Identification of circulatory microRNA based biomarkers for early pregnancy diagnosis in buffalo.

Introduction: The most crucial factor in improving animal reproduction efficiency is early pregnancy diagnosis. Early diagnosis not only reduces the time interval between two calvings but also aids farmers in identifying open animals, thereby preventing significant milk production losses. Therefore, the objective of this study was to discover circulatory miRNAs that would be useful for early pregnancy diagnosis in buffalo. Material and methods: Blood samples were taken on 0, 6th, 12th, and 18th day after artificial insemination from pregnant animals (n = 30) and non-pregnant animals (n = 20). During these stages of pregnancy, total RNA was extracted, and a small RNA library was subsequently generated and sequenced on the Illumina platform. Subsequently, Real-time PCR was used to validate the findings. Results and discussion: There were 4,022 miRNAs found during the pregnancy, with 15 of those lacking sequences and 4,007 having sequences already in the database. From the beginning of pregnancy until the 18th day, 25 of these miRNAs showed a substantial shift in expression levels in the maternal blood, with a change more than two logs. Furthermore, based on qPCR results, 19 miRNAs were found to be more abundant in pregnant animals than in non-pregnant animals. We used target prediction analysis to learn how maternally expressed miRNAs relate to fetal-maternal communication. In conclusion, miRNA based biomarkers that could be associated with the diagnosis of pregnancy were identified including miR-181a and miR-486 highly upregulated on the 18th day of pregnancy. This study also provides a comprehensive profile of the entire miRNA population in maternal buffalo blood during the early stages of pregnancy.

Development of a multiplexed Luminex assay for simultaneous detection of enteric viruses in cattle.

Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 105, 1 × 104, and 1 × 105 RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

Rapid detection of porcine sapelovirus by reverse transcription recombinase polymerase amplification assay.

BACKGROUND: Porcine Sapelovirus (PSV) infection has been confirmed in pigs worldwide, mostly asymptomatic, but in some cases, it can lead to significant issues in the gastrointestinal, respiratory, neurological, or reproductive systems. PSV is considered an emerging pathogen of porcine species. Recombinase polymerase amplification (RPA) is a simple and fast isothermal technique that uses three enzymes for amplification without the use of any sophisticated equipment. METHODS AND RESULTS: The reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and optimized for field based detection of PSV. The assay was developed by targeting 5´UTR region of PSV genome and optimized for reaction time, temperature, primer and MgOAc concentration. The analytical sensitivity and specificity of assay was determined. The assay was evaluated on 85 porcine faecal samples collected from field. In addition to conventional format, this assay was also optimized for visual dye-based detection format and lateral flow strips based detection (in combination with probe). The developed assay works at constant temperature of 35 °C for 20 min with forward primer concentration 20pm, reverse primer concentration 10pm and MgOAc concentration of 14mM. This assay is highly sensitive and detects up to 28 copies of viral nucleic acid both in the conventional as well as in fluorescent dye based detection format. Using the newly developed assay 21 samples out of 85 samples were found positive, showing positivity rate of 24.7%. The positivity rate of RT-RPA assay corroborated with the gold standard RT-PCR test. CONCLUSIONS: This study presented the development of an RT-RPA isothermal assay for rapid and accurate detection of PSV. The assay is highly sensitive, specific, works at a low and constant temperature, does not require any high-end instrument and can be a potential diagnostics tool for pen-side testing of PSV in the field conditions. The newly developed RT-RPA assay could successfully detect PSV circulating in swine population of Haryana, India. This is a first report of this kind from the region.

High throughput Luminex beads based multiplex assay for identification of six major bacterial pathogens of mastitis in dairy animals.

