Molecular Epidemiology and Phylogeny of Theileria ovis and Theileria lestoquardi in Sheep and Goats from Southern Punjab, Pakistan.

Background: Theileria spp. are responsible for ovine and caprine theileriosis, leading to significant morbidity and mortality in small ruminants. The present study aims to investigate Theileria spp. infections in small ruminants from Southern Punjab in Pakistan, and genetic characterize revealed Theileria spp. isolates. Methods: A total of 93 sheep and 107 goats were sampled between May and August 2022. Blood smears were examined microscopically, and PCR amplification targeting the 18S rRNA gene was performed to detect Theileria spp. Additionally, specific PCR assays targeting 18S rRNA and ms1 partial sequences were used to identify Theileria ovis and T. lestoquardi, respectively.  Results: The prevalence of Theileria spp. was significantly higher using PCR (13.5%) compared to microscopic screening (5%). Sheep showed a higher prevalence rate (19.4%) compared to goats (8.4%) (p = 0.024). Young sheep aged ≤ 1 year were more commonly infected with Theileria spp. (41%) compared to older sheep (p = 0.006). The prevalence of Theileria spp. was higher in sheep-only herds (37.3%) compared to goat-only herds (18%) or mixed-species herds (8.1%) (p = 0.015). The prevalence rates of T. ovis and T. lestoquardi were 9% and 2.5%, respectively, with four animals (2 goats and 2 sheep) showing co-infection. Phylogenetic analysis revealed that our T. ovis 18S rRNA sequence clustered with previously reported sequences from sheep in Turkey, China, Spain, and goats in Tanzania. The obtained T. lestoquardi ms1 partial sequence formed a distinct cluster from other T. lestoquardi isolates in Pakistan and neighboring countries.  Conclusion: Theileria spp. co-circulation in Pakistani small ruminants, particularly the presence of T. ovis and T. lestoquardi, highlights the need for attention from animal health decision-makers due to their financial and health impacts.

Computational investigation of turmeric phytochemicals targeting PTR1 enzyme of Leishmania species.

In this study, we used in silico techniques to identify available parasite treatments, representing a promising therapeutic avenue. Building upon our computational initiatives aimed at discovering natural inhibitors for various target enzymes from parasites causing neglected tropical diseases (NTDs), we present novel findings on three turmeric-derived phytochemicals as inhibitors of Leishmania pteridine reductase I (PTR1) through in silico methodologies. PTR1, a crucial enzyme in the unique folate metabolism of trypanosomatid parasites, holds established therapeutic significance. Employing MOE software, a molecular docking analysis assesses the efficacy of turmeric phytochemicals against Leishmania PTR1. Validation of the docking protocol is confirmed with an RMSD value of 2. Post-docking, compounds displaying notable interactions with critical residues and binding affinities ranging between -6 and -8 kcal/mol are selected for interaction pattern exploration. Testing twelve turmeric phytochemicals, including curcumin, zingiberene, curcumol, curcumenol, eugenol, bisdemethoxycurcumin, tetrahydrocurcumin, tryethylcurcumin, turmerones, turmerin, demethoxycurcumin, and turmeronols, revealed binding affinities ranging from -5.5 to -8 kcal/mol. Notably, curcumin, demethoxycurcumin, and bisdemethoxycurcumin exhibit binding affinities within -6.5 to -8 kcal/mol and establish substantial interactions with catalytic residues. These phytochemicals hold promise as lead structures for rational drug design targeting Leishmania spp. PTR in future applications. This work underscores the potential of these identified phytochemicals in the development of more effective inhibitors, demonstrating their relevance in addressing neglected tropical diseases caused by parasites.

Current perspectives and difficulties in the design of acaricides and repellents from plant-derived compounds for tick control.

Ticks and tick-borne diseases have gained increasing attention in recent years due to their impact on public health and significant losses in livestock production. The use of synthetic compounds for tick control is becoming problematic, mainly due to the resistance to commercially available products as well as their toxicity. Therefore, new alternative control methods are required. For this purpose, plant-derived extracts may be considered as effective repellents and/or acaricides. The present literature review focuses on studies evaluating the acaricidal and repellent activity of plant-derived extracts and plant secondary metabolites. We also noted recent advances in protein-ligand-docking simulation to examine the possible toxic effect of natural chemical compounds on ticks. In conclusion, plant-derived repellents/acaricides can be effective against ticks, especially in rural areas and livestock farms.

