data. Interestingly, xanthan obtained from sucrose and glucose showed the highest yield stress (τ0) of 12.50 ± 0.31 and 7.17 ± 0.21. Moreover, the highest xanthan molecular weight of 3.53 × 107 and 3.25 × 107 g/mol were also found with sucrose and glucose. At last, the proposed mechanism of sugar metabolism and xanthan biosynthesis pathway were described. Conclusively, maltose appeared as the best carbon source for maximum xanthan production: while sucrose and glucose gave qualitatively best results. In short, this systematically modelled approach maximizes the potential output and provides a solid base for continuous cultivation of xanthan at large-scale production.

Animal and Human Brucellosis in Pakistan.

Brucellosis is a bacterial zoonotic disease that affects many animal species and can be transmitted to humans via direct contact or via contaminated food. Although brucellosis is a serious health hazard, its public health concern has been neglected in many countries. In some developing countries, such as Pakistan, where brucellosis is endemic, this disease continues to be of importance. A literature search for the past 11 years (2011-2021) provided a comprehensive insight into brucellosis in Pakistan. In this review, particular emphasis was placed on occurrence, diagnostic tests used, and prevention, treatment, and control in the context of the "One Health" approach.

Rationally optimized generation of integrated Escherichia coli with stable and high yield lycopene biosynthesis from heterologous mevalonate (MVA) and lycopene expression pathways.

The stability and high productivity of heterogeneous terpenoid production in Escherichia coli expression system is one of the most key issues for its large scale industrialization. In the current study on taking lycopene biosynthesis as an example, an integrated Escherichia coli system has been generated successfully, which resulted into stable and high lycopene production. In this process, two modules of mevalonate (MVA) pathway and one module of lycopene expression pathway were completely integrated in the chromosome. Firstly, the copy number and integrated position of three modules of heterologous pathways were rationally optimized. Later, a strain DH416 equipped with heterogeneous expression pathways through chromosomal integration was efficiently derived from parental strain DH411. The evolving DH416 strain efficiently produced the lycopene level of 1.22 g/L (49.9 mg/g DCW) in a 5 L fermenter with mean productivity of 61.0 mg/L/h. Additionally, the integrated strain showed more genetic stability than the plasmid systems after successive 21st passage.

In-House Developed ELISA Indicates High Prevalence of Anti-Echinococcus granulosus IgG in Sheep Population-An Update from Pakistan.

Cystic echinococcosis (CE) is a World Health Organization (WHO)-listed neglected tropical farm economy jeopardizing and public health concern disease. This study was aimed at furnishing sero-epidemiological baseline data of CE in sheep in Pakistan, where data are non-existent. For this purpose, two sheep-rich provinces of Pakistan were selected, and 728 sheep sera were collected using probability proportional to size (PPS) statistical technique. Epidemiological information was recorded on a questionnaire for the estimation of potential risk factors. The serum samples were analyzed for IgG antibodies against Echinococcus granulosus using an in-house-developed EgAgB-based ELISA kit. The overall seroprevalence recorded was 21.98% (160/728) in the tested sheep, suggesting higher seropositivity in sheep from Punjab (23.73%) as compared to Khyber Pakhtunkhwa (KPK) (19.04%). The overall apparent prevalence observed by this ELISA method was almost similar to the calculated true prevalence (21.77%). Prevalence was significantly different (p < 0.05) among sheep from different districts. Higher prevalence was found in females (22.54%, OR 1.41), age group > 5 years (29.66%, OR 1.64), crossbreeds (42.85%, OR 2.70), and sheep with pasture access (25.96%, OR 3.06). Being in age group > 5 years and having pasture access were the factors significantly associated with seropositivity (p < 0.05). This study provides serological evidence of E. granulosus infection in sheep and can be used as a model for ante-mortem screening of the sheep globally.

Epidemiology and Associated Risk Factors for Brucellosis in Small Ruminants Kept at Institutional Livestock Farms in Punjab, Pakistan.

Brucellosis is reportedly endemic in ruminants in Pakistan. Both Brucella abortus and B. melitensis infections have been decumented in domestic animals and humans in the country. This study aimed to identify the burden of anti-Brucella antibodies in small ruminants as well as associated potential risk factors with its occurrence at nine institutional livestock farms in Punjab, Pakistan. The sera collected from equal number of sheep and goats (500 from each species) were screened by indirect-ELISA for anti-smooth-Brucella antibodies followed by a serial detection by real-time PCR. Overall, 5.1% (51/1000) seropositivity was registered corresponding to 5% (25/500) prevalence in goats and 5.2% (26/500) in sheep. Brucella-DNA could not be detected in any of the tested sera by real-time PCR. Multiple logistic regression model indicated that farm location (OR 34.05), >4 years of age (OR 2.88), with history of reproductive disorders (OR 2.69), and with BCS of ≤ 3 (OR 12.37) were more likely to test positive for brucellosis at these farms. A routine screening, stringent biosecurity, and quarantine measures are warranted for monitoring and eradication of the infection. Similarly, isolation and molecular investigation of the etiologic agent(s) are needed to understand the relationship of epidemiology and out-breaks of brucellosis in the country.