Introduction: Bovine mastitis is caused by over 150 different microorganisms. Specific identification and quantification of multiple bacteria in a single milk sample becomes essential for rapid intervention. Methods: In the present study a Luminex beads based multiplex assay emphasizing on the precise identification of six major bacterial pathogens of mastitis was developed. Assay was developed in two triplex sets, triplex 1 comprised of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis while triplex 2 consisted of Staphylococcus aureus, E. coli and Klebsiella pneumoniae. Results: The analytical sensitivity was 10 6 copies per reaction mixture for all the six bacteria. A 100% analytical specificity was observed for simultaneous detection of these bacteria. Clinical milk samples from 100 bovine quarters were tested for validation. Discussion: The analytical sensitivity was similar to the findings reported earlier in real time PCR multiplex assay targeting the DNA of the 11 most common bacterial species or groups in mastitis. The analytical specificity of the optimized assay was 100% similar to reported earlier for simultaneous detection of Mycoplasma spp. and for seven entric viruses of humans.The developed assay indicates a concept proof of a rapid, cost effective high throughput diagnostic tool for identification of major bacteria causing mastitis.

Temporal analysis of circulating nucleic acid in early days of pregnancy in buffalo.

Improving reproductive efficiency in livestock relies mainly on the ability to detect pregnancy quickly and accurately. Recently, circulating nucleic acids (CNAs) have been exploited for prenatal diagnosis in humans and animals. In the current investigation, serum samples were collected from pregnant animals (n = 30) and non-pregnant animals (n = 20) on 0th, 6th, 12th, and 18th day post artificial insemination. Total DNA was isolated from these serum samples. Two CNA tags (Bov-B and ART2A) derived from repetitive sections of the bovine genome were amplified using DNA extracted from serum samples. The expression analysis of these CNAs was done using real-time polymerase chain reaction assay, and copy number of each tag was calculated in pregnant and non-pregnant animals. The average number of copies of Art2A increased approximately threefold (P < 0.01) from day zero of pregnancy (7,000 copies) to the day 18 of pregnancy (> 21,000). Similarly, BovB levels in the pregnant group increased significantly (approximately 2.9-fold) from day zero (93,900 copies) till day 18 (> 2, 72,310 copies) (P < 0.01). There was no significant change observed on the 6th and 12th day of pregnancy and on the 18th day in the non-pregnant animals. In conclusion, based on these findings, the defined cut-off value can distinguish between pregnant and non-pregnant animals with a sensitivity of nearly 80% and specificity of nearly 70%. It is possible to employ these two CNA tags as biomarkers for early detection of pregnancy in buffaloes.

Exploring the potential of invariable surface glycoprotein (ISG65) as promising antigen for diagnosis of Trypanosoma evansi infection.

Trypanosoma evansi, a hemoflagellate protozoan, leads to wasting disease, surra in livestock animals causing huge economic losses. Currently, the preferred assay for surra diagnosis is whole cell lysate (WCL) based ELISA, which requires the use of rodents for WCL preparation. To avoid use of laboratory animals, we used recombinant DNA technology to express T. evansi invariable surface glycoprotein (ISG) in E. coli. The potential of recombinant ISG65 (rISG65) as a diagnostic antigen was investigated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The results indicated that rISG65 reacted with horse T. evansi positive serum giving two bands of approximately 48 kDa and 96 kDa. T. evansi-specific antibodies were detected as early as 10 and 14 days post infection using immunoblot and indirect ELISA, respectively using rISG65 antigen. No cross-reactivity was observed in ELISA and immunoblot with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions were observed between 30 and 100 kDa in T. evansi isolate of horse origin indicating the existence of multiple copies of ISG protein in a single trypanosome. The recombinant ISG has proven to be good candidate antigen to be used in ELISA for serodiagnosis of T. evansi infection in different animals.

Prevalence of porcine viral respiratory diseases in India.

The pig industry is growing rapidly in India and contributes a major share of growth in the livestock sector. Over the last few years, there is a gradual increase in the adoption of pigs for production by economically weaker sections of the country. However, this production is affected by many respiratory diseases which are responsible for significant economic loss. The occurrence and impact of these diseases are still under-documented. The four important pathogens including porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza A viruses (SIV) and classical swine fever virus (CSFV) are documented here. These diseases are highly devastating in nature and frequent outbreaks have been reported from different parts of the country. The rapid and specific diagnosis, effective prevention and control measures are required for the eradication of these diseases which is urgently required for the growth of the pig industry. This review highlights the prevalence, epidemiology, diagnostics and information gaps on important respiratory viral pathogens of pigs reported from different parts of India. This review also emphasizes the importance of these viral diseases and the urgent need to develop vaccines and effective measures for the eradication of these diseases.