First report of Anaplasma spp., Ehrlichia spp., and Rickettsia spp. in Amblyomma gervaisi ticks infesting monitor lizards (Varanus begalensis) of Pakistan.

Ticks pose significant health risks to both wildlife and humans due to their role as vectors for various pathogens. In this study, we investigated tick infestation patterns, tick-associated pathogens, and genetic relationships within the tick species Amblyomma gervaisi, focusing on its prevalence in monitor lizards (Varanus bengalensis) across different districts in Pakistan. We examined 85 monitor lizards and identified an overall mean intensity of 19.59 ticks per infested lizard and an overall mean abundance of 11.98 ticks per examined lizard. All collected ticks (n = 1019) were morphologically identified as A. gervaisi, including 387 males, 258 females, 353 nymphs, and 21 larvae. The highest tick prevalence was observed in the Buner district, followed by Torghar and Shangla, with the lowest prevalence in Chitral. Lizard captures primarily occurred from May to October, correlating with the period of higher tick infestations. Molecular analysis was conducted on tick DNA, revealing genetic similarities among A. gervaisi ticks based on 16S rDNA and ITS2 sequences. Notably, we found the absence of A. gervaisi ITS2 sequences in the NCBI GenBank, highlighting a gap in existing genetic data. Moreover, our study identified the presence of pathogenic microorganisms, including Ehrlichia sp., Candidatus Ehrlichia dumleri, Anaplasma sp., Francisella sp., Rickettsia sp., and Coxiella sp., in these ticks. BLAST analysis revealed significant similarities between these pathogenic sequences and known strains, emphasizing the potential role of these ticks as vectors for zoonotic diseases. Phylogenetic analyses based on nuclear ITS2 and mitochondrial 16S rDNA genes illustrated the genetic relationships of A. gervaisi ticks from Pakistan with other Amblyomma species, providing insights into their evolutionary history. These findings contribute to our understanding of tick infestation patterns, and tick-borne pathogens in monitor lizards, which has implications for wildlife health, zoonotic disease transmission, and future conservation efforts. Further research in this area is crucial for a comprehensive assessment of the risks associated with tick-borne diseases in both wildlife and humans.

Toxoplasma gondii infection in small ruminants from Khyber Pakhtunkhwa, Pakistan: Seroprevalence, spatial distribution and associated risk factors.

The primary aim of this investigation was to assess the occurrence of Toxoplasma gondii infection among sheep and goats in five districts within the southern region of Khyber Pakhtunkhwa province, Pakistan. Additionally, the study aimed to pinpoint potential factors associated with the spread of this infection. Serum samples were gathered from a total of 3505 small ruminants, comprising 1810 sheep and 1695 goats. These samples were then subjected to testing for anti-Toxoplasma antibodies using the Toxo-Latex slide Agglutination test. Concurrently, a predetermined questionnaire was employed to gather data on possible risk factors. The collected data underwent bivariate frequency analysis and multivariable logistic regression analyses. The findings revealed that 20.08% of the examined animals had positive results for anti-Toxoplasma antibodies. Notably, the prevalence of T. gondii was notably higher among sheep, with 21.8% of the sheep testing positive, as opposed to the 18% of tested goats (P = 0.007). The seroprevalence percentages were comparable across the five districts for both types of animals. For goats, the prevalence ranged from 16.8% to 20.1%, while for sheep, it ranged from 20.2% to 24%. Among the younger population (≤1 year), the seroprevalence was statistically lower at 17.7% compared to the older population (>1 year) at 23.1% (P < 0.001). Female animals demonstrated a higher prevalence (21.9%) in contrast to their male counterparts (17.8%) (P = 0.002). Animals reared for breeding and milking purposes displayed a greater seroprevalence (23.9%) compared to those intended for sale (17.7%) (P < 0.001). Furthermore, animals kept under a transhumant production system displayed the lowest prevalence (18.9%), followed by sedentary (21.7%) and nomadic (31.2%) herds (P < 0.001). The outcomes of this study highlight the importance of implementing management interventions to curtail T. gondii infection among animals within the Khyber Pakhtunkhwa province of Pakistan. Such measures hold the potential to reduce associated public health risks and enhance the profitability of farmers.

