An appraisal of traditional knowledge of plant poisoning of livestock and its validation through acute toxicity assay in rats.

Background: Kashmir Himalaya hosts the most diverse and rich flora in the world, which serves as grazing land for millions of small ruminants in the area. While most plant species are beneficial, some can be poisonous, causing economic losses and animal health issues for livestock. Consequently, this study is the first comprehensive report on the traditional phyto-toxicological knowledge in District Muzaffarabad and the assessment of its authenticity through experimental studies in rats. Methods: The data regarding traditional knowledge was gathered from 70 key respondents through semi-structured interviews, which was quantitatively analyzed and authenticated through plant extract testing on Wistar female rats and comparison with published resources. Results: A total of 46 poisonous plant species belonging to 23 families and 38 genera were reported to be poisonous in the study area. Results revealed that leaves were the most toxic plant parts (24 species, 52.1%), followed by the whole plant (18 species, 39.1%), stem (17 species, 36.9%), and seeds (10 species, 21.7%). At the organ level, liver as most susceptible affected by 13 species (28.2%), followed by the gastrointestinal tract (15 species, 32.6%), nervous system (13 species, 8.2%), dermis (8 species, 17.3%), renal (7 species, 15.2%), respiratory (4 species, 8.7%), cardiovascular system (3 species, 6.5%), and reproductive system (2 species, 4.3%). The poisonous plant species with high Relative frequency citation (RFC) and fidelity level (FL) were Nerium oleander (RFC, 0.6; FL, 100), Lantana camara (RFC, 0.6; FL, 100), and Ricinus communis (RFC, 0.6; FL, 100). Experimental assessment of acute toxicity assay in rats revealed that Nerium oleander was the most toxic plant with LD50 of (4,000 mg/kg), trailed by Ricinus communis (4,200 mg/kg), L. camara (4,500 mg/kg), and Datura stramonium (4,700 mg/kg); however, other plants showed moderate to mild toxicity. The major clinical observations were anorexia, piloerection, dyspnea, salivation, tachypnea, constipation, diarrhea, tremor, itchiness, and dullness. Conclusion: This study showed that numerous poisonous plants pose a significant risk to the livestock industry within Himalayan territory, leading to substantial economic losses. Consequently, it is of utmost importance to conduct further comprehensive studies on the phytotoxicity of plants.

Omicron variants breakthrough infection elicited higher specific memory immunity than third dose booster in healthy vaccinees.

Homologous booster, heterologous booster, and Omicron variants breakthrough infection (OBI) could improve the humoral immunity against Omicron variants. Questions concerning about memory B cells (MBCs) and T cells immunity against Omicron variants, features of long-term immunity, after booster and OBI, needs to be explored. Here, comparative analysis demonstrate antibody and T cell immunity against ancestral strain, Delta and Omicron variants in Omicron breakthrough infected patients (OBIPs) are comparable to that in Ad5-nCoV boosted healthy volunteers (HVs), higher than that in inactivated vaccine (InV) boosted HVs. However, memory B cells (MBCs) immunity against Omicron variants was highest in OBIPs, followed by Ad5-nCoV boosted and InV boosted HVs. OBIPs and Ad5-nCoV boosted HVs have higher classical MBCs and activated MBCs, and lower naïve MBCs and atypical MBCs relative to both vaccine boosted HVs. Collectively, these data indicate Omicron breakthrough infection elicit higher MBCs and T cells against SARS-CoV-2 especially Omicron variants relative to homologous InV booster and heterologous Ad5-nCoV booster.

Molecular mechanisms of resistance and treatment efficacy of clofazimine and bedaquiline against Mycobacterium tuberculosis.

Clofazimine (CFZ) and bedaquiline (BDQ) are currently used for the treatment of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains. In recent years, adding CFZ and BDQ to tuberculosis (TB) drug regimens against MDR Mtb strains has significantly improved treatment results, but these improvements are threatened by the emergence of MDR and extensively drug-resistant (XDR) Mtb strains. Recently, CFZ and BDQ have attracted much attention for their strong clinical efficacy, although very little is known about the mechanisms of action, drug susceptibility test (DST), resistance mechanisms, cross-resistance, and pharmacokinetics of these two drugs. In this current review, we provide recent updates on the mechanisms of action, DST, associated mutations with individual resistance and cross-resistance, clinical efficacy, and pharmacokinetics of CFZ and BDQ against Mtb strains. Presently, known mechanisms of resistance for CFZ and/or BDQ include mutations within the Rv0678, pepQ, Rv1979c, and atpE genes. The cross-resistance between CFZ and BDQ may reduce available MDR-/XDR-TB treatment options. The use of CFZ and BDQ for treatment in the setting of limited DST could allow further spread of drug resistance. The DST and resistance knowledge are urgently needed where CFZ and BDQ resistance do emerge. Therefore, an in-depth understanding of clinical efficacy, DST, cross-resistance, and pharmacokinetics for CFZ and BDQ against Mtb can provide new ideas for improving treatment outcomes, reducing mortality, preventing drug resistance, and TB transmission. Along with this, it will also help to develop rapid molecular diagnostic tools as well as novel therapeutic drugs for TB.