Molecular detection of respiratory avian mycoplasmosis associated bacterial and viral concurrent infections in the poultry flocks.

Respiratory tract infections are of serious concern to the poultry industry. The present study was aimed to delineate the extent of respiratory avian mycoplasmosis associated bacterial and viral concurrent infections in the poultry flocks. A total of 146 poultry flocks of Haryana and Rajasthan, India, suspected for chronic respiratory disease (CRD) were screened for avian mycoplasmas, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and avian pathogenic Escherichia coli (APEC) by conventional polymerase chain reaction (PCR) assays. A total of 49.31% (72/146) flocks were found positive for Mycoplasma infection. Of the Mycoplasma-positive flocks, 80.55% (58/72) represented pathogenic avian mycoplasmas (MG and/or MS), while 19.44% (14/72) flocks were positive for commensal avian mycoplasmas (other than MG and MS). A correlation was deduced between avian mycoplasmosis and bacterial and/or viral co-infections. The results revealed that 17.24% (10/58) flocks had only avian mycoplasmosis infection. However, in the remaining flocks, the avian mycoplasmosis was associated either with APEC infection [17.24% (10/58)], IBV infection [43.10% (25/58)], or both APEC and IBV infections [22.41% (13/58)], respectively. Further epidemiological studies on respiratory avian mycoplasmosis associated concurrent infections with other pathogens are recommended to assess circulating strains, risk factors, and economic losses.

In vitro and in vivo evaluation of kinase and protease inhibitors against Trypanosoma evansi.

Trypanosoma evansi is a causative agent of chronic wasting and fatal disease of livestock and wild animals known as surra. In this study, repurposing approach based on drug target was used to investigate the efficacy of kinase inhibitors (Barasertib-HQPA, BAR and Palbociclib isethionate, PAL) and protease inhibitors (Z-pro-prolinal, Z-PRO and Leupeptin hemisulphate, LEU) against T. evansi in HMI-9 medium. BAR, PAL and Z-PRO exhibited IC50 values of 13.52 µM, 0.6375 µM and 63.20 µM against T. evansi in terms of growth inhibition, in the contrary, LEU failed to exhibit a significant growth inhibition at any time interval. Furthermore, oligopeptidase B and aurora kinase genes of T. evansi were targeted to determine the effect of these drugs on quantitative mRNA expression, which showed significant (p < 0.01) up-regulation of both genes in the BAR and PAL-exposed population at 12 h of exposure, whereas, Z-PRO showed only significant (p < 0.05) up-regulation of aurora kinase gene at 12 h interval. In cytotoxicity assay, BAR exhibited 52% and 41% cytotoxicity at 50 µM concentration (about five folds the IC50 value) on equine PBMC's and Vero cell line, respectively. Similarly, the cytotoxicity of 25% and 24% were recorded at 10 µM concentration (about ten folds to the IC50 value) of PAL in equine PBMC's and Vero cell line, respectively. Of these, BAR and PAL, which were found effective under in vitro trials, raised the longevity of mice at higher doses during in vivo trials. Data generated showed that kinase inhibitors have higher potential to explore therapeutic molecules against surra organism.

Development of real-time RT-PCR systems for detection and quantitation of bovine enteric viral pathogens.

