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.

Structure-based in silico design and in vitro acaricidal activity assessment of Acacia nilotica and Psidium guajava extracts against Sarcoptes scabiei var. cuniculi.

Infestation by Sarcoptes scabiei var. cuniculi mite causes scabies in humans and mange in animals. Alternative methods for developing environmentally friendly and effective plant-based acaricides are now a priority. The purpose of this research was the in silico design and in vitro evaluation of the efficacy of ethanol extracts of Acacia nilotica and Psidium guajava plant leaves against S. scabiei. Chem-Draw ultra-software (v. 12.0.2.1076.2010) was used to draw 36 distinct compounds from these plants that were employed as ligands in docking tests against S. scabiei Aspartic protease (SsAP). With docking scores of - 6.50993 and - 6.16359, respectively, clionasterol (PubChem CID 457801) and mangiferin (PubChem CID 5281647) from A. nilotica inhibited the targeted protein SsAP, while only beta-sitosterol (PubChem CID 222284) from P. guajava interacted with the SsAP active site with a docking score of - 6.20532. Mortality in contact bioassay at concentrations of 0.25, 0.5, 1.0, and 2.0 g/ml was determined to calculate median lethal time (LT50) and median lethal concentration (LC50) values. Acacia nilotica extract had an LC50 value of 0.218 g/ml compared to P. guajava extract, which had an LC50 value of 0.829 g/ml at 6 h. These results suggest that A. nilotica extract is more effective in killing mites, and these plants may have novel acaricidal properties against S. scabiei. Further research should focus on A. nilotica as a potential substitute for clinically available acaricides against resistant mites.

Molecular detection of small ruminant piroplasmosis and first report of Theileria luwenshuni (Apicomplexa: Theileridae) in small ruminants of Pakistan.

Theileriosis is a widespread and economically important disease of small ruminants in Pakistan. Ruminants are the intermediate hosts in the lifecycle of Theileria spp., with ticks of the family Ixodidae being the definitive hosts. To better understand the distribution and prevalence of theileriosis in Pakistan, a molecular survey was performed in small ruminants from the Lower Dir district of the Khyber Pakhtunkhwa province. A total of 200 healthy sheep and goats were screened from Maidan, Samar Bagh and Munda districts of district Dir Lower, Pakistan during December (2017) to April (2018). DNA samples were screened through nested PCR using universal primers. The amplified 492-498 bp amplicon was subjected to RLB analysis which was based on the hypervariable of the 18S rRNA gene to test for the presence of genotypes of Theileria in blood samples. A phylogeny was constructed to determine the species of Theileria genotypes. Nested PCR results indicated 53.5% prevalence of one or more Theileria genotypes in the blood of the host animal. From RLB assay, 27 animals (13.5%) showed infection with only a single species of Theileria while 80 animals (40%) showed coinfection by multiple Theileria spp. Based on the 18S rRNA phylogeny, the unknown genotype is of the species Theileria luwenshuni and is closely related to Chinese isolates. The present finding is the first report on molecular diagnosis of Theileria luwenshuni in small ruminants in Pakistan.

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.

Fasciola hepatica in some Buffaloes and cattle by PCR and microscopy.

Fasciolosis is the burning problem of the livestock rearing community having huge morbidity, mortality, and economic losses to livestock industries in our country Pakistan. The faecal and liver biopsy samplings were examined by polymerase chain reaction (PCR) and microscopy technique during the entire study. A total of 307 samples including 149 samples from Karak and 158 samples from Kohat abattoirs were examined by PCR method and overall prevalence of fasciolosis was 5.86% (18/307), amongst theses 8.05% (12/149) in liver biopsy and 3.79% (6/158) in feacal samples of cattle and Buffaloes were recorded. Similarly the microscopy based detection was 3.58% (11/307) including 4.61% (7/149) in liver biopsy and 2.5% (4/158) in faecal samples accordingly. Furthermore the areawise prevalence of fasciolosis in abattoirs by PCR method was found to be 7.59% (12/158) in Kohat and 4.02% (6/149) in Karak. A 618 pb DNA was amplified in 2% agarose gel electrophoreses. It is concluded from the study that prevalence of fasciolosis was higher in abattoir of district Kohat and PCR was a more sensitive method of diagnosis than microscopy.

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.

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.

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.

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.

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.

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.

