Sporadic Outbreaks of Equine Herpes Myeloencephalopathy in Punjab, India: A virological, serological and molecular investigation

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Data may be preliminary. 17 April 2025 V1 Latest version Share on Sporadic Outbreaks of Equine Herpes Myeloencephalopathy in Punjab, India: A virological, serological and molecular investigation Authors : Harnoor Kaur Dawra , Ashwani Sharma 0000-0002-9540-0503 [email protected] , Baldev Raj Gulati , Kuldip Gupta , and Adil Majid Bhat Authors Info & Affiliations https://doi.org/10.22541/au.174490966.68718242/v1 220 views 170 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background Equine Herpes Myeloencephalopathy (EHM) is a rare manifestation of Equine Herpesvirus-1 (EHV-1) infection. EHM outbreaks have been documented in Europe, North America, and New Zealand, no confirmed cases have been reported in India. Objectives The study presents the virological, clinic-pathological, and outcome data from two EHM outbreaks in Punjab, India. Study design Descriptive epidemiology Methods Two outbreaks of Equine Herpes Myeloencephalopathy (EHM) were reported from distinct agro-climatic zones of Punjab, India. This study documents the clinical, serological, molecular, and histopathological findings of the reported outbreaks. Blood and serum samples were used for virus neutralization test (VNT) to assess serological responses, and quantitative PCR (qPCR) for molecular detection of EHV-1. Additionally, nasal swabs were collected for virus isolation, and tissue samples from necropsied horses were tested for both virus isolation and qPCR. Relative quantification of EHV-1 DNA was performed using real-time PCR targeting gB gene (ORF33) Results In the first outbreak, three stallions showed variable clinical signs, including facial nerve paralysis, hind limb paresis, prolapsed penis, fever, hind limb swelling, and episodic seizures. The outbreak was confirmed serologically, with VNT titers greater than 1:4. The second outbreak began with a 7-year-old stallion showing fever, ataxia, and paresis, which collapsed. EHV-1 was detected in nasal and ocular swabs, and tissue samples of the dead animal. qPCR amplification using specific primers for the EHV-1 gB gene confirmed EHV-1 infection, distinguishing it from EHV-4. No other horses on the farm showed clinical signs of EHM, but one of four horses, a 6-year-old stallion, tested positive for EHV-1 with a VNT titer of 1:8. Main Limitation Small sample size Conclusion This study describes two sporadic outbreaks of Equine Herpesvirus Myeloencephalopathy (EHM) in India, which were limited to a small number of horses. Serological, molecular, virus isolation, and histopathological analyses confirmed the presence of EHV-1. Sporadic Outbreaks of Equine Herpes Myeloencephalopathy in Punjab, India: A virological, serological and molecular investigation AUTHORS AND AFFILIATIONS Harnoor Kaur Dawra 1,2 , Ashwani Kumar Sharma 2* , Baldev Raj Gulati 3 , Kuldip Gupta 4 , Adil Majid Bhat 2 Author Affiliations 1 Veterinary Officer, The Mahalaxmi Race Course, Mumbai, India - 400034 2 Department of Veterinary Medicine,Guru AngadDev Veterinary and Animal Sciences University, Ludhiana, Punjab-141004 3 Department of Veterinary Micobiology, National Research Center on Equines, Hisar, Haryana-125001 4 Department of Veterinary Pathology,Guru AngadDev Veterinary and Animal Sciences University, Ludhiana, Punjab-141004 KEYWORDS Horse, Herpes Virus-1, VNT, gB-qPCR, ataxia, nervous signs FUNDING INFORMATION There was no specific funding source for the study ACKNOWLEDGEMENTS The authors are highly thankful to the veterinary microbiology department of Indian Council of Agricultural Research – National Research Centre on equines, Hisar, Haryana, India for providing necessary laboratory support for diagnosis of EHV infection. CONFLICT OF INTERESTS . The authors declare no conflict of interest DATA INTEGRITY STATEMENT Ashwani Kumar Sharma had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of data analysis ETHICAL ANIMAL RESEARCH Not required for outbreak investigation INFORMED CONSENT Not applicable Were any of the horses described in this manuscript given quinolones, extended spectrum beta-lactam antimicrobials (such as 3rd/4th generation cephalosporins) or macrolides? No Does this study aim to investigate any aspect of quinolones, extended spectrum beta-lactam antimicrobials (such as unlicensed 3rd/4th generation cephalosporins) or macrolides? No DATA AVAILABILITY STATEMENT Open data sharing is not applicable to this article due to the qualitative nature of the data in this study. Background Equine Herpes Myeloencephalopathy (EHM) is a rare manifestation of Equine Herpesvirus-1 (EHV-1) infection. EHM outbreaks have been documented in Europe, North America, and New Zealand, no confirmed cases have been reported in India. Objectives The study presents the virological, clinic-pathological, and outcome data from two EHM outbreaks in Punjab, India. Study design Descriptive epidemiology Methods Two