Factors affecting superovulation induction in goats (Capra hericus): An analysis of various approaches.

Goats are generally called a "poor man's cow" because they not only provide meat and milk but also other assistance to their owners, including skins for leather production and their waste, which can be used as compost for fertilizer. Multiple ovulation and embryo transfer (MOET) is an important process in embryo biotechnology, as it increases the contribution of superior female goats to breeding operations. The field of assisted reproductive biotechnologies has seen notable progress. However, unlike in cattle, the standard use of superovulation and other reproductive biotechnologies has not been widely implemented for goats. Multiple intrinsic and extrinsic factors can alter the superovulatory response, significantly restricting the practicability of MOET technology. The use of techniques to induce superovulation is a crucial step in embryo transfer (ET), as it accelerates the propagation of animals with superior genetics for desirable traits. Furthermore, the conventional superovulation techniques based on numerous injections are not appropriate for animals and are labor-intensive as well as expensive. Different approaches and alternatives have been applied to obtain the maximum ovarian response, including immunization against inhibin and the day-0 protocol for the synchronization of the first follicular wave. While there are several studies available in the literature on superovulation in cattle, research on simplified superovulation in goats is limited; only a few studies have been conducted on this topic. This review describes the various treatments with gonadotropin that are used for inducing superovulation in various dairy goat breeds worldwide. The outcomes of these treatments, in terms of ovulation rate and recovery of transferrable embryos, are also discussed. Furthermore, this review also covers the recovery of oocytes through repeated superovulation from the same female goat that is used for somatic cell nuclear transfer (SCNT).

In vitro and in vivo development of interspecies Asian elephant embryos reconstructed with pig enucleated oocytes.

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant-pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant-pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 µm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant-pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant-pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant-pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.

Development of RAG2 -/- IL2Rγ -/Y immune deficient FAH-knockout miniature pig.

Human hepatocyte transplantation for liver disease treatment have been hampered by the lack of quality human hepatocytes. Pigs with their large body size, longevity and physiological similarities with human are appropriate animal models for the in vivo expansion of human hepatocytes. Here we report on the generation of RAG2-/-IL2Rγ-/YFAH-/- (RGFKO) pigs via CRISPR/Cas9 system and somatic cell nuclear transfer. We showed that thymic and splenic development in RGFKO pigs was impaired. V(D)J recombination processes were also inactivated. Consequently, RGFKO pigs had significantly reduced numbers of porcine T, B and NK cells. Moreover, due to the loss of FAH, porcine hepatocytes continuously undergo apoptosis and consequently suffer hepatic damage. Thus, RGFKO pigs are both immune deficient and constantly suffer liver injury in the absence of NTBC supplementation. These results suggest that RGFKO pigs have the potential to be engrafted with human hepatocytes without immune rejection, thereby allowing for large scale expansion of human hepatocytes.

Small Ruminant Farming in Tribal Areas of Dera Ghazi Khan, Punjab, Pakistan.

Provincially Administered Tribal Areas (PATA) of Punjab-Pakistan are comprised of hilly mountains with small ruminants as a sole source of income. In this study, farming practices, productivity, health and the economic value of sheep were evaluated in PATA through a survey of farmers (n = 138) holding 11,558 heads of sheep. Out of a total population, 87% were non-descriptive flocks, and 9% and 4% were purebred flocks belonging to the Kajli and Thali populations, respectively. Sheep flocks were mainly (86%) reared under the traditional production system and had a delayed onset of puberty. There was low influence of season on the reproduction, and the majority of flocks (78%) were bred throughout the year. The lack of proper vaccination and poor management exposed the flocks to bacterial, viral and parasitic infections, which lead to high mortality in lambs (~22%) and adults (~32%). The share of sheep in farmers livelihood was 42%, and only 20% of producers' living standard was improved with sheep farming, but the rise in rearing more sheep was quite low (20%). Although the livestock department arranged farmers' training, the majority of farmers (83%) never participated in training and had no knowledge of modern technologies. Collectively, the traditional sheep production systems, poor management, lack of vaccination, marketing channels and farmers training hampered the sheep rearing and producers' livelihood in the PATA of Punjab-Pakistan. However, developing model livestock farms, conducting farmer training, establishing a viable market for dairy products, and introducing subsidy policy interventions can improve the sheep farming in these areas.