Epidemiological survey, molecular profiling and phylogenetic analysis of cutaneous leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Cutaneous leishmaniasis (CL), an emerging vector-borne ailment in Khyber Pakhtunkhwa (KPK), Pakistan, exhibits diverse spread patterns and outbreaks. METHODS: To comprehend its epidemiology and identify parasite species, we conducted an active survey on suspected CL cases (n=8845) in KPK. RESULTS: Microscopy and internal transcribed spacer-1 PCR-restriction fragment length polymorphism (RFLP) molecular techniques detected Leishmania spp. in blood samples. Phylogenetic analysis gauged genetic affinities with other areas. District Bannu displayed the highest CL impact (14.58%), while Swat had the lowest impact (4.33%) among cases. Annual blood examination rate, parasite incidence and slide positivity rate were 4.96 per 1000 people, 0.0233 and 0.047%, respectively. CL infections were prevalent in 1- to 20-y-olds, with males (57.17%) more vulnerable than females (42.82%). Single lesions occurred in 43.73% of patients, while 31.2% people had two lesions, 17.31% had three lesions and 7.74% had more than three lesions. Most had sand-fly exposure but lacked preventive measures like repellents and bed nets. Leishmania tropica was confirmed via RFLP analysis in amplified samples. Phylogenetic analysis unveiled genetic parallels between L. tropica of KPK and isolates from China, Iran, Afghanistan, India, Syria and Morocco. CONCLUSIONS: Urgent comprehensive control measures are imperative. Early detection, targeted interventions and raising awareness of CL and sand-fly vectors are vital for reducing the disease's impact. International collaboration and monitoring are crucial to tackle Leishmania spp.'s genetic diversity and curtail its cross-border spread.

Molecular detection of Babesia ovis and blood parameters' investigation reveal hematological and biochemical alterations in babesiosis-infected Lohi sheep in Multan, Pakistan.

Background: Babesia infections in sheep can cause a wide range of clinical and laboratory presentations. Changes in blood parameters are a meaningful manifestation of physiological and pathological changes in an organism. Aim: Therefore, the present study was conducted to analyze and compare hematological and biochemical parameters between blood profiles of Lohi sheep naturally infected and uninfected with Babesia ovis, the main causative agent of ovine babesiosis. Methods: Initially, blood and serum samples from 67 Lohi sheep were collected, DNA was extracted and babesial infection was detected through polymerase chain reaction. The overall infection rate of B. ovis was 37% (25/67). Sixteen infected (experiment group) and 16 uninfected (control group) sheep that were apparently healthy with no history of previous treatment for babesiosis, were selected for hemato-biochemical analysis. Blood samples were analyzed through an automatic CBC analyzer, while serum collected from gel vacutainers was analyzed for blood urea, blood urea nitrogen (BUN), creatinine, and total bilirubin. Each parameter was compared between infected and uninfected animals using a paired t-test in Minitab Express™ software for statistical analyses. Results: Erythron comparison showed a highly significant ( p < 0.0001) decrease in RBC, hemoglobin, and Hct. A nonsignificant increase in mean corpuscular volume (MCV), red cell distribution width (RDW), and RDW-standard deviation (RDW-SD), while a nonsignificant decrease in mean corpuscular haemoglobin (MCH) and MCH concentration (MCHC) values was recorded in infected sheep. Leukon comparison showed a significantly low level of total leukocyte (p < 0.001) in infected sheep. Platelet (Plt) along with platelet crit (Pct) and platelet distribution width (PDW) were nonsignificantly higher, whereas a nonsignificant decrease in mean Plt volume was recorded in infected sheep as compared to uninfected animals. Among biochemical parameters, blood urea, BUN, and total bilirubin showed significant differences (p < 0.05), while creatinine showed a nonsignificant difference. Conclusion: To the best of our knowledge, this is the first report on hemato-biochemical changes associated with babesiosis in the Lohi breed. Consistent with hemolytic anemia, these data would justify physical examination and, together with the medical history, would provide an excellent basis for the diagnosis of babesiosis.

Molecular prevalence and phylogeny of Anaplasma marginale, Anaplasma ovis and Theileria ovis in goats and sheep enrolled from a hill station in Punjab, Pakistan.