Serological and Molecular Investigation of Brucellosis in Breeding Equids in Pakistani Punjab.

Brucellosis is an important zoonosis worldwide. Equines are susceptible to the infection when in close contact with infected animals. The objective of our study was to update the existing knowledge and detect and differentiate the causative agent of brucellosis in breeding equines in Punjab, Pakistan. A cross-sectional study was designed to evaluate the occurrence and etiology of the infection in the equine population in three districts. A total of 448 equine sera were collected from three prefectures viz. Sahiwal, Khanewal, and Okara of the Punjab Province of Pakistan. Ninety-six (21.4%) samples were found positive by RBPT, 3.56% (16/448) by iELISA, and 4.24% (19/448) by CFT. Real-time PCR demonstrated the presence of Brucella abortus-DNA in sero-positive samples. Age and location were found as risk factors. The study concludes equine brucellosis seroprevalence in the country where Brucella abortus as the main etiology. Fistulous withers and poll evil cases should be treated with care as they could be hazardous and a source of zoonotic transmission. Routine screening at an early age, vaccination in ruminants, and consumption of pasteurized dairy milk in humans is recommended for prevention of the infection. Specific tests need to be standardized and validated.

Serologic Evidence of Severe Fever with Thrombocytopenia Syndrome Virus and Related Viruses in Pakistan.

We describe the seroprevalence of severe fever with thrombocytopenia syndrome virus (SFTSV) and the association of antibody occurrence with location, sex, and age among the human population in Pakistan. Our results indicate substantial activity of SFTSV and SFTSV-related viruses in this country.

Genetic survey of cystic echinococcosis in farm animals in Oman.

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis (CE) in animals and humans with a worldwide distribution affecting pastoral and agro-pastoral communities. Little is known about the genetic diversity and public health significance of E. granulosus s.l. among animals and human in Oman. This study was undertaken to investigate the circulating genotypes of E. granulosus s.l. in farm animals (camels, cattle, goats and sheep) by using multiplex PCR (mPCR) and sequence analysis of a fragment of the NADH dehydrogenase subunit 1 (NADH-1) gene. In this study, 39 hydatid cyst isolates from dromedary camels (n = 17), cattle (n = 12), goats (n = 9) and sheep (n = 1) from five governorates in Oman were collected. These isolates were analysed genetically to classify E. granulosus s.l. using a single-tube mPCR and further subjected to sequence analysis of mitochondrial NADH-1 gene. The results of mPCR revealed that most of the cyst isolates (71.8%) belonged to E. granulosus sensu stricto G1/G2/G3 genotypes, whereas 28.2% were linked to E. canadensis G6/G7 genotypes. However, sequencing of these isolates has confirmed the existence of the two genotypes E. canadensis G6 and E. granulosus sensu stricto G1 genotype. This study provides a molecular evidence of E. granulosus s.l. genotypes in Oman and confirms the predominance of the sheep and camel strains and their role in the transmission dynamics of CE in animals. The study will serve as a foundation for future planning and implementation for CE control program in Oman.

Susceptibility survey of Ommatissus lybicus (de Bergevin) populations against deltamethrin and fenitrothion in Oman.

Despite of extensive application of insecticides, severe infestation of date palms is reported with Ommatissus lybicus every year. The development of insecticide resistance in the field strains receiving heavy pesticide regimes can be a reason for unsuccessful control. Seventeen field strains of O. lybicus from Oman were appraised for resistance against deltamethrin and fenitrothion in years 2017 and 2018. Eleven field strains depicted susceptibility (RR < 3-fold) while six strains expressed minor (RR = 3-5-fold) to low level (RR = 5-10-fold) of resistance against deltamethrin when compared with lab-susceptible strain. In case of fenitrothion, fourteen field strains exhibited minor to low level of resistance and only two showed susceptibility. Intermediate resistance (RR = 10-40-fold) was also reported in one field strain against fenitrothion. A significant but low resistance (RR = 7.86-fold) was observed in a lab strain selected with deltamethrin for resistance development mechanism studies. The deltamethrin-selected strain exhibited minor resistance development (RR = 3.13-fold) against fenitrothion. Significant reduction in resistance was observed with higher toxicity values when the two pesticides were tested along with piperonyl butoxide (PBO) against all the field collected dubas bug strains. Higher susceptibility of field strains to deltamethrin suggest it a suitable alternate candidate to deter resistance development in O. lybicus.