Production of Triple-Gene (GGTA1, B2M and CIITA)-Modified Donor Pigs for Xenotransplantation.

Activation of human immune T-cells by swine leukocyte antigens class I (SLA-I) and class II (SLA-II) leads to xenograft destruction. Here, we generated the GGTA1, B2M, and CIITA (GBC) triple-gene-modified Diannan miniature pigs, analyzed the transcriptome of GBC-modified peripheral blood mononuclear cells (PBMCs) in the pig's spleen, and investigated their effectiveness in anti-immunological rejection. A total of six cloned piglets were successfully generated using somatic cell nuclear transfer, one of them carrying the heterozygous mutations in triple genes and the other five piglets carrying the homozygous mutations in GGTA1 and CIITA genes, but have the heterozygous mutation in the B2M gene. The autopsy of GBC-modified pigs revealed that a lot of spot bleeding in the kidney, severe suppuration and necrosis in the lungs, enlarged peripulmonary lymph nodes, and adhesion between the lungs and chest wall were found. Phenotyping data showed that the mRNA expressions of triple genes and protein expressions of B2M and CIITA genes were still detectable and comparable with wild-type (WT) pigs in multiple tissues, but α1,3-galactosyltransferase was eliminated, SLA-I was significantly decreased, and four subtypes of SLA-II were absent in GBC-modified pigs. In addition, even in swine umbilical vein endothelial cells (SUVEC) induced by recombinant porcine interferon gamma (IFN-γ), the expression of SLA-I in GBC-modified pig was lower than that in WT pigs. Similarly, the expression of SLA-II DR and DQ also cannot be induced by recombinant porcine IFN-γ. Through RNA sequencing (RNA-seq), 150 differentially expressed genes were identified in the PBMCs of the pig's spleen, and most of them were involved in immune- and infection-relevant pathways that include antigen processing and presentation and viral myocarditis, resulting in the pigs with GBC modification being susceptible to pathogenic microorganism. Furthermore, the numbers of human IgM binding to the fibroblast cells of GBC-modified pigs were obviously reduced. The GBC-modified porcine PBMCs triggered the weaker proliferation of human PBMCs than WT PBMCs. These findings indicated that the absence of the expression of α1,3-galactosyltransferase and SLA-II and the downregulation of SLA-I enhanced the ability of immunological tolerance in pig-to-human xenotransplantation.

Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage.

As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and other organs is still unknown. In this study, we successfully generated PIK3C3 transgenic pigs through somatic cell nuclear transfer (SCNT) by designing a specific vector for the overexpression of PIK3C3. Plasmid identification was performed through enzyme digestion and transfected into the fetal fibroblasts derived from Diannan miniature pigs. After 2 weeks of culturing, six positive colonies obtained from a total of 14 cell colonies were identified through PCR. One positive cell line was selected as the donor cell line for SCNT for the construction of PIK3C3transgenic pigs. Thirty single blastocysts were collected and identified as PIK3C3 transgenic-positive blastocysts. Two surrogates became pregnant after transferring the reconstructed embryos into four surrogates. Fetal fibroblasts of PIK3C3-positive fetuses identified through PCR were used as donor cells for SCNT to generate PIK3C3 transgenic pigs. To further explore the function of PIK3C3 overexpression, genotyping and phenotyping of the fetuses and piglets obtained were performed by PCR, immunohistochemical, HE, and apoptosis staining. The results showed that inflammatory infiltration and vacuolar formation in hepatocytes and apoptotic cells, and the mRNA expression of NF-κB, TGF-ß1, TLR4, TNF-α, and IL-6 significantly increased in the livers of PIK3C3 transgenic pigs when compared with wild-type (WT) pigs. Immunofluorescence staining showed that LC3B and LAMP-1-positive cells increased in the livers of PIK3C3 transgenic pigs. In the EBSS-induced autophagy of the porcine fibroblast cells (PFCs), the accumulated LC3II protein was cleared faster in PIK3C3 transgenic (PFCs) thanWT (PFCs). In conclusion, PIK3C3 overexpression promoted autophagy in the liver and associated molecular mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR, causing liver damage in pigs. Therefore, the construction of PIK3C3 transgenic pigs may provide a new experimental animal resource for liver diseases.