Anaplasma marginale (A. marginale), Anaplasma ovis (A. ovis) and Theileria ovis (T. ovis) are among the most commonly reported intracellular tick borne pathogens that infect ruminants across the globe causing huge economic losses. This study aims to report the prevalence and phylogenetic evaluation of these three pathogens infecting sheep and goats (n = 333) that were enrolled from Fort Munro region in Pakistan by using msp1b, msp4 and 18S rRNA genes for A. marginale, A. ovis and T. ovis respectively. Results revealed almost similar infection rates in sheep and goats with an overall prevalence of 11% for A. marginale, 28% for A. ovis and 3% for T. ovis. Concurrent infection was also recorded, however, the number of animals infected with two pathogens (n = 24; 7.2%) was higher than infection with three pathogens (n = 2; 0.6%). Risk factor analysis revealed that sheep reared in small herds had higher A. marginale (P = 0.03) and A. ovis (P = 0.04) infection rates compared to those from large herds. In addition, it was observed that bucks (P ≤ 0.05) and tick-free goats (P ≤ 0.05) exhibited higher A. ovis infection rates than nannies. Phylogenetic analysis of all three pathogens showed that Pakistani isolates were clustered together and were closely related to previously deposited Pakistani isolates as well as with those that were reported from worldwide countries. In conclusion, we are reporting that Pakistani sheep and goats have A. marginale, A. ovis and T. ovis mediated infections and control measures should be taken against them to improve the productivity of the livestock sector.

Homozygous Mutations in GDAP1 and MFN2 Genes Resulted in Autosomal Recessive Forms of Charcot-Marie-Tooth Disease in Consanguineous Pakistani Families.

Charcot-Marie-Tooth disease (CMT) is a heritable neurodegenerative disease of peripheral nervous system diseases in which more than 100 genes and their mutations are associated. Two consanguineous families Dera Ghazi Khan (PAK-CMT1-DG KHAN) and Layyah (PAK-CMT2-LAYYAH) with multiple CMT-affected subjects were enrolled from Punjab province in Pakistan. Basic epidemiological data were collected for the subjects. Nerve conduction study (NCS) and electromyography (EMG) were performed for the patients. Whole-exome sequencing (WES) followed by Sanger sequencing was applied to report the genetic basic of CMT. The NCS findings revealed that sensory and motor nerve conduction velocities for both families were <38 m/s. EMG presented denervation, neuropathic motor unit potential, and reduced interference pattern of peripheral nerves. WES identified that a novel nonsense mutation (c. 226 G>T) in GADP1 gene and a previously known missense mutation in MFN2 gene (c. 334 G>A) cause CMT4A (Charcot-Marie-Tooth disease type 4A) in the PAK-CMT1-DG KHAN family and CMT2A (Charcot-Marie-Tooth disease type 2A) in the PAK-CMT2-LAYYAH family, respectively. Mutations followed Mendelian pattern with autosomal recessive mode of inheritance. Multiple sequence alignment by Clustal Omega indicated that mutation-containing domain in both genes is highly conserved, and in situ analysis revealed that both mutations are likely to be pathogenic. We reported that a novel nonsense mutation and a previously known missense mutation in GAPD1 gene and MFN2 gene, respectively, cause CMT in consanguineous Pakistani families.

Integrating In Vitro and In Silico Approaches to Assess Monotheca buxifolia Plant Extract against Rhipicephalus (Boophilus) microplus and Sarcoptes scabiei.

Tick and mite infestations pose significant challenges to animal health, agriculture, and public health worldwide. The search for effective and environmentally friendly acaricidal agents has led researchers to explore natural alternatives. In this study, we investigated the acaricidal potential of the Monotheca buxifolia plant extract against Rhipicephalus microplus ticks and Sarcoptes scabiei mites. Additionally, we employed a computational approach to identify phytochemicals from the extract that could serve as drug candidates against these ectoparasites. The contact bioassay results demonstrated that the M. buxifolia plant extract exhibited significant efficacy against R. microplus and S. scabiei, with higher concentrations outperforming the positive control acaricide permethrin in terms of mite mortality. Time exposure to the extract also showed a positive correlation with better lethal concentration (LC50 and LC90) values. Similarly, the adult immersion test revealed a notable inhibition of tick oviposition via the plant extract, especially at higher concentrations. The two-protein primary structure, secondary structure and stability were predicted using the Expasy's ProtParam server, SOPMA and SUSUI server, respectively. Using Homology modeling, the 3D structure of the protein was obtained and validated through the ERRAT server, and active sites were determined through the CASTp server. The docking analysis revealed that Alpha-Amyrenyl acetate and alpha-Tocopherol exhibited the highest docking scores for S. scabiei and R. microplus aspartic protease proteins, respectively. These phytochemicals demonstrated strong binding interactions, suggesting their potential as acaricidal drug candidates. In conclusion, the M. buxifolia plant extract displayed significant acaricidal activity against R. microplus and S. scabiei. Moreover, the computational approach identified promising phytochemicals that could serve as potential drug candidates for controlling these ectoparasites.

In Silico Exploration and Experimental Validation of Camellia sinensis Extract against Rhipicephalus microplus and Sarcoptes scabiei: An Integrated Approach.