outbreaks of Equine Herpes Myeloencephalopathy (EHM) were reported from distinct agro-climatic zones of Punjab, India. This study documents the clinical, serological, molecular, and histopathological findings of the reported outbreaks. Blood and serum samples were used for virus neutralization test (VNT) to assess serological responses, and quantitative PCR (qPCR) for molecular detection of EHV-1. Additionally, nasal swabs were collected for virus isolation , and tissue samples from necropsied horses were tested for both virus isolation and qPCR . R elative quantification of EHV-1 DNA was performed using real-time PCR targeting gB gene (ORF33) Results In the first outbreak, three stallions showed variable clinical signs, including facial nerve paralysis, hind limb paresis, prolapsed penis, fever, hind limb swelling, and episodic seizures. The outbreak was confirmed serologically, with VNT titers greater than 1:4. The second outbreak began with a 7-year-old stallion showing fever, ataxia, and paresis, which collapsed. EHV-1 was detected in nasal and ocular swabs, and tissue samples of the dead animal. qPCR amplification using specific primers for the EHV-1 gB gene confirmed EHV-1 infection, distinguishing it from EHV-4. No other horses on the farm showed clinical signs of EHM, but one of four horses, a 6-year-old stallion, tested positive for EHV-1 with a VNT titer of 1:8. Main Limitation Small sample size Conclusion This study describes two sporadic outbreaks of Equine Herpesvirus Myeloencephalopathy (EHM) in India, which were limited to a small number of horses. Serological, molecular, virus isolation, and histopathological analyses confirmed the presence of EHV-1. Introduction Equine herpesvirus-1 (EHV-1) is reported worldwide in equine population and is cause of significant economic losses to the equine breeders 1.2 . The clinical manifestations of the infection by EHV-1 occur in respiratory, reproductive and nervous form 3 . The nervous form is the rare manifestation of EHV-1 virus infection in equines and is termed as Equine Herpes Myeloencephlopathy (EHM) 4 . EHM was first reported in 1966 and in the recent years, there has been an unexpected increase in the outbreaks of EHM 5.6. 7.8.9. and is therefore classified as a potentially emerging disease of equines by the US Department of Agriculture 10 Neurological signs of EHM are due to vestibule-cochlear and spinal cord lesions 11 and the clinical presentation of disease depends on focal or diffuse distribution of lesions in the CNS 12.13.14 . The prognosis of disease is directly related to the length of recumbency with an increase in development of complications in recumbent horses 15 . Due to variable clinical presentation, laboratory diagnosis remains central to the diagnosis of EHM. At present EHV-1 is detected in nasal swabs, buffy coat, blood and in post-mortem tissues of affected animals by PCR and virus isolation 10 . Recently there has been resurgence of EHM cases with outbreaks reported from different parts of the world 16.17.18.19.20.21 . EHM has been classified as emerging disease as it satisfies the requirement of emerging disease based on its more virulent nature and increased incidence with high case fatality rate 22 . To the authors knowledge there is only one report on EHM hafling horses in India that had been imported from Austria in 1982 23 . Due to emerging nature of EHM and its significance for equine industry more data is required to study the risk factors and latent infection in the equine population of Indian sub-continent. The present study reports the virological, clinic-pathological and outcome data of two outbreaks of EHM in different agro-climatic zones of Punjab province of India. Materials and Methods 2.1 Demography of study area and equine population The state of Punjab is located in North-western part of Indian sub-continent and shares its border with neighbouring state of Pakistan. The state is spread over an area of 50,363 square km and is located between latitude 31°04’18.48” North and longitude 75°24’16.92”. It comprises of five agro-climatic zones (Fig. 1) and has a rich biodiversity of equine population (34,000 horses) that are at risk of developing EHV-1 infection and due to movement of animals across the border these animals serve as potential source of transboundary transmission of disease 24 . 2.2 Outbreak I The first outbreak was reported in February 2019 at a farm in village Dhira Patra district Ferozepur, located in the Western Plain agro-climatic zone (Fig.1). At the time of outbreak the farm housed 22 non-descript stallions, including yearlings, adolescents, and adults ranging from 2 to 15 years of age. All the animals at the time of outbreak shared the paddocks and were in close contact. None of the horses were vaccinated against EHV-1 or any other disease. All the horses were kept under stall-fed conditions and were primarily used for riding. They were relatively mobile, moving between three adjacent districts that span the central and western plain zones of the state. 