The enteric viruses in animals are responsible for severe and devastating losses to the livestock owners with a profound negative impact on animal, health, welfare, and productivity. These viruses are usually transmitted via the feco-oral route and primarily infect the digestive tract of the humans, bovines and different mammals as well as birds. Some of the important enteric viruses in ruminants are: Rotavirus A (RVA), Peste des petits virus (PPRV), Norovirus (NV), Bovine corona virus (BoCV) and Bluetongue virus (BTV). In the present study, sensitive, specific and reliable TaqMan probe-based RT-qPCRs were developed and standardized for the rapid detection and quantification of enteric viruses from fecal samples. The assays result in efficient amplification of the RVA, BTV and BoCV RNA with a limit of detection (LoD) of 5, 5 and 4 copies, respectively, which is 1000 times more sensitive than the traditional gel-based RT-PCR. The reproducibility of each assay was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. In conclusion, real time PCR developed for these viruses are highly specific and sensitive technique for the detection of diarrheic viral pathogens of cattle and buffalo.

An Insight on RNA Based Therapeutics and Vaccines: Challenges and Opportunities.

In this era, RNA molecules have provided a unique opportunity to researchers all over the world for expanding their range of targets in the development of drugs. Due to the unique pharmacological as well as physicochemical characteristics of different RNA molecules such as aptamers, small interfering RNAs (siRNA), antisense oligonucleotides (ASO) and guide RNAs (gRNA), they have emerged recently as a new class of drugs. They are used for selective action on proteins and genes that were not possible to target by conventional drug molecules. These RNA molecules like guide RNAs are also components of novel gene editing mechanisms which can modify the genome nearly in all cells. Vaccines based on RNA molecules have also provided a promising alternative to conventional live attenuated vaccines. RNA based vaccines have high potency, can be rapidly developed, and have potential for manufacturing at a cheaper rate and safe administration. However, the application of these RNAs has been restricted by the high instability and inefficient in vivo delivery. Technological advancement needs to overcome these issues so that RNA based drugs targeting several diseases can be developed. This article emphasizes the potential of RNA based drugs and the major barriers associated with the development of RNA therapeutics. Additionally, the role of RNA based vaccines and their challenges in advancing this promising vaccine platform for the prevention of infectious diseases have been discussed.

Comparative evaluation of indirect-ELISA and DOT blot assay for serodetection of Mycoplasma gallisepticum and Mycoplasma synoviae antibodies in poultry.

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is an economically important disease of the poultry industry. The present study was aimed to develop whole cell based indirect-ELISA (i-ELISA) and DOT blot assay (DOT-ELISA) as rapid, sensitive, specific and economical sero-detection tests for MG and MS. A total of 306 blood samples were collected from birds slaughtered at local meat shops of different districts of Haryana, India to detect MG and MS antibodies. Sonicated antigens prepared from freshly grown culture of MG and MS were used to develop i-ELISA and DOT blot assay. In i-ELISA, 50.32% and 61.76% serum samples were found to be positive for MG and MS antibodies, respectively. However in DOT blot assay, 41.83% and 53.92% serum samples were found positive for MG and MS antibodies, respectively. The relative diagnostic sensitivity and specificity of DOT-ELISA were measured considering i-ELISA as a reference test. The relative diagnostic sensitivity of the DOT blot assay was found to be 69.48% and 82.01%; whereas relative diagnostic specificity was 86.18% and 91.45% for the detection of MG and MS antibodies, respectively. The developed serological assays may be used as rapid and economical diagnostic tools for large scale screening of poultry sera for MG and MS antibodies.

In-silico Analysis of Angiotensin-converting Enzyme 2 (ACE2) of Livestock, Pet and Poultry Animals to Determine its Susceptibility to SARS-CoV- 2 Infection.

BACKGROUND: Novel coronavirus SARS-CoV-2 is responsible for the COVID-19 pandemic. It was first reported in Wuhan, China, in December 2019, and despite the tremendous efforts to control the disease, it has now spread almost all over the world. The interaction of SARSCoV- 2spike protein and its acceptor protein ACE2 is an important issue in determining viral host range and cross-species infection, while the binding capacity of spike protein to ACE2 of different species is unknown. OBJECTIVE: The present study has been conducted to determine the susceptibility of livestock, poultry and pets to SARS-CoV-2. METHODS: We evaluated the receptor-utilizing capability of ACE2s from various species by sequence alignment, phylogenetic clustering and protein-ligand interaction studies with the currently known ACE2s utilized by SARS-CoV-2. RESULT: In-silico study predicted that SARS-CoV-2 tends to utilize ACE2s of various animal species with varied possible interactions. The probability of the receptor utilization will be greater in horse and poor in chicken, followed by ruminants. CONCLUSION: Present study predicted that SARS-CoV-2 tends to utilize ACE2s of various livestock and poultry species with greater probability in equine and poor in chicken. The study may provide important insights into the animal models for SARS-CoV-2 and animal management for COVID- 19 control.