Knowledge, attitude, and practices towards cutaneous leishmaniasis in referral cases with cutaneous lesions: A cross-sectional survey in remote districts of southern Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Cutaneous leishmaniasis is a neglected tropical disease caused by Leishmania spp. and transmitted by female sandflies. Terrorism and counter-insurgency military operations in Federally Administered Tribal Areas (FATA) lead to a large-scale migration of internally displaced persons (IDPs) in Khyber Pakhtunkhwa and thus, new outbreaks of several infectious diseases such as cutaneous leishmaniasis occurred. This study intended to find the prevalence of cutaneous leishmaniasis in people with cutaneous lesions suspected of having cutaneous leishmaniasis in four remote districts of Khyber Pakhtunkhwa and to assess the participant's knowledge, attitude, and practices about the infection and its control. METHODS: A cross-sectional study was carried out in four remote districts of Khyber Pakhtunkhwa including Karak, Lakki Marwat, Tank, and Dera Ismail Khan (D. I. Khan) and a total of 1,674 participants were recruited using a convenience sampling technique. RESULTS: The prevalence of cutaneous leishmaniasis among the participants with cutaneous lesions was 50.4% and the infection was comparatively more prevalent in district Karak. Among participants, 56.8% were male and mostly, 53.8% were under the age of 16 years with 52.8% living in kutcha houses and were from rural areas. Multiple skin lesions were more common, and the face was frequently affected body part. The ratio of participants with lesions older than a month was higher and the majority confronted infections with blood protozoan parasites for the first time. Most participants were unaware of the signs/symptoms of the disease, basic knowledge of the vectors, anthroponotic spread, preventive measures, secondary infections, and reservoir hosts. The use of wood/animal dung as fuel, closeness with reservoir animals, and no use of insect repellents were some of the notable risk factors. CONCLUSION: Cutaneous leishmaniasis is highly prevalent in the study area and a very low level of awareness was reported among the participants. This study necessitates the planning and execution of regulations and preventive programs, public health education, awareness campaigns, and disease management practices to overcome future incidence of cutaneous leishmaniasis.

Changes in the Prevalence of Natural Paramphistomum Cercariae Infection in Indoplanorbis and Lymnaea Intermediate Hosts Influenced by Meteorological Factors.

Paramphistomosis is a neglected ruminant parasitic disease caused by trematodes known as Paramphistomum, which has a diheteroxenic life cycle involving freshwater snail genera, i.e., Planorbis and Lymnaea as the intermediate host and mammals as the definitive host. Snail vector distribution, infection with Paramphistomum spp. cercariae, preferred habitat, and their relationship with certain meteorological factors were not investigated in the province Khyber Pakhtunkhwa of Pakistan. Therefore, this study is designed to evaluate the effects of meteorological factors on the occurrence and severity of Paramphistomum spp. cercariae in Indoplanorbis and Lymnaea intermediate snail hosts. For this purpose, a cross-sectional survey was conducted from October 2018 to September 2019, and snails were collected and then identified using snail shell morphology; their infection with Paramphistomum spp. cercariae was determined through microscopy; and descriptive statistics were used to estimate the prevalence of infection and evaluate their occurrence relationship with a certain meteorological factors including temperature, humidity, rainfall, and pan evaporation in different districts of the above-mentioned province of Pakistan, i.e., adjacent areas of Bara and Kabul rivers in district Nowshehra, Kabul River (Sardaryab) of district Charsadda, Kalpani River of district Mardan, and Indus River (Hund) of district Swabi. A total of 2,706 Indoplanorbis (1539) and Lymnaea (1167) snails were collected, in which overall 10.30% shed Paramphistomum spp. cercariae. The highest infection rate was found in the river of district Swabi (13.20%), while the lowest in adjacent rivers of district Nowshehra (8.19%). Meteorological factors play an essential role in the causation of Paramphistomum spp. infection, parasitic reproduction, vector growth, and survival. Due to these factors, high significant prevalence was found in the summer season (11.83), followed by autumn (11.25), which might be due to optimum temperature, relative humidity, and rainfall (p < 0.05). It was concluded from the study that meteorological factors contribute to the prevalence of Paramphistomum species in the Indoplanorbis and Lymnaea, which act as vectors for the disease paramphistomosis, which may lead to the increased intensity of infection outbreaks of the parasite population in humans and domestic animals.

In silico approaches to develop herbal acaricides against R. (Boophilus) Microplus and In vitro Anti-Tick activities of selected medicinal plants.

In tropical and sub-tropical areas of the world the most damaging pest of the livestock sector are cattle tick, Rhipicephalus microplus. The current study was aimed to generate phytochemical derived acaricides to control Rhipicephalus microplus populations, to maintain livestock herd production, minimize economic losses and to reduce uses of man-made chemicals acaricides. To achieve this goal, Adult immersion and larval package test were used to determine the feasibility of Berberium lyceum and Tamarixa aphylla against Rhipicephalus microplus ticks. Further, an In silico technique was employed to discover biologically active substances from both plants using docking method. Berberium lyceum and Tamarixa aphylla exhibited a reasonably high fatal effect at 40.0 mg/L on egg laying (index of egg laying = 0.19 and 0.19) respectively, thus inhibiting the oviposition (49.5 and 45.1, respectively) and the larval mortality (97% and 93%, respectively). Further, we also used Chem-Draw ultra-software (v. 12.0.2.1076. 2010) to illustrate different structures of38 known bioactive phytochemicals which are discovered in the PubChem database and verify the hypothesis that tick inhibition was linked to acetylcholinesterase (AChE). Barbamunine and rutin from Berberium lyceum showed remarkable interaction with RmAChE1 active site residues with docking scores of -9.11 to -8.71 while phytol and dehydrodigallic acid from Tamarix aphylla showed comparable docking scores of -7.17 and -7.14 respectively against Rhipicephalus microplus acetylcholinesterase protein. Based on obtained result, we believe that Berberium lyceum and Tamarixa aphylla bioactive components could be potential candidates in the control and management of Rhipicephalus microplus and should be studied further as a supplement or replacement for synthetic acaricides.