Sarcoptes scabiei is an ectoparasite of humans and animals that causes scabies. The Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite that transmits various pathogens. These two parasites have caused great losses to a country's dairy and agriculture sectors. The aim of this study was to determine the in vitro and in silico efficacy of Camellia sinensis plant extracts. Different concentrations of C. sinensis ethanolic plant extracts were prepared using the maceration method and were used against mites and ticks (in adult immersion test AIT and larval packet test LPT) to evaluate their in vitro acaricidal activity. Additionally, in silico molecular docking was performed to investigate the inhibitory interactions between the phytochemicals of the plant and S. scabiei and R. microplus glutathione transferase proteins (SsGST and RmGST). This study observed that the plant extract showed high efficacy in vitro against mites and different tick stages in adult immersion and larval packet tests. Additionally, the in silico study revealed a strong binding interaction between ellagic acid and SsGST protein, with a binding energy of -7.3 kcal/mol, with respect to permethrin (-6.7 kcal/mol), whereas quercetin and RmGST resulted in a docking score of -8.6 kcal/mol compared to deltamethrin (-8.2 kcal/mol). Overall, this study explored the potential of C. sinensis as a natural alternative for controlling tick and mite infestations and provided insights into the inhibitory mechanisms of its phytochemicals.

Acaricidal assessment of the fungal extract of Pleurotus ostreatus against Rhipicephalus microplus: Role of in vitro and in silico analysis.

The Pleurotus ostreatus mushroom, commonly referred to as the oyster mushroom, is a widely consumed edible mushroom that grows in clusters on dead or dying trees. In addition to its culinary uses, research has found that the mushroom may also have potential medicinal properties. The current study investigated the potential use of a fungal extract from P. ostreatus as a natural acaricide against Rhipicephalus microplus, a major vector of economically significant infections and one of the most significant bovine ectoparasites. The study used the adult immersion test (AIT) and the larvae packet test (LPT) to evaluate the effectiveness of the extract against ticks. To evaluate the reproductive effect of the fungal extract on the reproduction of R. microplus engorged females, the eggs were tested for weight and egg-laying index. The results of the study showed that P. ostreatus extract had a significant acaricidal effect, with a 40 mg/mL concentration causing 89 ± 2.64% mortality in R. microplus larvae compared to the 91.00 ± 3.60% mortality by the positive control at 48 h interval. Whereas in the AIT, the extract inhibited 39.86 ± 4.13% of oviposition. The study also used in silico approach to further examine the binding mechanisms of the compounds in the fungal extract to the target protein R. microplus Triosephosphate isomerase RmTIM, using molecular docking in AutoDock Vina software. Docking scores of -8.3, -7.7 and -6.9 kcal/mol, respectively, showed that rutin, naringin and myricitin had significant interactions with the active site residues of the target protein. Our results suggest that P. ostreatus extract may be a potential alternative to traditional acaricides for controlling R. microplus in livestock.

Epidemiology and Phylogeny of Anaplasma ovis with a Note on Hematological and Biochemical Changes in Asymptomatic Goats Enrolled from Four Districts in Punjab, Pakistan.

Background: Anaplasma ovis is an intra-erythrocytic gram negative rickettsial bacterium that infects small ruminants, resulting in huge economic losses worldwide. Materials and Methods: The present investigation aims at reporting the molecular prevalence of A. ovis in 1200 asymptomatic goats that were enrolled from 4 districts (Layyah, Lohdran, Dera Ghazi Khan, and Rajanpur) in Punjab, Pakistan by targeting the msp4 gene of bacterium. Risk factors associated with the prevalence of A. ovis and phylogeny of bacterium were also documented. Results: 184 out of 1200 (15%) goat blood samples were infected with A. ovis. The prevalence of the pathogen varied with the sampling sites (p = 0.005), and the highest prevalence was detected in goats from Layyah (19%) followed by Rajanpur (17%), Dera Ghazi Khan (15%), and Lohdran district (9%). The represented partial msp4 gene amplicon was confirmed by Sanger sequencing and deposited to GenBank (OP225957-59). Phylogenetic analysis revealed that the amplified isolates resembled the msp4 sequences reported from Iran, Mangolia, Sudan, and the United States. Sex and age of goats, herd composition and size, and the presence of ticks on goats and dogs associated with herds were the rick factors associated with the prevalence of A. ovis. Red blood cells, lymphocytes (%), neutrophils (%), hemoglobin, and hematocrit levels in blood and Aspartate amino transferase, urea, and creatinine levels in serum were disturbed in A. ovis infected goats when compared with uninfected animals. Conclusion: We are reporting the prevalence of A. ovis in Pakistani goats from four districts of Punjab and these data will help in developing the integrated control policies against this tick-borne pathogen that is infecting our goat breeds.