Towards Improving the Outcomes of Multiple Ovulation and Embryo Transfer in Sheep, with Particular Focus on Donor Superovulation.

Considerable improvements in sheep multiple ovulation and embryo transfer (MOET)protocols have been made; however, unlike for cattle, MOET is poorly developed in sheep, and thus has not been broadly applicable as a routine procedure. The tightly folded nature of the ewe cervix, the inconsistent ovarian response to various superovulatory treatments, and the requirement of labor to handle animals, particularly during large-scale production, has limited the implementation of successful MOET in sheep. Moreover, several extrinsic factors (e.g., sources, the purity of gonadotrophins and their administration) and intrinsic factors (e.g., breed, age, nutrition, reproductive status) severely limit the practicability of MOET in sheep and other domestic animals. In this review, we summarize the effects of different superovulatory protocols, and their respective ovarian responses, in terms of ovulation rate, and embryo recovery and transfer. Furthermore, various strategies, such as inhibin immunization, conventional superovulation protocols, and melatonin implants for improving the ovarian response, are discussed in detail. Other reproductive techniques and their relative advantages and disadvantages, such as artificial insemination (AI), and donor embryo recovery and transfer to the recipient through different procedures, which must be taken into consideration for achieving satisfactory results during any MOET program in sheep, are also summarized in this article.

Inhibition of FOXO1­mediated autophagy promotes paclitaxel­induced apoptosis of MDA­MB­231 cells.

Triple­negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it often becomes resistant to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX­induced tumor cell death, and targeting autophagy has been promising for improving the efficacy of tumor chemotherapy in recent years. The aim of the present study was to clarify the mechanism of PTX inducing autophagy in TNBC cells to provide a potential clinical chemotherapy strategy of PTX for TNBC. The present study reported that PTX induced both apoptosis and autophagy in MDA­MB­231 cells and that inhibition of autophagy promoted apoptotic cell death. Furthermore, it was found that forkhead box transcription factor O1 (FOXO1) enhanced PTX­induced autophagy through a transcriptional activation pattern in MDA­MB­231 cells, which was associated with the downstream target genes autophagy related 5, class III phosphoinositide 3­kinase vacuolar protein sorting 34, autophagy related 4B cysteine peptidase, beclin 1 and microtubule associated protein 1 light chain 3ß. Knocking down FOXO1 attenuated the survival of MDA­MB­231 cells in response to PTX treatment. These findings may be beneficial for improving the treatment efficacy of PTX and to develop autophagic targeted therapy for TNBC.

Delayed body development with reduced triglycerides levels in leptin transgenic pigs.

Leptin is a well-known adipokine that plays critical role in adiposity. To further investigate the role of leptin in adiposity, we utilized leptin overexpressing transgenic pigs and evaluated the effect of leptin on growth and development, fat deposition, and lipid metabolism at tissue and cell level. Leptin transgenic pigs were produced and divided into two groups: elevated leptin expression (leptin ( +)) and normal leptin expression group (control). Results indicated that leptin ( +) pigs had elevated leptin protein and mRNA expression levels and exhibited sluggish growth and development followed by decreased subcutaneous fat thickness, low serum triglycerides, saturated, unsaturated fatty acids and high cholesterol esters (p < 0.05). There were differences in the lipid metabolism related genes at different fat depots, including upregulation of PPARγ, AGPAT6, PLIN2, HSL and ATGL in subcutaneous, PPARγ in perirenal, and FAT/CD36 and PLIN2 in mesenteric adipose tissues and downregulation of AGPAT6 and ATGL in perirenal and AGPAT6 in mesenteric adipose tissues (p < 0.05). Additionally, in-vitro cultured leptin ( +) preadipocytes exhibited upregulation of PPARγ, FAT/CD36, ACACA, AGPAT, PLIN2, ATGL and HSL as compared to control (p < 0.05). These findings suggested that homeostasis imbalance in lipolysis and lipogenesis at adipose tissue and adipocytes levels led to low subcutaneous fat depots in leptin overexpression pigs. These pigs can act as model for obesity and related metabolic disorder.

