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Tick sialome is comprised of a rich cocktail of bioactive molecules that function as a tool to disarm host immunity, assist blood-feeding, and play a vibrant role in pathogen transmission. The adaptation of the tick’s blood-feeding behavior has lead to the evolution of bioactive molecules in its saliva to assist them to overwhelm hosts’ defense mechanisms. During a blood meal, a tick secretes different salivary molecules including vasodilators, platelet aggregation inhibitors, anticoagulants, anti-inflammatory proteins, and inhibitors of complement activation; the salivary repertoire changes to meet various needs such as tick attachment, feeding, and modulation or impairment of the local dynamic and vigorous host responses. For instance, the tick’s salivary immunomodulatory and cement proteins facilitate the tick’s attachment to the host to enhance prolonged blood-feeding and to modulate the host’s innate and adaptive immune responses. Recent advances implemented in the field of “omics” have substantially assisted our understanding of host immune modulation and immune inhibition against the molecular dynamics of tick salivary molecules in a crosstalk between the tick–host interface. A deep understanding of the tick salivary molecules, their substantial roles in multifactorial immunological cascades, variations in secretion, and host immune responses against these molecules is necessary to control these parasites. In this article, we reviewed updated knowledge about the molecular mechanisms underlying host responses to diverse elements in tick saliva throughout tick invasion, as well as host defense strategies. In conclusion, understanding the mechanisms involved in the complex interactions between the tick salivary components and host responses is essential to decipher the host defense mechanisms against the tick evasion strategies at tick-host interface which is promising in the development of effective anti-tick vaccines and drug therapeutics.

While Cutaneous leishmaniasis (CL) is not a life-threatening disease, it leads to devastating effects on local community. CL is widely scattered manifesting a noticeable epidemiological pattern around the globe. The present study was planned to address the role of Geographic Information System (GIS) using CL clinico-epidemiological data to determine the high-risk areas of CL. Recorded data (2014-2018) of 3630 positive individuals was collected from Basic Health Units of the Upper and Lower Dir Districts, Khyber Pakhtunkhwa, Pakistan. Descriptive and statistical analysis was used for clinico-epidemiological characterization. For spatial analysis, ArcGIS V.10.3 was used and the CL average incidence was tagged on the proportional, choropleth, and digital elevation model maps. For focal transmission and high-risk zones, Inverse Density Weight (IDW) spatial interpolation, focal statistics, hot spot, cluster and outlier, and Bayesian geostatistical analysis were used. The trend of CL cases was elevated from 2014 to 2016 except for 2017 and 2018. Individuals of both genders younger than 20 years old were highly susceptible. Single lesions were more prominent (56%) and frequently affected facial parts (51%). The size and pretreatment duration of the CL lesion was significantly associated. Spatially, a choropleth map displayed the maximum CL incidences in Tehsil Balambat, Khal, and Termergara (31%-13%) located within a range of 948-1947m elevation in the central regions with proximal CL transmissions. Hot spot and cluster and outlier analysis affirmed that Tehsil Khal was the high-risk CL foci. The Bayesian geostatistical analysis revealed high temperature, less altitude, and annual precipitation as important risk factors. An increasing trend in CL transmission has become evident, affecting both genders and20 years old children. GIS resolute the concealed CL hubs in the least elevated central regions which warrant the control strategies to restrict future epidemics.

The genetic diversity of Leishmania spp. in North Eastern Pakistan remains undetermined despite increased cases of cutaneous leishmaniasis (CL). This study was designed to decipher the molecular characterization and genetic diversity of Leishmania spp. in North Eastern Pakistan. Out of 13761 CL suspected cases, 567 cases were microscopically positive and confirmed as Leishmania spp. by internal transcribed spacer (ITS) gene amplification through the PCR- RFLP technique. Further, isolates were directly sequenced to conduct phylogenetic analysis for genetic diversity. Among suspected CL cases, Mirpur showed the highest proportion of CL infection with 4.85% (118/2431) of the cases, while the Neelum district showed the lowest percentage at 3.29% (9/273). The slide positivity rate, annual blood examination rate, and annual parasite incidence rate were 3.84, 0.27, and 0.01% respectively, and the incidence of CL in the age group 1–20 years old was higher in males (50.92%) than females (25.75%). The RFLP analysis and sequencing confirmed the occurrence of Leishmania tropica, Leishmania major, and Leishmania infantum. Leishmania tropica (p = 0.02) confirmed significantly higher nucleotides variation than L. major (p = 0.05). Current findings confirmed the prior assumption that anthroponotic CL is the primary CL form present in North Eastern Pakistan. Moreover, this is the first report based on molecular identification of L. major, and L. infantum from North Eastern Pakistan. This remarkable heterogeneity in the Leishmania spp. is the leading cause of treatment failure and emergence of new haplotypes. Therefore more extensive investigations are recommended from all geographical regions of North Eastern Pakistan, especially those using a large sample size.