In-vivo (Albino Mice) and in-vitro Assimilation and Toxicity of Zinc Oxide Nanoparticles in Food Materials.

Purpose: Recent advances in nanotechnology have given rise to the potential utilization of nanoparticles as food, nano-medicine/biomedicines. Patient: The study aimed to investigate the effects of nano-zinc oxide (nano-zinc) on the bio-assimilation of mineral (Zn) in mice, aged 3-6 weeks. Methods: ZnO nanoparticles were added to the basal diet as a supplement at amounts of 0.07, 0.14 and 0.21 mg/kg. The synthesized material was characterized by Fourier transform infrared spectrophotometer, particle size, scanning electron microscope, Thermogravimetric Analysis Thermal, X-ray diffraction spectrophotometer and Zeta potential. Results: In-vitro bioavailability of synthesized group ZnO (120 nm) was 43%, whereas for standard group ZnO (50 nm) was reported as 55%. In-vivo bioavailability of zinc oxide illustrated the maximum absorption level compared with the control. In-vivo toxicity was characterized as damage done to the liver and spleen tissues with a high dose of 0.21 mg/kg, while smaller doses indicated no toxic effects. Conclusion: The study provided important insights on the toxicological effects of ZnO nanoparticles, depending on dose rate and bio-assimilation, as well as particles, under various conditions (in-vitro and in-vivo). These findings will motivate further detailed research on nano-based medicine for alleviating malnutrition conditions.

Phytotoxicological study of selected poisonous plants from Azad Jammu & Kashmir.

Poisonous plants cause tremendous economic losses to the livestock industry. These economic losses are deterioration in their health, decreased productivity, deformed offspring, and reduced longevity. The current study is the first comprehensive report on poisonous plants of Azad Jammu and Kashmir which systematically documents the phytotoxicological effect and mode of action in livestock. The information was gathered from 271 informants including 167 men and 104 women through semi-structured interviews and literature search through available databases. The data collected through interviews was analyzed with quantitative tools viz. the factor informant consensus and fidelity level. A total of 38 species of flowering plants belonging to 23 families and 38 genera were reported. Family Asteraceae (5 spp) was the most dominant, followed by Solanaceae (4 spp), Fabaceae (4 spp), Euphorbiaceae (4 spp) and Convolvulaceae (3 spp). Among all the species collected, herbs were the dominant life form (22 spp, 57.89%), trailed by shrubs (11 spp, 28.95%), and trees (5 spp, 13.16%). Whole plant toxicity was reported to be the highest (15 spp, 39.47%), followed by leaf toxicity (12 spp, 31.58%), seed toxicity (4 spp, 7.89%), fruit toxicity (3 spp, 10.53%), latex toxicity (2 spp, 5.26%), flowers toxicity (1 spp, 2.63%), and berries toxicity (1 spp, 2.63%). The most toxic route of administration was found oral (39 spp, 40.63%), followed by intraperitoneal (24 spp, 25%), and intravenous (21 spp, 21.88%). The most commonly affected organ was found liver (20.41%), followed by gastrointestinal tract (20.341%), CNS (16.33%), skin (14.29%), kidneys (12.24%), lungs (4.04%), reproductive organs (2.04%), spleen (1.75%), blood (1.75%), heart (1.75%), urinary tract (1.75%), and pancreas (1.75%). The maximum Fic value was found for dermatological disorders (0.91), followed by the endocrine system (0.90), gastrointestinal (0.82), neurology (0.77), nephrology (0.67), cardiovascular (0.67), urinary (0.67), respiratory (0.60), sexual (0.60) disorders. Senecio vulgaris, and Ageratum conyzoides were the most important plants with fidelity level (0.95) and (0.87). Nerium oleander, Lantana camara, Leucaena leucocephala, and Ricinus communis were the important poisonous plant with maximum fidelity level (100%). Ricinus communis with reported lowest LD50 (<20 mg/kg) was the top-ranked poisonous plant followed by Lantana camara and Justicia adhatoda (25-50 mg/kg), Nerium Oleander (157.37 mg/kg), and Datura innoxia (400 mg/kg). We found that knowledge about poisonous plants is less prevailing in the rural areas of Azad Kashmir compared to the knowledge about medicinal plants and poisonous nature of reported plants is due to production of toxic substances and presence of essential oils.