Detection of Leptospira in urine of apparently healthy dogs by quantitative polymerase chain reaction in Haryana, India.

BACKGROUND AND AIM: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira. The organism can spread through the urine of infected animals, which can get into water or soil and can survive there for weeks to months. The study was undertaken to detect the pathogenic Leptospira in healthy dogs' urinary shedding by real-time polymerase chain reaction (qPCR). MATERIALS AND METHODS: Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira. To detect the pathogenic Leptospira organisms in dogs' urinary shedding, 239 urine samples were collected from healthy dogs from April 2018 to March 2019 from different areas of Haryana. All the urine samples were processed for DNA extraction and qPCR technique was used to detect the presence of Leptospira. RESULTS: Out of 239 urine samples of dogs, none of the samples resulted in the detection of DNA of pathogenic Leptospira organisms. CONCLUSION: The present study indicated low risk of transmission of Leptospira organisms from dogs' urine to human beings in Haryana.

Drug-induced reactive oxygen species-mediated inhibitory effect on growth of Trypanosoma evansi in axenic culture system.

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)-mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC50 (50% inhibitory concentration) values 6.12 µM and 0.33 µM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system.

Bioengineered Polymer/Composites as Advanced Biological Detection of Sorbitol: An Application in Healthcare Sector.

Bioengineered polymers and nanomaterials have emerged as promising and advanced materials for the fabrication and development of novel biosensors. Nanotechnology-enabled biosensor methods have high sensitivity, selectivity and more rapid detection of an analyte. Biosensor based methods are more rapid and simple with higher sensitivity and selectivity and can be developed for point-of-care diagnostic testing. Development of a simple, sensitive and rapid method for sorbitol detection is of considerable significance to efficient monitoring of diabetes-associated disorders like cataract, neuropathy, and nephropathy at initial stages. This issue encourages us to write a review that highlights recent advancements in the field of sorbitol detection as no such reports have been published till the date. The first section of this review will be dedicated to the conventional approaches or methods that had been playing a role in detection. The second part focused on the emerging field i.e. biosensors with optical, electrochemical, piezoelectric, etc. approaches for sorbitol detection and the importance of its detection in healthcare application. It is expected that this review will be very helpful for readers to know the different conventional and recent detection techniques for sorbitol at a glance.

Molecular characterization and phylogenetic analysis of Trypanosoma evansi from Northern India based on 18S ribosomal gene.

Six Trypanosoma evansi isolates were collected from ponies (PH1 and PK6), camel (CB2), donkeys (DJ3 and DH4) and cattle (CK5) from different States of Northern India (Haryana, Rajasthan, Uttar Pradesh and Gujarat) for molecular characterization based on 18S rRNA gene. The 18S rRNA gene (2251 bp) of different isolates was amplified, cloned and custom sequenced separately. Based on sequence and phylogenetic analysis of all six isolates, collected from different hosts as well as geographical areas, were having high identity among Indian T. evansi strains (99.7%) and with other strains of T. evansi (99.2%) distributed worldwide. There is less genetic diversity among different salivarian strains of T. evansi except few nucleotide changes at significant locations in one Indian isolate of camel origin (CB2). All Indian T. evansi isolates were grouped in salivarian clade with high bootstrap values and remained far away from stercorarian clade having 88-90% nucleotide identity. The study will be helpful in understanding the evolutionary relationship, molecular epidemiology and variation in disease pathogenesis among different T. evansi strains. Further, more studies are required on large number of isolates collected from diverse host and geographical areas to reaffirm the present finding.