Differential diagnosis of theileriosis through blood smear examination and polymerase chain reaction in small ruminants from Pakistan.

Background: Ovine and caprine theileriosis is a tick-borne hemoprotozoan disease, caused by Theileria spp., responsible for heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of ovine theileriosis is primarily based on clinical symptoms, microscopic screening of stained blood smears, and lymph node biopsy smears, but the limitations of these detection methods against Theileria spp. infection limits their specificity. Aim: To overcome these limitations, the current study reports the differential diagnosis of theileriosis through a blood smear examination and polymerase chain reaction (PCR) in small ruminants from Pakistan. Methods: The study was conducted on 1,200 apparently healthy small ruminants (737 sheep and 463 goats). First, blood smears were screened for the presence of Theileria piroplasms in red blood cells. Second, PCR amplification based on 18S rRNA gene was performed by using primers specific to Theileria spp. Results: Out of the 1,200 samples of examined blood smears, 100 animals (8.33%) were found positive for Theileria species, which showed intra-erythrocytic bodies in the form of dot and comma shapes. Amplification of the isolated DNA from randomly collected blood samples of 737 sheep and 463 goats showed that an amplicon size of 1,098 bp was positive for Theileria spp. In total, 315 out of the 1,200 small ruminants examined in this study were found positive for Theileria spp. DNA through PCR amplification. Notably, out of the 885 blood samples negative by PCR amplification, only 15 blood samples were found positive by the blood smear test. Conversely, 230 blood samples that tested negative in the smear technique produced a specific band through PCR amplification. Overall, the sensitivity and specificity rates were 26.98% and 98.31% for the blood smear method and 73.01% and 100% for the PCR assay, respectively. Conclusion: Our finding suggests that PCR is the gold standard method compared to the conventional method of smear examination for the diagnosis of ovine and caprine theileriosis in Pakistan.

Seasonality, epidemiology and phylogeny of Theileria ovis with a note on hematological and biochemical changes in asymptomatic infected goats from Pakistan.

Caprine theileriosis, caused by Theileria ovis is a serious production issue, especially in the areas that depend on goats and sheep for milk, meat, and other economic benefits. Pakistan has a large goat population, but few reports have been documented from this country regarding PCR-based detection of T. ovis. The molecular prevalence of T. ovis, on a seasonal basis, in various goat breeds enrolled from Muzaffar Garh district of Punjab in Pakistan was determined from October 2018 to September 2019. In this study, 1084 goat blood samples were screened for the detection of T. ovis DNA through PCR-based amplification of 18S rRNA gene. Out of 1084 goats, 12 (1.11%) were infected with T. ovis. The parasite prevalence varied with the sampling seasons (Chi square test, P = 0.008), and the parasite prevalence was highest in goat blood samples collected in summer (2.39%) followed by winter (1.88%). DNA sequencing and BLAST analysis confirmed the presence of T. ovis, and the amplified isolates from the 18S rRNA gene of T. ovis were found to be highly conserved during phylogenetic analysis. Young goats (Fischer exact test, P = 0.022) were found more infected with T. ovis during the winter season. Infected goats had elevated white blood cell counts (Two-sample t-test, P = 0.04), blood urea nitrogen to Creatinine ratio (Two-sample t-test, P = 0.02) and decreased serum Creatinine (Two-sample t-test, P = 0.001) as compared to T. ovis negative goats. We report a relatively low molecular prevalence of T. ovis in goats from the Muzaffar Garh district. However, it is recommended that control measures to eradicate T. ovis infection in goats in this area should be taken.

Diversity, prevalence and risk factors associated to gastrointestinal tract parasites in wild and domestic animals from Pakistan.