The interactions of flaviviruses with cellular receptors: Implications for virus entry.

Flaviviruses are a group of enveloped viruses that enter the host cells through receptor-mediated endocytosis. The entry of flaviviruses into the cells is a multi-step process which involves several host factors that trigger the uptake of the virus. The initial step in the virus life cycle is the interactions between viral envelope proteins and the specific receptors on the surface of host cell. To date, several receptors have been identified such as glycosaminoglycans, tight junction proteins, laminin receptor and phosphatidylserine receptors. Moreover, the viruses may utilize integrins and C-type lectin receptors on the surface of host cells as the initial attachment factors. This mini-review will focus on recent progresses in the understanding of virus attachment, internalization, and membrane fusion with specific emphasis on the cellular receptors.

Immunity against Delta and Omicron variants elicited by homologous inactivated vaccine booster in kidney transplant recipients.

Background: A third mRNA vaccine booster is recommended to improve immunity against SARS-CoV-2 in kidney transplant recipients (KTRs). However, the immunity against SARS-CoV-2 Ancestral strain and Delta and Omicron variants elicited by the third dose of inactivated booster vaccine in KTRs remains unknown. Methods: The blood parameters related to blood cells count, hepatic function, kidney function, heart injury and immunity were explored clinically from laboratory examinations. SARS-CoV-2 specific antibody IgG titer was detected using an enzyme-linked immunosorbent assay. Cellular immunity was analyzed using interferon-γ enzyme-linked immunospot assay. Results: The results showed that there were no severe adverse effects and apparent changes of clinical laboratory biomarkers in KTRs and healthy volunteers (HVs) after homologous inactivated vaccine booster. A third dose of inactivated vaccine booster significantly increased anti-Ancestral-spike-trimer-IgG and anti-Ancestral-receptor binding domain (RBD)-IgG titers in KTRs and HVs compared with the second vaccination. However, the anti-Delta-RBD-IgG and anti-Omicron-RBD-IgG titers were significantly lower than anti-Ancestral-RBD-IgG titer in KTRs and HVs after the third dose. Notably, only 25.6% (10/39) and 10.3% (4/39) of KTRs had seropositivity for anti-Delta-RBD-IgG and anti-Omicron-RBD-IgG after booster, which were significantly lower than HVs (anti-Delta-RBD-IgG: 100%, anti-Omicron-RBD-IgG: 77.8%). Ancestral strain nucleocapsid protein and spike specific T cell frequency after booster was not significantly increased in KTRs compared with the second dose, significantly lower than that in HVs. Moreover, 33.3% (12/36), 14.3% (3/21) and 14.3% (3/21) of KTRs were positive for the Ancestral strain and Delta and Omicron spike-specific T cells, which were significantly lower than HVs (Ancestral: 80.8%, Delta: 53.8%, and Omicron: 57.7%). Conclusions: A third dose of inactivated booster vaccine may significantly increase humoral immunity against the Ancestral strain in KTRs, while humoral and cellular immunity against the Delta and Omicron variants were still poor in KTRs.

A recombinant selective drug-resistant M. bovis BCG enhances the bactericidal activity of a second-line anti-tuberculosis regimen.