2.3 Outbreak II The second outbreak was reported 200 km east to first outbreak (Ferozpur district) in city of Chandigarh in the month of March 2019 (Fig. 1). The farm comprised of 34 Indian Thoroughbreds (24 stallions and 12 mares) that were between 7 to 20 years of age and were reared under stall fed conditions. These animals were used for various equestrian events like show jumping, tent pegging etc. None of the horses were vaccinated against EHV-1 or any other disease. Blood samples were collected randomly from five animals at the farm. 2.3 Sampling framework Following these outbreaks, four other randomly selected farms (Jalandhar, Ludhiana, Barnala and Hoshiarpur) from other three agro-climatic zones of Punjab, with different herd size and breed, were clinically and serologically investigated to ascertain the spread of the disease. There was no prior history of any type of infectious disease outbreaks in the last 5 years on these farms. Two of these farms comprised of athletic horses that were kept for participation in various equestrian events and were not used for breeding purposes. None of the horses were vaccinated against EHV (Fig. 1). 2.4 Sampling and processing protocol A total of 100 blood samples were collected aseptically from the jugular vein, with samples placed in EDTA-coated tubes for DNA isolation and glass vials for serum separation. The extracted DNA and serum samples were transported on ice to the National Research Center on Equines (NRCE) in Hisar, Haryana, India, and stored at -70°C for further processing. In addition, nasopharyngeal and ocular swabs were taken from the screened animals. Tissue samples, including the brain, spinal cord, lungs, liver, spleen, stomach, and kidneys, were collected from the horse that died of disease, and transported in a viral transport medium containing phosphate-buffered saline (pH 7.2-7.6) with antibiotics. 2.5 Virus isolation Virus isolation from swabs (nasal and ocular) and tissue suspension (10% w/v) was performed on confluent rabbit kidney (RK 13) cells cultured in Eagle’s Minimal Essential Medium (EMEM; Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% antibiotic-antimycotic solution containing 100 I.U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin-B (Sigma Aldrich, St. Louis, MO, USA) by incubation at 37 ◦ C in an atmosphere containing 5% CO2 for two days. The tissue culture was examined for appearance of cytopathic effects for 7 consecutive days. When CPE was not detected, the cells were passaged blindly three times before the samples were declared negative. 2.6 Virus Neutralization Test (VNT) Serum samples were submitted for initial virus neutralizing antibody (VNA) titres for EHV-1.The RK-13 cells suspension containing 10 5 cells/ml in growth medium was distributed, 100 μl per well, into entire 96-well tissue culture plates.The plate was incubated at 37 ◦ C in 5% CO2 for 4 days and results of CPE were recorded for the entire plate.The titer of the virus in absence and presence of EHV1 serum was calculated 25 and virus identity was confirmed if the virus titer differed more than 2 log10 using virus neutralization test. 2.7 qPCR for detection of EHV-1gB (gB-qPCR) Equine herpesvirus-1 DNA was extracted from 200 µl of secretions (nasopharyngeal and ocular), whole blood samples and cell culture supernatant using a DNeasy blood and tissue DNA extraction kit (Qiagen, India) according to the manufacturer’s instructions with a final DNA elution volume of 60 µl. The relative quantification of EHV-1 specific DNA was done by real time PCR (gB-qPCR) assay targeting gB gene (ORF33) using aQuantitect multiplex PCR NoRox Kit (Qiagen, Hilden, Germany) 26 . The reactions were conducted in a total volume of 20 µl. The reaction mixture included 2x Quantitect multiplex mastermix (10.5 µl), gB-qPCR/for (1.0 µl), gB-qPCR/rev (1.0 µl), gB-qPCR/probe (1.0 µl), template DNA (2.0 µl) and nuclease free water (5.0 µl). The reaction was set in automatic thermocycler with following conditions: initial denaturation at 95 ◦ C for 10 minutes; 40 cycles of denaturation 95 ◦ C(15 sec) and 40 cycles of annealing at 60 ◦ C (60 sec). 2.8 Histopathology Complete necropsy and histopathological examinations were conducted following the death of one horse. The formalin-fixed tissues were processed, embedded in paraffin wax, sectioned at 4 μm, stained with hematoxylin and eosin using standard techniques, and examined under a light microscope. 2.9 Ethical clearance No ethical clearance required to study the outbreak Results 3.1 Description of EHV-1 outbreaks and clinical presentation 3.1.1 Outbreak I The index case of the outbreak was retrospectively traced to a stallion that died six months (August 2018) ago due to development of neurological signs that progressed to complete paralysis. Another stallion at the same farm developed similar signs in December 2018) and collapsed. Since there were no reported cases of Equine Herpesvirus (EHV) in the local equine population, veterinarians and equine breeders did not consider EHV as a potential cause. As a result, affected animals were managed symptomatically without further diagnostic investigation. Subsequently in the month of February 2019 the farm again reported the cases with similar signs. Concerned about the situation, the local veterinarian referred the cases to the referral hospital at Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. 