Synergy in penicillin, cephalosporin, amphenicols, and aminoglycoside against MDR S. aureus isolated from Camel milk

Abstract This study investigated the synergy testing of penicillin, cephalosporin, amphenicols, and aminoglycoside in the camel milk (n=768 samples), subsequently used for isolation of MDR S. aureus targeting mecA gene. Antibiotic susceptibility of S. aureus showed >90% isolates were sensitive to ciprofloxacin and trimethoprim and resistant against oxacillin, ampicillin, and cefoxitin. Further, 50-85% of the S. aureus were sensitive to gentamicin, oxytetracycline, and chloramphenicol and resistant against cefotaxime, vancomycin, and cefixime. Minimum inhibitory concentration (MIC) of cefotaxime, (C) and ampicillin (A) in combination with gentamicin (G) was reduced by 99.34% and 70.46%, respectively, while with chloramphenicol (Ch), reduction was 57.49% and 60%, respectively. In addition, the Fractional Inhibitory Concentration Index (FICI) of G+A, Ch+C and Ch+G combinations showed synergy against 80%, 60%, and 30% of MDR S. aureus, respectively. Similarly, C+A and Ch+G displayed indifferent interaction against 70 % and 30% of isolates, respectively, while the later showed additive interaction against 10% of MDR S. aureus. Altogether, our results described effective combination of gentamicin and chloramphenicol with ampicillin and cefotaxime to combat MDR S. aureus

The effect of GnRH administration/insemination time on follicular growth rate, ovulation intervals, and conception rate of Nili Ravi buffalo heifers in 7 -day-CIDR Co-synch.

The present study aimed to compare two different insemination times (72 vs 84 h) associated with an ovulation induction (GnRH) in a 7-day CIDR Co-synch to improve the conception rate of Nili Ravi buffalo heifers. Forty Nili Ravi buffalo heifers were randomly separated into two treatments based on artificial insemination (AI) timing (72 vs 84 h). All heifers were subjected to controlled internal drug release (CIDR), containing 1.38 g of progesterone for 7 days. On CIDR removal, both treatments received 150 µg of prostaglandin intramuscularly. In 7-day CIDR Co-synch (n = 20), animals were injected 100 µg of GnRH administration intramuscularly and inseminated concurrently at 72 h after CIDR removal. The remaining half (n = 20) were injected and inseminated concurrently at 84 h of CIDR removal. Pregnancy diagnosis was performed on day 40 of timed artificial insemination (TAI) with ultrasound. The follicular growth rate between 72 h after PGF2α/CIDR removal to pre-ovulatory follicle in 7-day CIDR Co-synch was more (0.102 ± 0.005 mm vs 0.079 ± 0.003 mm; P = 0.01) at 84 than 72 h. The interval from GnRH administration/TAI to ovulation was high (26.8 ± 1.64 h vs. 15.1 ± 1.25 h, P = 0.01) in 72 than 84 h. Conception rates were considerably higher in buffalo heifers inseminated at 84 h (65%) than 72 h (25%) in 7-day CIDR Co-synch protocol. In conclusion that in Nili Ravi buffalo heifers, GnRH administration/TAI after 84 h of CIDR removal allows greater follicular growth rate and shortens interval from AI to ovulation compared to the GnRH administration/TAI after 72 h of CIDR removal in 7-day CIDR-Co-synch protocol.

Improving porcine SCNT efficiency by selecting donor cells size.

Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 µm), group M (medium, 13 µm 18 µm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 µm are more suitable donor cells for SCNT in pigs.Abbreviations: DMEM: Dulbecco's modified Eagle's medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.

Gold nanoparticles improve the embryonic developmental competency of artificially activated mouse oocytes.

Currently, artificial oocyte activation has attracted wide attention in assisted reproduction due to extensive range of applications, particularly in somatic cell nuclear transfer and deriving pluripotent stem cell lines and it is the unique model to determine the role of paternal genome. Numbers of artificial activating agents have been used extensively to induce the oocytes activation; however, embryos developmental competency of artificially activated oocytes is still very low. In the present study, we determined the functional impact of strontium chloride supplementation with gold nanoparticles (AuNPs) in artificial oocytes activation and subsequent embryonic development. Oocytes were activated artificially in the culture medium containing 250 nM AuNPs with constant concentration of strontium chloride 10.00 mM. We found that adding 250 nM AuNPs with constant concentration of strontium chloride (10.00 mM for 3 hr) in culture medium improves the proportion of embryos reaching to the morula and blastocyst stages from 61.00% and 42.00% (controls) to 75.00% and 58.00% (250 nM AuNPs), respectively. In addition, foster mothers receiving AuNPs-treated embryos showed more implantation percentage and pregnancy rate relative to females received control embryos. Finally, embryos treated with 250 nM AuNPs concentration showed no toxic effect in term of blastocyst development. Collectively, our findings suggest the potential role of AuNPs in early embryonic development for mouse oocytes activated artificially and provide new insights in the field of animal biotechnology and assisted reproduction in humans.