Drug-resistant tuberculosis (DR-TB) poses a new threat to global health; to improve the treatment outcome, therapeutic vaccines are considered the best chemotherapy adjuvants. Unfortunately, there is no therapeutic vaccine approved against DR-TB. Our study assessed the therapeutic efficacy of a recombinant drug-resistant BCG (RdrBCG) vaccine in DR-TB. We constructed the RdrBCG overexpressing Ag85B and Rv2628 by selecting drug-resistant BCG strains and transformed them with plasmid pEBCG or pIBCG to create RdrBCG-E and RdrBCG-I respectively. Following successful stability testing, we tested the vaccine's safety in severe combined immune deficient (SCID) mice that lack both T and B lymphocytes plus immunoglobulins. Finally, we evaluated the RdrBCG's therapeutic efficacy in BALB/c mice infected with rifampin-resistant M. tuberculosis and treated with a second-line anti-TB regimen. We obtained M. bovis strains which were resistant to several second-line drugs and M. tuberculosis resistant to rifampin. Notably, the exogenously inserted genes were lost in RdrBCG-E but remained stable in the RdrBCG-I both in vitro and in vivo. When administered adjunct to a second-line anti-TB regimen in a murine model of DR-TB, the RdrBCG-I lowered lung M. tuberculosis burden by 1 log10. Furthermore, vaccination with RdrBCG-I adjunct to chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG-I showed almost the same virulence as its parent BCG Tice strain in SCID mice. Our findings suggested that the RdrBCG-I was stable, safe and effective as a therapeutic vaccine. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.

TB47 and clofazimine form a highly synergistic sterilizing block in a second-line regimen for tuberculosis in mice.

Multidrug-resistant tuberculosis (MDR-TB) remains a serious public health threat worldwide. To date, the anti-TB activity of TB47 (T), an imidazopyridine amide class of antibiotics targeting QcrB in the electron transport chain, has not been systematically evaluated, especially in a new regimen against MDR-TB. This study employed both macrophage infection and a mouse model to test the activity of T alone or in combination with other antimicrobial agents. Different regimens containing amikacin (A), levofloxacin (L), ethambutol (E), and pyrazinamide (Z) + clofazimine (C)/T were evaluated in the mouse model. The bacterial burdens of mice from different groups were monitored at different time points while relapse was assessed 6 months after treatment cessation. Colonies obtained at relapse underwent drug susceptibility testing. We found that T exhibited highly synergistic bactericidal activity with C in all models. Adding T to ALEZC might shorten the MDR-TB treatment duration from ≥ 9 months to ≤ 5months, as five months of treatment with ALEZCT achieved zero relapse rates in 2 animal experiments. These findings indicate that T exhibits a highly synergistic sterilizing activity when combined with C. All isolates from relapsing mice remained sensitive to each drug, suggesting that the relapse was not due to drug resistance but rather associated with the type of regimen.

Rapid, serial, non-invasive quantification of Pseudomonas aeruginosa in live mice with a selectable marker-free autoluminescent strain.

Pseudomonas aeruginosa is an increasingly prevalent pathogen that has become a serious health concern due to an increasing incidence of multidrug-resistant (MDR) hospital-acquired infections. The emergence of MDR-P. aeruginosa coupled with shrinking antibiotic pipelines has increased the demand for new antimicrobials and therapeutics. An effective tool for drug screening both in vitro and in vivo can facilitate the discovery of drugs and regimens for treating P. aeruginosa infection. Here, for the first time, we combined the mini-Tn7 system and Xer/dif recombinase system to construct a stable and selectable marker-free autoluminescent P. aeruginosa (SfAlPa) by one step. Afterwards, in vitro and in vivo activities of several antibiotics including amikacin, biapenem, levofloxacin and polymyxin B were assessed using SfAlPa. This study demonstrated that the use of SfAlPa could significantly facilitate rapid real-time evaluating the activities of compounds. Compared to prevailing methods, this method reduces the time, effort, animals and costs consumed in the discovery of new drugs against P. aeruginosa. Additionally, the methodology described in this study could be easily modified for construction of selectable marker-free reporter strain in other Gram-negative bacteria.

In vitro Impact of Yeast Expressed Hybrid Peptide CATH-2TP5 as a Prophylactic Measure Toward Sepsis and Inflammation.

CATH-2TP5 is a linear cationic hybrid peptide, consequent from naturally occurring antimicrobial peptide (AMPs) Cathelicidin-2 (CATH-2) and Immunomodulatory peptide Thymopentin (TP5) having dynamic and potent anti-inflammatory activities without hemolytic effect. The biocompatible mechanism of CATH-2TP5 is favored to explore new methodologies in the direction of biomedical applications. In this retrospectively study, an antiendotoxin and anti-inflammatory hybrid peptide CATH-2TP5 was emulated into pPICZα-A and successfully expressed in Pichia pastoris (P. pastoris). The recombinant CATH-2TP5 was purified through the Ni-affinity column and reversed-phase HPLC. The purified CATH-2TP5 peptide exhibited robust anti-endotoxin activity and significantly (p < 0.05) neutralized the effect of lipopolysaccharide (LPS). Furthermore, the down-regulated effect of CATH-2TP was more pronounced (p < 0.05) on LPS-induced cytotoxic effects, nitric oxide secretion and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in murine RAW264.7 macrophages. As associated to control and parental peptide the number of apoptotic cells was also contracted with the treatment of CATH-2TP5. Thus, we concluded that CATH-2TP5 peptide may be used in various biomedical applications as a therapeutic drug.