The gastrointestinal tract (GIT) of animals contains various helminth parasites, responsible for subclinical and clinical parasitism. The health situation of animals is seriously affected by these parasites and leads massive financial losses for the livestock trade. The present study was conducted to determine the prevalence and associated risk factors of GIT parasites in small and large ruminants of Mardan district and zoo animals of Golden Life Safari Park Khyber Pakhtunkhwa. A total of 419 fecal samples were collected including 117 from goats, 110 from cattle, 80 from sheep, 66 from buffaloes, 40 from birds, four from deer, and two from leopards. The fecal samples were subjected to sedimentation and floatation to identify parasite eggs. Of a total of 419 fecal samples, 215 (51.31%) were found positive for GIT parasites eggs. The prevalence of GIT parasites was high in sheep (65%), followed by goats (62.39%), cattle (42.70%), and buffaloes (37.87%). In birds, GIT parasite eggs were identified in 11 (45.83%) and 7 (43.75%) fecal samples of Pheasant and Peacock, respectively. However, an absence of infection was noted in fecal samples from deer and leopards. Eggs of GIT Parasites belonging to one superfamily and four different genera were recognized in fecal samples of domestic animals and buffaloes, which were Trichostrongyloidea (19.81%), Strongyloides (9.31%), Trichuris (7.77%), Monezia (6.44%), and Fasciola (4.53%). For bird, the coccidia group comprises the highest representation of parasites (2.63%), followed by the Trichuris genus (1.67%). Settlement, management system, anthelminthic medications and body condition score were significantly associated with the occurrence of GIT parasites in feces (P < 0.05). Animals from rural settlements, rearing under poor management systems, with thin (poor) body condition and under warmed were more affected by GIT parasites. The diversity and the high prevalence of GIT parasites reported in this study highlights the urgent need for intervention through treatment and modification of management practices in the investigated areas of Pakistan.

Simultaneous molecular detection of Anaplasma marginale and Theileria annulata in cattle blood samples collected from Pakistan-Afghanistan boarder region.

Theileria annulata (T. annulata) and Anaplasma marginale (A. marginale) are among the most extensively reported tick borne pathogens and are associated with huge economic losses worldwide. A total of 298 cattle blood samples were screened to report the presence of these two pathogens. The samples were collected from apparently healthy cattle (Achai, n = 155, Jersy, n = 88 and crossbred, n = 55) in Bajaur district of Khyber Pakhtunkhwa (KPK) during June and July of 2022. A total of 31 out of 298 cattle (10.4%) were found infected with T. annulata as PCR amplified a 156 base pair fragment from Tams-1 gene of T. annulata from their blood. While 16/298 animals (5.4%) were found infected with A. marginale as they amplified a 382 base pair fragment specific for msp5 gene of this bacterium. Three animals (1%) were found co infected. Cattle susceptibility to T. annulata infection was significantly higher than A. marginale infection (P < 0.001). Phylogenetic analysis revealed that Pakistani isolates of both detected pathogen clustered together and were closely related isolates from worldwide countries. Prevalence of T. annulata varied significantly among the sampling sites (P = 0.05) while no such association was observed for A. marginale among the tested cattle. Epidemiological data analysis revealed that none of the studied risk factors was found associated either with the prevalence of T. annulata or A. marginale (P > 0.05) among enrolled cattle. In conclusion, our study has revealed a relatively higher prevalence of T. annulata than A. marginale in cattle from the Bajaur district in KPK. This information is important for improving the productivity of the livestock sector, which is one of the main sources of income in the country. It is recommended that this data be taken into account for the development and implementation of effective tick control programs, as well as for the improvement of livestock management practices to prevent and manage TBDs in Pakistan.

Individual and Community-Level Risk Factors for Giardiasis in Children under Five Years of Age in Pakistan: A Prospective Multi-Regional Study.

OBJECTIVES: This study aimed to estimate the prevalence of Giardia lamblia infection and identify associated risk factors at both individual and community levels in a pediatric population in different agroecological zones of Khyber Pakhtunkhwa, Pakistan. METHODS: A community-based cross-sectional study was conducted from March to December 2022. Using stratified sampling, 1026 households were recruited from nine agroecological zones. Stool samples were collected from 1026 children up to the age of five years and processed for detection of Giardia using a commercial ELISA kit. Data on potential risk factors were collected using a pre-structured questionnaire. A multivariable logistic regression model was used to identify risk factors associated with giardiasis. RESULTS: The study found that the prevalence of giardiasis in the study area was 3.31%. Children aged 13-24 months were found to be at higher risk for giardiasis. Illiterate mothers and fathers attending daycare institutions/kindergartens, mothers not practicing hand washing during critical times, households with companion animals, and homes where stray dogs/cats enter were identified as predictors of giardiasis at the individual level. Children living in sub-mountain valleys use un-piped water, inadequate domestic water storage vessels, drink un-boiled or unfiltered water, live near rubbish heaps or un-paved streets/pathways, and have unimproved latrine facilities were identified as risk factors of giardiasis at the community level. CONCLUSIONS: The study highlights the need for integrated intervention approaches at both individual and community levels to reduce the incidence of giardiasis in Khyber Pakhtunkhwa, Pakistan. Interventions aimed at promoting behavioral change and providing safe and adequate water sources, combined with individual-level interventions such as hand washing and awareness of giardiasis prevention methods, would be critical to addressing this health concern. Inter-sectoral collaboration between the health sector and other sectors would also be necessary to achieve meaningful progress in reducing the incidence of giardiasis in resource-limited areas.