Recombinant interferon stimulated protein 15 (rISG15) as a molecular marker for detection of early pregnancy in Bubalus bubalis.

Early and accurate diagnosis of pregnancy in animals is important for improving the reproductive management of livestock. The buffalo (Bubalus bubalis) is the most important dairy animal in India, but there are reproductive problems resulting from extended calving interval and ovulation occurring in the absence of behavioral estrus. The lack of simple methods for early pregnancy diagnosis intensifies these problems. The present study, therefore, was conducted to ascertain the role of the interferon-stimulated gene, (ISG), 15 in pregnancy detection. The anti-ISG15 Mab based ELISA was developed that could be used for detecting pregnancy at 18 to 20 days after artificial insemination (AI). The ISG15 protein was isolated from a pregnant buffalo and was amplified, and cloned in Escherichia coli by using coding region primers. The ISG15 gene was expressed in the host Escherichia coli BL21 (DE3), and the protocol was standardized for optimum gene expression. Using immortal hybridoma (fused myeloma and B cells) cells, a highly specific and sensitive antibody, anti-ISG15 mAb, for detecting ISG15 (protein) in the serum of pregnant buffaloes was obtained. A blocking ELISA was developed using the anti-ISG15 mAb to detect pregnancy in buffalo within 18 to 21 days after AI. The ISG15 gene was upregulated (P < 0.05) in pregnant buffalo at 18 to 21 days of pregnancy. This assay has an overall diagnostic accuracy of 75.0%. It, therefore, is concluded that recombinant ISG15 retains the potential for detecting pregnancy in B. bubalis and may have applications in ELISA kits for pregnancy detection in closely related species.

Unraveling cryptic epizootiology of equid trypanosomosis in Punjab state of India by parasitological and sero-molecular techniques.

To unravel equid trypanosomosis caused by Trypanosoma evansi in Punjab state of India, a cross sectional study was designed by utilizing parasitological and sero-molecular tools with objective to assess the prevalence of T. evansi in association with various risk factors in all agroclimatic zones of Punjab state of India. Parasitological Romanowksy stained thin blood smears (RSTBS) to detect patent infection, molecular techniques polymerase chain reaction I (PCR I; TBR 1/2 primers; targeting minichromosomal satellite DNA of T. evansi), polymerase chain reaction II (PCR II; TR 3/4 primers; targeting variable surface glycoprotein region DNA of T. evansi) & LAMP (Loop mediated isothermal amplification) assay to detect latent infection and serological assays card agglutination test (CATT/T. evansi) & ELISA (Enzyme linked immunosorbent assay) to detect exposure status of trypanosomosis were utilized in the present study. A total 429 equid blood and serum samples from all the five agroclimatic zones of Punjab state tested by these techniques showed a prevalence of 1.39% (CL: 0-15.28) by RSTBS, 6.52% (10.94-45.09) by both TBR 1/2 PCR and LAMP assay, 5.82% (11.57-38.42) by TR 3/4 PCR, 15.15% (36.57-135.42) with CATT/T. evansi and 22.84% (17.77-840.22) with ELISA. Interpretation of various risk factors revealed that the donkey/mules population (RR = 5.46, 95% [CI] = 0.15-15.56) was found to be at higher risk of T. evansi infection predominantly at 'unorganized' farms (RR = 4.06, 95% [CI] = 0.12-4.51). Animal used for commercial purposes (RR = 3.25, 95% [CI] = 0.06-7.42), rearing of equids with other domestic animals (RR = 2.36, 95% [CI] = 0.10-17.11) and farms without application of fly repellant/insecticides/net (RR = 3.68, 95% [CI] = 0.08-5.94) made them more prone to the disease. This comprehensive report utilizing the classical, serological and molecular diagnostic tools for epidemiology of T. evansi establishes the endemic stability of this infection in all agro climatic zones of Punjab with LAMP assay to be a promisingly sensitive and specific technique for the diagnosis of T. evansi under isothermal conditions in field situations.