Molecular typing and genomic characteristic of human adenoviruses in Datong, Northern China.

The molecular prevalence of human adenoviruses (HAdVs) in Datong city and molecular evolution of HAdV-C species is still obscure. Here, we explored the molecular prevalence of HAdVs by simultaneous sequencing of hexon and fiber. Then, the penton gene fragments of HAdV-C species were determined by sequencing. Finally, genomic and proteotyping analysis were performed for exploration of molecular evolution of unique HAdV-6. Our results showed that dominant molecular types of HAdVs were HAdV-3, HAdV-2, and HAdV-1 based on the hexon and fiber genotype. Among H2F2 isolates, P1H2F2 was most common, followed by P2H2F2 and HAdV-89. The clinical symptoms of HAdV-1 or HAdV-2 infected patients were more severe than HAdV-3 infected patients, the prognosis of HAdV-1, HAdV-2, and HAdV-3 infected patients was indifference. Genomic and proteotyping analysis demonstrated that DT15 was different from HAdV-6 prototype, with high-discrepant sequences localized in the E3 region. In conclusion, HAdV-1 and HAdV-2 have a high affinity to infect younger children and cause more severe symptoms than HAdV-3. The E3 gene of HAdV-C species was considered as highly recombination and mutation region.

A Hybrid Peptide DEFB-TP5 Expressed in Methylotrophic Yeast Neutralizes LPS With Potent Anti-inflammatory Activities.

DEFB-TP5 is a novel auspicious health-beneficial peptide derivative from two naturally occurring peptides, ß-Defensin (DEFB) and thymopentin (TP5), and shows strong anti-inflammatory activity and binds to LPS without cytotoxicity and hemolytic effect. Furthermore, the application of DEFB-TP5 peptide is inadequate by its high cost. In the current study, we developed a biocompatible mechanism for expression of the DEFB-TP5 peptide in Pichia pastoris. The transgenic strain of hybrid DEFB-TP5 peptide with a molecular weight of 6.7kDa as predictable was obtained. The recombinant DEFB-TP5 peptide was purified by Ni-NTA chromatography, estimated 30.41 mg/L was obtained from the cell culture medium with 98.2% purity. Additionally, The purified DEFB-TP5 peptide significantly (p< 0.05) diminished the release of nitric oxide (NO), TNF-α, IL-6, IL-1ß in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner. This study will not only help to understand the molecular mechanism of expression that can potentially be used to develop an anti-endotoxin peptide but also to serve as the basis for the development of antimicrobial and anti-inflammatory agents as well, which also provides a potential source for the production of recombinant bioactive DEFB-TP5 at the industrial level.

Dysregulated NF-κB signal promotes the hub gene PCLAF expression to facilitate nasopharyngeal carcinoma proliferation and metastasis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is common in Southern China. The molecular mechanism underlying NPC genesis and progression has been comprehensively investigated, but the key gene (s) or pathway (s) pertaining to NPC are unidentified. METHODS: We explored some key genes and pathways involved in NPC through using meta-analysis of deposited expression of microarray data of NPC. The expression of proliferating cell nuclear antigen clamp associated factor (PCLAF) was determined by real-time PCR and western blots. CCK-8 assay, colony formation assay, transwell migration assay, cell wound healing assay, cell cycle analysis and cell apoptosis were carried out to assess biological behaviors caused by downregulation and overexpression of PCLAF in vitro. CHIP was utilized to determine the direct upstream regulatory transcription factors of PCLAF. RESULTS: PCLAF was the key gene of NPC, which was significantly up-regulated in NPC cell line compared to the normal nasopharyngeal cell line. Additionally, in vitro assay has demonstrated the down-regulation and overexpression of PCLAF, resulted in significantly suppressed and enhanced NPC proliferation, metastasis and invasion respectively. Furthermore, the up-regulation of PCLAF in NPC is induced by direct binding of dysregulated NF-κB p50/RelB complex to the promoter of PCLAF. CONCLUSION: Our results offer a strategy for re-using the deposited data to find the key genes and pathways involved in pathogenesis of cancer. Our study has provided evidence of supporting the role of PCLAF in NPC genesis and progression.