A cross-sectional seroepidemiological study of camel (Camelus dromedarius) brucellosis and associated risk factors in the Sultanate of Oman.

Brucellosis is a globally distributed and economically devastating zoonotic disease of multiple species, including camels. Human and livestock brucellosis is prevalent in Oman, especially in southern Dhofar governorates of Oman, where camels share habitat and have close contact with other susceptible species. We conducted a randomized cross-sectional sero-epidemiological study to investigate the seroprevalence of brucellosis in camels of Oman. The sera from 2,250 camels from 552 geographically marked farms were screened through Rose Bengal plate agglutination test and later confirmed by the competitive ELISA (COMPLIZA, VLA, UK). In total, nine [0.4%, confidence interval (CI) 0.4, 0.8] camels from eight (1.5%, CI 0.6, 2.8) herds were tested positive for brucellosis. The highest prevalence was recorded in Dhofar (3.7%, CI 1.4, 7.9) and the lowest in Sharqiyah (1.3%, CI 0.0, 7.2) governorate (p = 0.052). All seropositive camels were of local breed and females. Seroprevalence was higher (0.5%, CI 0.2, 1.0) in adults (>4 yr of age) as compared with young (≤4 yr of age) camels (0.2%, CI 0.0, 0.8). The results of binary logistic regression indicated that camel herds located in south (Dhofar) [odds ratio (OR) 6.39, CI 1.01, 40.67], practice of open replacement system (OR 14.49, CI 1.83, 114.51) and with history of abortions (OR 8.01, CI 1.34, 47.77) were more likely to test positive for brucellosis. We conclude that brucellosis is endemic at a low level in camels of Oman and a control strategy based upon test-and-slaughter/inclusion of camels in the current vaccination program after carefully planned vaccine evaluation studies could be considered to control it.

Seroepidemiology of foot and mouth disease (FMD) virus non-structural protein (NSP) antibodies in the livestock of Oman.

Foot and mouth disease (FMD) is a highly contagious acute viral disease that causes severe economic losses to the affected countries. To estimate the serological prevalence of the local livestock to the natural infection with FMD virus, a cross-sectional study was conducted from 2015 to 2017 in Oman. Sera from 5807 randomly selected animals (1792 cattle, 2119 goats and 1896 sheep) belonging to 884 herds were tested for the presence of antibodies against 3ABC non-structural protein (NSP) of the FMD virus by a Competition ELISA. Prevalence along with confidence intervals (CI) and odds ratio (OR) was calculated, and the data were further analysed through univariable and multivariable techniques. The herd-level seroprevalence of NSP (41.4%) varied significantly among 11 governorates of Oman. At herd-level seroprevalence varied significantly among cattle (55.2%), goats (38.2%) and sheep (37.7%). At animal-level, the significantly higher prevalence was recorded in cattle (26.8%) followed by sheep (17.9%) and goats (17.3%). Binary logistic regression analysis at the individual level indicated that cattle (OR: 1.88), livestock above one year of age (OR: 3.47), imported breeds (OR: 1.82) and females (OR: 1.27) were the risk factors associated with higher prevalence. Whereas, sedentary (OR: 2.63) farming system, vaccination against FMD (OR: 5.98) and previous history of FMD (OR: 5.55) were the variables found associated with higher seroprevalence at the herd-level. We conclude that natural infection of FMDV is widespread in Oman and in future molecular typing of FMD virus outbreaks strains and vaccine matching studies should be employed to develop an effective control program in Oman.

Concurrent vaccination of goats with foot and mouth disease (FMD) and peste des petits ruminants (PPR) booster vaccines.

Foot and mouth disease (FMD) remains subclinical and self-limiting in small ruminants, but risk of spread of infection to susceptible cohorts is of great epidemiological significance; therefore, small ruminants must be included in vaccination campaigns in FMD endemic regions. Three groups of goats already immunized against peste des petits ruminants (PPR) were vaccinated with FMD and PPR vaccines alone or concurrently. The specific antibody response against three FMD virus strains and PPR virus were evaluated by competitive enzyme-linked immunosorbent assay (cELISA). Goats concurrently vaccinated with PPR + FMD vaccines had significantly (p < 0.05) higher antibody titers to two serotypes of FMD virus at 28, 45, and 60 days post-immunization compared to goats vaccinated with FMD vaccine alone, while goats vaccinated with PPR vaccines alone or PPR + FMD vaccines concurrently showed similar antibody kinetics against PPR virus up till 60 days post-vaccination. Overall, antibody kinetic curves for all three tested strains of FMD virus and PPR virus were similar in vaccinated groups during the course of experiment.