Density Functional Theory Calculations and Molecular Docking Analyses of Flavonoids for Their Possible Application against the Acetylcholinesterase and Triose-Phosphate Isomerase Proteins of Rhipicephalus microplus.

Ticks and tick-borne diseases constitute a substantial hazard to the livestock industry. The rising costs and lack of availability of synthetic chemical acaricides for farmers with limited resources, tick resistance to current acaricides, and residual issues in meat and milk consumed by humans further aggravate the situation. Developing innovative, eco-friendly tick management techniques, such as natural products and commodities, is vital. Similarly, searching for effective and feasible treatments for tick-borne diseases is essential. Flavonoids are a class of natural chemicals with multiple bioactivities, including the inhibition of enzymes. We selected eighty flavonoids having enzyme inhibitory, insecticide, and pesticide properties. Flavonoids' inhibitory effects on the acetylcholinesterase (AChE1) and triose-phosphate isomerase (TIM) proteins of Rhipicephalus microplus were examined utilizing a molecular docking approach. Our research demonstrated that flavonoids interact with the active areas of proteins. Seven flavonoids (methylenebisphloridzin, thearubigin, fortunellin, quercetagetin-7-O-(6-O-caffeoyl-ß-d-glucopyranoside), quercetagetin-7-O-(6-O-p-coumaroyl-ß-glucopyranoside), rutin, and kaempferol 3-neohesperidoside) were the most potent AChE1 inhibitors, while the other three flavonoids (quercetagetin-7-O-(6-O-caffeoyl-ß-d-glucopyranoside), isorhamnetin, and liquiritin) were the potent inhibitors of TIM. These computationally-driven discoveries are beneficial and can be utilized in assessing drug bioavailability in both in vitro and in vivo settings. This knowledge can create new strategies for managing ticks and tick-borne diseases.

In Vitro and In Silico Protocols for the Assessment of Anti-Tick Compounds from Pinus roxburghii against Rhipicephalus (Boophilus) microplus Ticks.

Pinus roxburghii, also known by the name "Himalayan chir pine," belongs to the Pinaceae family. Rhipicephalus (Boophilus) microplus tick is one of the most significant bovine ectoparasites, making it a major vector of economically important tick-borne diseases. The researchers conducted adult immersion tests (AIT) and larval packet tests (LPT) to investigate the acaricidal effect of P. roxburghii plant extract on R. (B.) microplus and its potential modulatory function when used with cypermethrin. Eggs were also assessed for their weight, egg-laying index (IE), hatchability rate, and control rate. After exposure to essential extract concentrations ranging from 2.5 to 40 mg/mL for 48 h, adult female ticks' oviposition inhibition and unfed R. (B.) microplus larvae's mortality rates were analyzed. Engorged females exposed to P. roxburghii at 40 mg/mL had reduced biological activity (oviposition, IE) compared to positive and negative controls. A concentration of 40 mg/mL of P. roxburghii caused 90% mortality in R. (B.) microplus larvae, whereas cypermethrin (the positive control) caused 98.3% mortality in LPT. In AIT, cypermethrin inhibited 81% of oviposition, compared to the 40 mg/mL concentration of P. roxburghii, which inhibited 40% of the ticks' oviposition. Moreover, this study assessed the binding capacity of selected phytocompounds with the targeted protein. Three servers (SWISS-MODEL, RoseTTAFold, and TrRosetta) recreated the target protein RmGABACl's 3D structure. The modeled 3D structure was validated using the online servers PROCHECK, ERRAT, and Prosa. Molecular docking using Auto Dock VINA predicted the binding mechanisms of 20 drug-like compounds against the target protein. Catechin and myricetin showed significant interactions with active site residues of the target protein, with docking scores of -7.7 kcal/mol and -7.6 kcal/mol, respectively. In conclusion, this study demonstrated the acaricidal activity of P. roxburghii extract, suggesting its potential as an alternative natural acaricide for controlling R. (B.) microplus.