3.1.1.1Clinical presentation The three stallions, aged 2, 3, and 7 years, were affected and underwent clinical examination. The first stallion had developed facial nerve paralysis (flaccid lip, rotation of the nostril (Fig 2) and unilateral blindness. The second horse had facial nerve deficit (ptosis), hind quarter paresis and prolapsed of penis (underwent penectomy later). The third stallion had fever, swelling of hind limbs and episodic seizures (Fig. 2). 3.1.1.2 Diagnosis Diagnosis of EHV infection was confirmed by serum antibody titers in VNT. The antibody titer in three affected horses was greater than 1:4 (1/8; 1/8 and 1/12). No other horse in the farm had antibody titer in VNT ( Table 1) . 3.1.2 Outbreak II The index case in this outbreak was found to be a stallion aged 7 years that had clinical signs of fever, ataxia and paresis. 3.1.2.1 Diagnosis In this outbreak ante-mortem diagnosis was not possible as the animal collapsed before sample collection. However, virus was isolated from nasopharyngeal and ocular swabs, and tissue (brain, spinal cord, liver, kidney, stomach) samples of the dead horse. Virus isolation revealed EHV-1 specific cytopathic effects, such as rounding, increase in refractility and detachment of cells in cell monolayers (Fig. 3). Further tissue samples from different organs and nasopharyngeal swabs from dead horse showed amplification on qPCR using specific primers of EHV-1 gB, that allowed the discrimination between EHV-1 and EHV-4 (Fig 4). No other horse in the farm had clinical sign of EHM, however, out of four random horses tested one 6-year stallion was detected positive for EHV-1 with a titre of 1/8 by VNT ( Table 1) . 3.1.2.2 Histopathology Gross pathology did not reveal any specific lesions. However congestion was found in serosal blood vessels of stomach wall, cerebrum and cerebellum and cortico-medullary junction of both kidneys. The liver was enlarged and congested. Haemorrhages were present in the lungs (Fig. 5, 6, 7, 8,9). Histopathological examination revealed accumulation of glial cells around degenerating neuron (satellitosis) in the brain along with encephalomalacia. The liver was congested and also showed fatty changes along with coagulative necrosis. Interstitial pneumonia was seen in the lungs whereas superficial necrosis was evident in the stomach. Moreover, characteristic intra-nuclear inclusion bodies were seen in hepatocytes (Fig. 10, 11,12, 13, 14,15). 3.2 Screening of associated farms Four other equine farms from nearby areas were randomly selected to assess the spread of infection. Blood and serum samples were collected from 72 horses for qPCR and VNT, respectively. Three horses from (one each from district Mohali, Jalandhar and Hoshiarpur) tested positive on VNT with a titer of 1:8, 1:16 and 1:8, respectively ( Table 1) . 3.3 Haematological findings The hemoglobin, packed cell volume, total erythrocyte count, total leukocyte count and absolute neutrophilic count were significantly ( P <0.05) decreased in EHV positive horses as compared with healthy animals. There was significant (P<0.05) increase in mean platelet count and absolute lymphocytic count in EHV-1 affected horses (Table 2). Discussion Equine herpesvirus-1 is the most prevalent virus in horse populations throughout the world that causes substantial losses to equine breeders. It has been suggested that up to 80% of horses worldwide are latently infected with either EHV-1 or EHV-4 11 . Various outbreaks have been described in veterinary literature from Europe, North America and recently from New Zealand 27 however, there are no confirmed reports of EHM from Iceland, Africa, South America (except Argentina) or Asia with the exception of Japan 28 . In this study, we describe two outbreaks of EHM in equine population of Punjab province that is located in the North-western Indian sub-continent. The outbreaks were reported during the months of February and March. The outbreaks observed in our study were sporadic in nature affecting only limited farms in the region. EHM outbreaks have been reported in sporadic form from other parts of the world; in Europe thirteen outbreaks of EHM were reported in 11 years from 2009 to 2020 29 and nine outbreaks of EHM were reported in equine population of Neitherland during a period of four years (2009-2013) 27 . Similar results have been reported by other workers 30 . In contrast to previous studies 27.31 , which reported clustering of new EHM cases over time, in our study while the first outbreak exhibited cases developing over a period of time, no such clustering was observed during the second outbreak. The clinical signs were observed in 3 out of 22 horses (13.63%) during the first outbreak and in 1 out of 36 horses (2.78 %) during the second outbreak. The proportion of horses developing clinical signs of EHM varies significantly, with studies reporting a wide range of prevalence. For instance, in an investigation on two outbreaks of EHM the proportion of affected horses was found to be 15% in one outbreak and 3.1% in the other 30 . Similarly in an outbreak of EHM reported at show jumping competition 60 out of 752 (8%) exposed horses developing clinical signs of EHM 9 . Other studies have reported the incidence of EHM in exposed horses ranging from 2.2 to 78 %. 