Characterization of content and source of heavy metals in soils are necessary to establish quality standards on a regional level. In relation to this, two zones, (sampling zone-1) and (sampling zone-2) depending on nature and intensity of wastewater disposal along the peri-urban area of Peshawar, Pakistan were selected. Thirty-six samples of wastewater and topsoil each were collected to determine the content of Pb, Cr, Cd, Cu, Zn, Ni, Fe and Mn, and physico-chemical parameters like pH, electrical conductivity, total solids, total dissolved solids, total suspended solids, and organic matter. Analytical determinations were performed by atomic absorption spectroscopy after microwave sample digestion in acid solution. Chemometric techniques which include hierarchical cluster analysis, principal component analysis, correlation analysis, and tukey test were applied. Concentrations of physico-chemical properties in wastewater and soil were higher in sampling zone-2. Concentrations of six heavy metals in wastewater and two in soil exceeded permissible limits of World Health Organization (Guidelines for drinking water quality, 4th edition, 2011), European Union (Heavy metals in wastes, European commission on environment. http://ec.europa.eu/environment/waste/studies/pdf/heavymetalsreport , 2002). Hierarchical cluster analysis grouped eight heavy metals into two clusters for wastewater and five clusters for soils. Principal component analysis describes four factors possessing Eigenvalues greater than 1.0 and explained the cumulative total variance of 84% with factor 1, having positive loading of anthropogenic metals (Cd, Cu, and Ni). Significant correlation was found between anthropogenic metals like Ni and Cd in water and between Cu and Cr in soil. Further research in other agricultural lands in peri-urban region would improve the basis for proposing such soil quality standards.

Documented reports are limited, showing cutaneous leishmaniasis (CL) as a severe threat to schoolchildren in Pakistan. The present study aimed to investigate the clinico-epidemiology and associated risk factors of CL in local and Afghan male schoolchildren between 6 and 16 years of age. The experimental strategy involved a questionnaire for the collection of information and clinical diagnosis (microscopy and semi-nested PCR) of 113 CL symptomatic schoolchildren out of 8,833 schoolchildren (7,175 local and 1,658 Afghan refugees) studying in nineteen schools of the Upper and Lower Dir Districts, Khyber Pakhtunkhwa, Pakistan. Previous records of CL in Pakistan was studied, and spatial analysis was performed on elevation and agro-ecological maps using Arc-GIS v10.3.1. Active lesions were found predominant (n=113, 1.2%: cutaneous lesions, 97, 86%, and lesions with mucosal involvement, 16, 14%) than scars (20, 0.25%). Active lesions of both local (100, 88%) and Afghan refugees (13, 12%), and infected age groups were found significantly different. Majority of the lesions were dry crusted (98, 86.7%), single (83, 73%), and frequently infecting facial region (59, 52%). Avoiding bed nets, living in mud houses and animal shelters were highly associated with CL infection. Temergara (30, 26.5%) and Rabath (14, 12.3%) were hyperendemic CL foci. Microscopically, 71 (63%) cases were positive, while the PCR assay revealed Leishmania tropica in 110 (97.3%) cases. Previous record revealed that L. tropica is dominant throughout Pakistan, and dry mountains and plateaus of northwestern and southwestern regions are spatially at high-risk. Measures should be taken to reduce CL infection by eliminating the associated risk factors, promoting PCR-based diagnosis and basic medical facilities.

Background: Paramphistomiasis (Rumen fluke disease) in ruminants is a major health problem, characterized by coarse hair, weakness, loss of appetite, weight retardations, intestine ulcers, inter-mandibular inflammation, causing substantial economic losses, and high mortality. In tropical and subtropical regions, the disease was neglected but has recently emerged as an important cause of production losses. While documented reports on Paramphistomum cervi, Paramphistomum ichikawai and Paramphistomum are limited in Asian countries and paramphistomosis has been considered the major health and economic problem in several countries. The present study aimed to identify paramphistomoid flukes that infects buffaloes with the goal of characterization of prevalence in Pakistan and its comparison with neighbor countries. Materials, Methods & Results: In 2018, a total of 178 slaughtered buffaloes aged four to six years were examined. After an immediate postmortem examination of each buffalo, flukes were collected from their infected rumen and reticulum using sterilized forceps and placed in a saline solution. DNA was extracted from adult Paramphistome species using the standard phenol chloroform method and used for amplification of partial fragment of 18S rRNA sequences using specific pair of primer. After amplification and sequencing of 18S rRNA partial fragment, the generated sequences were assembled and trimmed to remove any primer contaminations. Twenty-three randomly selected and morphologically identified adult Paramphistomum were used in species-level identification using specific primers for partial fragment of 18S rRNA sequences. The cleaned sequences (810 bp) were used to identify similar sequences using BLAST on the NCBI website. The GenBank retrieved sequences and new Paramphistomum species isolated sequences were aligned using CLUSTAL in the BioEdit Sequence Alignment Editor. In addition, a phylogenetic tree was constructed using maximum likelihood method in MEGA X. The 18S rRNA sequence was found 100% similar with Paramphistomum cervi of China and 98% with Paramphistomum epiclitum and other Paramphistomum species of India. The parasitic Pharamphistomum species was identified molecularly as Paramphistomum cervi. Discussion: Molecular studies provide insight into the biology and phylogenetic relationship among various parasites. These studies are reliable in the genetic-based identification and description of several disease causing agents. The 18S rRNA sequence of Paramphistomum cervi generated in this study was found closely identical to the P. cervi of the neighbor countries (China and India) which may be due to the similar geographical, environmental conditions and transboundary movement of infected hosts. This is the first nature of study which provides the molecular-based evidence of P. cervi existence in Pakistan and revealed the 18S rRNA as novel molecular marker for the identification and further characterization of Paramphistomum species across Pakistan. The submitted sequence of this study will provide a baseline for further molecular characterization and to compare with other Paramphistoma species from different regions of Pakistan.