Safety and efficacy of ketamine xylazine along with atropine anesthesia in BALB/c mice

Anesthetics are an indispensable prerequisite for surgical intervention and pharmacological animal studies. The objective of present study was to optimize the dose of ketamine (K) and xylazine (X) along with atropine sulfate (A) in order to achieve surgical tolerance in BALB/c mice. Several doses of ketamine (100, 150, 200 mg/kg) and xylazine (10, 15, 20 mg/kg) were mixed and combination of nine doses (K/X: 100/10, 100/15, 100/20, 150/10, 150/15, 150/20, 200/10,200/15,200/20) were evaluated (n=9 per combination). A constant dose of atropine (0.05 mg/kg) was also used to counter side effect. Time-related parameters were evaluated on the basis of reflexes. KX at dose 200/20 mg/kg produced surgical tolerance in all nine mice with duration 55.00±6.87 minutes. The induction time 0.97±0.09 minutes, sleeping time 90.67±5.81 minutes and immobilization time (102.23±6.83 minutes) were significantly higher than all combination. However, this combination was considered unsafe due to 11 % mortality. While, KX at dose 200/15 mg/kg results in none of the mortality, so was considered as safe. Moreover, this combination produces surgical tolerance in 89 % mice with duration (30.00±7.45 minutes). It was concluded that KX at dose 200/15 mg/kg along with atropine 0.05 mg/kg is safe for performing surgical interventions in BALB/c mice.

Epidemiology of Cutaneous Leishmaniasis Outbreak, Waziristan, Pakistan.

During 2013-2015, prevalence of cutaneous leishmaniasis in war-affected Waziristan areas was 3.61% by PCR. Youths (1-15 years of age) were more susceptible. Internal transcribed spacer 1 PCR followed by restriction fragment length polymorphism analysis identified Leishmania tropica in 215 samples and Leishmania major in 6 samples.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan.

OBJECTIVE: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only. METHODS: Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011-2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. RESULTS: Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples. CONCLUSIONS: This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

Epidemic outbreak of anthroponotic cutaneous leishmaniasis in Kohat District, Khyber Pakhtunkhwa, Pakistan.

An epidemiological and molecular study was carried out for the first time in Kohat District of Khyber Pakhtunkhwa (KP) province, Pakistan from April 2015 to May 2016 to determine the prevalence of Cutaneous Leishmaniasis (CL) in local population and Internally Displaced People (IDPs). In 13 different villages, a total of 1359 (out of 26,250 individuals belonging to local population) and 140 (out of 3615 IDPs residing in these villages) cases were recorded and 300 samples were collected. The total prevalence of CL in local population was 5.17% with active lesions and scar prevalence of 3.91% and 1.26% respectively. Similarly a prevalence of 3.86% for CL was recorded in IDPs. Highest number of IDPs having CL active lesions and scars were recorded in villages Sherkot, Surgul, and Jarma and their presence was positively correlated with CL in local population. Age wise prevalence was highest in young children of age group 1-15 years. The microscopic examination showed 64.33% (193/300) positive samples while kinteoplastic PCR showed 84.66% (254/300) positive. For the first time in KP province, 2/784 sandflies trapped from the study villages was found positive for Leishmania by PCR. Restriction Fragment Length Polymorphism of patients and sandflies samples revealed L. tropica as the prevalent Leishmania species in this district. The results of sequencing and RFLP identified L. tropica in Phlebotomus sergenti. This is the first ever report of molecular identification of L. tropica from sandflies of genus P. sergenti in Khyber Pakhtunkhwa province. This data can be helpful for health authorities in finding out new CL foci and to plan effective strategies for the provision of health facilities to poor people of this area.