Serologic Evidence for MERS-CoV Infection in Dromedary Camels, Punjab, Pakistan, 2012-2015.

Dromedary camels from Africa and Arabia are an established source for zoonotic Middle East respiratory syndrome coronavirus (MERS-CoV) infection among humans. In Pakistan, we found specific neutralizing antibodies in samples from 39.5% of 565 dromedaries, documenting significant expansion of the enzootic range of MERS-CoV to Asia.

Sero-epidemiology of cystic echinococcosis (Echinococcus granulosus) in the livestock of Oman.

A cross-sectional serological survey of cystic echinocossis was carried out on 2802 randomly collected sera of camels (n=706), cattle (n=687), goats (n=701) and sheep (n=708) from all governorates of Oman. The samples were analysed by in house indirect ELISA (iELISA) using Echinococcus granulosus antigen B (EgAgB) of naturally infected camels. The overall percentage of antibodies against EgAgB was found to be 14.6%. The highest percentage of positive was observed in sera from camels (22.4%) followed by cattle (12.9%), sheep (12.2%) and goats (10.9%). The highest percentage of seropositivity was observed in females (15.4%) as compared to male animals (10.6%). The imported livestock were found more seropositive (15.2%) as compared to local (14.7%) and crossbred livestock (14.1%). The highest exposure was observed in animals with the age group of above 5years (18.3%) followed by the age group of up to 2years (15.1%,) and those between 2 and 5years (12.4%). The univariate analysis has indicated that camels (OR:2.33, CI 1.74, 3.14), cattle (OR:1.21, CI 0.87, 1.67), sheep (OR:1.12, CI 0.81, 1.55) were more likely to test positive than goats. Furthermore, females (OR: 1.53, CI 1.11, 2.11) were more likely to test seropositive. Sera from animals above 5years of age (OR:1.58, CI 1.25, 2.01) and between 2 and 5years old (OR:1.30, CI 0.98, 1.71) were found more likely to test seropositive than those up to 2years of age. The multivariable analysis at individual level indicated that camels (OR: 2.07, CI 1.66, 2.56, p<0.001) and female (OR: 1.43, CI 1.04, 1.98, p=0.030) were more likely to acquire CE. At herd level, the final multivariable model indicated that herds located in Dofar and Musandam (OR: 4.48, CI 2.69, 7.45, p<0.001), in areas receiving seasonal rains (OR: 2.54, CI 1.09, 5.90, p<0.001) and practicing transhumance (OR: 4.22, CI 1.84, 9.65, p<0.001) and sedentary (OR: 2.07, CI 1.38, 3.12, p=0.001) farming system were more likely to acquire CE in Oman. The study documents the serological evidence of CE in livestock of Oman and a carefully planned control program should be devised after further epidemiological and molecular investigations in the intermediate and final hosts.

Seroprevalence and Risk Factors for Theileria equi Infection in Equines from Khyber Pakhtunkhwa Province, Pakistan.

BACKGROUND: Theileria equi is a tick borne protozoan parasite which causes piroplasmosis among equines worldwide. The present study was aimed to determine seroprevalence of T. equi in donkeys, horses, and mules from two equine populated districts (Peshawar and Charsadda) of Khyber Pakhtunkhwa (KPK), Pakistan. METHODS: A total of 393 equine (195 horses, 194 donkeys and 4 mules) serum samples were collected from five and four randomly selected localities in Charsadda (n = 193) and Peshawar (n = 200), respectively. The presence of antibodies to T. equi was determined using a commercially available competitive enzyme-linked immunosorbent assay. RESULTS: An overall seroprevalence of 38.2% (n=150) was observed among all the tested animals suggesting a higher seropositivity among equids belonging to Charsada (50.3%) as compared to Peshawar (27.5%). Binary logistic regression analysis revealed that being a donkey (OR 2.94), having tick infestation (OR 4.32), history of voiding red (i.e., blood containing) urine (OR 3.97) and anemia (OR 2.1) were the factors significantly associated with the seroprevalence of T. equi. For animals with higher anti-T. equi antibody titers, a strong association of seroprevalence for T. equi was recorded with species, age, sex, tick infestation, anemia and history of hematuria. CONCLUSION: The present study indicates a high level of exposure of working equids to T. equi in KPK region, Pakistan. Future studies should focus on tick vector identification and other factors responsible for spread of the disease.