Effects of Subcutaneous Ochratoxin-A Exposure on Immune System of Broiler Chicks.

Ochratoxin A (OTA), an immunosuppressive mycotoxin, can increase the risk of many infectious diseases and contribute to economic losses to the poultry industry. The immunosuppressive effect has mainly been investigated through oral exposure; however, birds may also be contaminated through skin absorption. The present study investigated the influence of OTA exposure on the defense system of broiler chicks through the subcutaneous route and including low doses. Groups of broiler chicks (Cobb), 05 days old, were exposed to subcutaneous inoculation of OTA at concentrations of 0.1; 0.5; 0.9; 1.3; and 1.7 mg OTA/kg body weight. The size of the lymphoid organs, circulating immune cells, and total IgY and IgA levels were evaluated 21 days post inoculation. Subcutaneous OTA exposure decreased the weight of the thymus, spleen, and bursa of Fabricius, and leukocytopenia (p < 0.05) was detected in chicks of the OTA treated groups. In a dose-dependent way, decreased levels of circulating lymphocytes and heterophils (p < 0.05), and increased levels of monocytes (p < 0.05) were detected. Decreased IgY and IgA serum concentrations were noted in the OTA treated groups (p < 0.05). In conclusion, subcutaneous OTA exposure induces immunosuppression even at low levels.

Dietary quercetin maintains the semen quality in rabbits under summer heat stress.

This study focused to determine beneficial impact of feeding quercetin supplemented diet on semen quality in summer heat imposed rabbits. Twelve heat stressed (HS) adult rabbits bucks were either fed with basal diet (HS; n = 06) or quercetin supplemented diet (QU-HS; n = 06) for a period of 56 days. Semen samples were collected and evaluated for volume, osmolality, morphology, concentration, motility, motion kinetics, viability, acrosome integrity, mitochondrial potential, and seminal plasma MDA level. Semen volume, concentration, motility and sperm kinetics parameters were affected by diet supplementation. Diet affected the sperm mitochondrial potential and day of treatment affected the viable sperm percentage. There was an effect of diet, day of treatment and diet by day interaction on acrosome reaction rate. Sperm head abnormalities were influenced by diet provision, sperm mid-piece abnormalities were affected by diet and day of treatment, whereas, the effect of diet and diet by day of treatment interaction were observed for total sperm abnormalities. There was an effect of diet and diet by day interaction for seminal plasma MDA level. In conclusions, quercetin reduces the damaging effects of HS and maintains the semen quality by lowering the oxidative stress in rabbits.

A study on prevalence and molecular characterization of trypanosomal species infecting equines in Lahore region, Pakistan.

Trypanosomiasis is an important protozoal disease with a diverse range of susceptible host including human. In the current study, molecular characterization of prevalent species was done through a pan-trypanosome polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). A total of three hundred (n = 300) equines including horses, donkeys and mules (100 each) were randomly selected and the equine blood samples were subjected to screening for trypanosomes through microhaematocrit centrifuge technique (MHCT), conventional PCR, semi-nested PCR and RFLP. Overall prevalence of trypanosomal species was 8% (24/300) as revealed by MHCT and species wise prevalence in horses, donkeys and mules was 4.33% (13/300), 1.33% (4/300) and 2.33% (7/300), respectively. Conventional and semi-nested PCR depicted an overall prevalence of 21% (63/300) and species wise prevalence in horses, donkeys and mules was 12% (36/300), 3.67% (11/300) and 5.33% (16/300), respectively. RFLP analysis of the semi-nested products, using Msp1 and Eco571 enzymes, negated the presence of T. congolense, T. brucei, T. vivax, T. theileri, and T. vivax in the positive samples and revealed that the animals might be suffering from T. evansi infection as the enzymes used were not able to detect this species. This hypothesis was further confirmed by using T. evansi specific primers which depicted all of the 63 samples were positive for T. evansi. It is inferred that T. evansi is the major trypanosome species prevalent in equines. Furthermore, PCR is more sensitive as compared to microscopic examination and the pan-trypanosome PCR-RFLP assay is suitable for carrying out laboratory diagnosis of field samples and epidemiological studies. Further studies on the possibilities of use of other restriction enzymes may help to improve the species specificity of the assay.