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.

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.

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.

The potential of Allium sativum and Cannabis sativa extracts for anti-tick activities against Rhipicephalus (Boophilus) microplus.

The efficacy of Allium sativum and Cannabis sativa against Rhipicephalus microplus ticks was evaluated using the adult immersion and the larval packet test. In addition, an in silico approach was utilized by performing a docking study in order to identify the active ingredients from both plants. Results showed a comparatively high lethal effect of A. sativum and C. sativa on egg laying (index of egg laying = 0.26 and 0.24, respectively), egg hatching (33.5 and 37.1, respectively), and total larval mortality (100%, both), at 40 mg/mL. When applied to cattle which had been inoculated with larvae ticks, it was observed that a 45% solution of both herbal extracts significantly reduced the number of ticks by 96 h post treatment. We analyzed in silico 27 known active molecules from both plants and identified in the PubChem database to explore the hypothesis that the effect found on ticks was based on inhibition of acetylcholinesterase (AChE). Vitamin E and cannabidiol are the most potent AChE inhibitors with docking scores of -15.85 and -14.38, respectively. Based on these findings, we conclude that A. sativum and C. sativa may potentially be used for the control of R. microplus, and should be further investigated as a potential supplement to or replacement of synthetic acaricides.

Acaricidal efficacy of Calotropis procera (Asclepiadaceae) and Taraxacum officinale (Asteraceae) against Rhipicephalus microplus from Mardan, Pakistan.

Medicinal plants are used by traditional folk healers, modern physicians, and veterinarians as an alternative to conventional drugs to treat a wide range of disorders including parasitic diseases. Some compounds from these plants have been shown to have acaricidal activity and repel arthropods. The cattle tick Rhipicephalus microplus is one of the most destructive pests to the livestock industry in tropical and subtropical parts of the world. The potential to develop herbal acaricides to control R. microplus infestations is critical in maintaining cattle herd productivity, reducing economic losses, and curtailing the overuse of synthetic chemical acaricides. Calotropis procera, the apple of Sodom, and Taraxacum officinale, the common dandelion, were evaluated for acaricidal activity against R. microplus larvae and adults in vitro. Both plant species tested are common indigenous species of Pakistan where R. microplus infestations are widespread across livestock species including cattle, sheep, and goats. Whole-plant extracts derived from C. procera and T. officinale significantly reduced the index of egg laying (P < 0.01) and increased the percent inhibition of oviposition of adult female ticks at a concentration of 40 mg/mL when assessed by the adult immersion test (AIT). Calotropis procera and T. officinale treatments at the same concentration also resulted in larval mortality of 96.0% ± 0.57 and 96.7% ± 0.88, respectively, as measured using the larval packet test (LPT). An increasing range of extract concentrations was tested to determine the LD50 and LD90 for C. procera, 3.21 and 21.15 mg/mL, respectively, and T. officinale, 4.04 and 18.92 mg/mL, respectively. These results indicate that further studies are warranted to determine the relative contribution of individual phytochemicals from whole-plant extracts on acaricidal activity. This information will guide the design of further acaricidal efficacy tests using livestock infested with R. microplus.

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.

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.

Infestation prevalence, spatio-temporal distribution, phylogenetic positioning, and pathogen investigation of Argas persicus ticks in domestic hens (Gallus gallus domesticus) from Pakistan.

Soft ticks pose significant health risks as vectors of various pathogens. This study explored the spatio-temporal distribution and genetic relationships of the soft tick species Argas persicus infesting domestic hens (Gallus gallus domesticus) across different districts in Pakistan. An examination of 778 hens revealed a notable tick infestation prevalence of 70.82%, with a total of 1299 ticks collected from 551 hens. The overall mean intensity was 2.19 soft ticks per infested chicken, and the overall mean abundance was 1.61 soft ticks per examined hen. Morphological identification confirmed all collected ticks (n = 1210) as A. persicus, comprising 719 males, 333 females, 121 nymphs, and 38 larvae. The Haveli, Muzaffarabad, and Kotli districts had the highest infestation rates, while Bagh had the lowest. Molecular analyses of tick DNA, focusing on 16S rDNA and 12S rDNA sequences, revealed genetic similarities among A. persicus soft ticks from Pakistan and other regions, providing insights into their evolutionary history. Importantly, no Babesia, Rickettsia, or Anaplasma infections were detected in the examined samples. These findings enhance the understanding of soft tick infestation patterns and the genetic diversity of A. persicus in the studied region.

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.

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.

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.

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.

Distribution and Risk of Cutaneous Leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

Cutaneous leishmaniasis (CL) is a zoonotic infection caused by obligate intracellular protozoa of the genus Leishmania. This study aimed to investigate CL in Khyber Pakhtunkhwa, Pakistan and to estimate the risk of epidemics. Clinico-epidemiological data of 3188 CL patients were collected from health facilities in 2021. Risk factors were analyzed using the chi-square test. ArcGIS V.10.7.1 was applied for spatial analysis. The association between CL occurrence and climatic variables was examined by Bayesian geostatistical analysis. The clinical data revealed males or individuals younger than 20 years old were more affected. Most patients presented with a single lesion, and the face was the most attacked body part. CL was prevalent in the southern region in winter. A proportional symbol map, a choropleth map, and a digital elevation model map were built to show the distribution of CL. Focal transmission was predicted by inverse distance weighting interpolation. Cluster and outlier analysis identified clusters in Bannu, Dir Lower, and Mardan, and hotspot analysis suggested Bannu as a high-risk foci. Bayesian geostatistical analysis indicated that increasing precipitation and temperature as well as low altitudes were associated with CL infection. The study has provided important information for public health sectors to develop intervention strategies for future CL epidemics.

Antidiarrheal Potential of Viola canescens: In Vivo and In Silico Approaches.

Viola canescens Wall. is an important medicinal plant with reported therapeutic benefits. The current work sought to investigate the antidiarrheal properties of V. canescens extracts both in vivo and in silico. This study applied molecular docking to unravel the molecular mechanism of V. canescens and to find the most effective phytocompounds with antidiarrheal effects. The antidiarrheal activity of V. canescens was assessed utilizing the castor oil-induced diarrhea assay and the charcoal meal assay. Antidiarrheal characteristics were evaluated by measuring parameters such as intestinal motility, fecal score, and hypersecretion. The V. canescens extract had a dose-dependent and statistically significant impact in the charcoal meal assay and castor oil-induced diarrhea assay. In the castor oil-induced diarrhea assay, the ethyl acetate fraction (65.96%) showed the highest percentage of defecation inhibition at the highest dose (300 mg/kg (bw)), followed by the uncorrected crystalline compound (63.83%), crude alkaloids (63.83%), chloroform fraction (63.83%), and crude flavonoids (55.32%), while the aqueous fraction (40.43%) and n-Hexane fraction (42.55%) revealed the lowest antidiarrheal potential. In addition, the molecular docking investigation showed emetine, quercetin, and violanthin, isolated chemicals of V. canescens, to have the highest binding affinity to the target µ and δ opioid receptors with significant inhibitory capacity. These pharmacologically active metabolites in V. canescens were effective in treating diarrhea. This study lends credence to the traditional usage of V. canescens in treating gastrointestinal disorders.

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.

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.

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.

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.

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.

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.