10.20.27.29.31.32. Clinical signs EHM is considered as a rare event that can occur as sequalae of EHV-1 respiratory form or in some outbreaks without respiratory signs 27.28 . In our study no prior history of respiratory signs were found. Most of the animals affected by EHV exhibited neurological signs, including hind limb ataxia and paresis, which progressed to posterior paralysis. The presence of hindquarter ataxia and paralysis is consistent with previous reports of EHV cases 28.33 . The clinical signs of ataxia, paresis and paralysis are due to damage to both the white and gray matter of the spinal cord. The damage could be symmetric to asymmetric, mild to severe and in some cases cause paralysis or tetraplegia 34 . The pelvic limbs are most commonly affected and may result in signs of urinary incontinence. In addition to the commonly observed neurological signs, other symptoms included facial nerve paralysis, which led to ptosis, drooping of the upper lip, and muzzle deviation. Facial nerve signs, however, are not consistently reported in all EHM outbreaks, and the reasons for this variability remain unclear. In an outbreak of EHM facial nerve paralysis was found in 2 out of 11 affected animals and ptosis in 1 animal during an EHM outbreak 33 . Signs of facial nerve paralysis have been observed in an reported outbreak of EHM in New Zealand 20 . Notably, fever was absent in all horses that developed neurological signs, as it is a nonspecific symptom of EHM. In an outbreak of EHM in aged equids fourteen horses developed an acute onset of neurological deficits, including ataxia, weakness, vestibular signs, urinary incontinence, and recumbency (EHM cases). Fever was not observed in any of the EHM cases 34 . In contrast, during an EHM outbreak in France fever was reported in all cases with neurological signs 10 . Others studies have also reported cases of EHM without fever 18.30 . Additionally, one horse experienced seizures before ultimately collapsing. Though rare, neurological signs such as seizures and blindness have been documented in EHM outbreaks and are attributed to encephalopathy 18 . Non-neurological signs were minimal, with only one horse exhibiting fever and limb edema. Previous studies on naturally occurring EHM outbreaks have reported variable non-neurological symptoms, including limb edema, coughing, and ocular discharge 10.30.31. Risk factors The age group of EHM affected equines was between 2 to 7 years. Our results are in concurrence with studies conducted by other workers 27.35.36 . Courouce et al. (2023) reported horses aged more than 9 years The resistance to infection in young animals could be associated with transfer of maternal virus neutralization antibodies 37 . There was higher proportions of males affected than females which is in contrast with what has been observed in other studies, where females (especially while pregnant or lactating) in naturally occurring EHM are at higher risk of developing neurological problems 27. 29.38.16). The outbreaks of EHM in our study occurred in winters. A strong seasonal clustering of outbreaks has been described previously with all outbreaks occurring between mid-November and mid-May 27 . Diagnostic testing Ante-mortem diagnosis of Equine Herpesvirus Myeloencephalopathy (EHM) is challenging, as no single test can provide a definitive diagnosis of EHV infection 26 . In our study, we utilized a combination of diagnostic methods to identify EHV infection, including quantitative PCR (qPCR), virus neutralization test (VNT), and viral isolation. VNT In the Virus Neutralization Test (VNT), a four-fold increase in titer between acute and convalescent serum is considered presumptive evidence of EHV-1 infection 13 . However, since the acute phase had already passed when the outbreak was reported, we collected paired serum samples from both clinically affected and healthy in-contact horses. This approach allowed us to detect seroconversion in both groups, providing indirect evidence of EHV-1 as the etiologic agent 13 . In the first outbreak, no healthy animal sero-converted; however, in the second outbreak, one out of four healthy animals showed sero-conversion. The lack of sero-conversion in the first outbreak may be attributed to the fact that the initial case occurred six months earlier, and in the rest of animals the antibody response might have waned. In our study, antibody titers ranged from 1/8 to 1/16. In contrast, during an EHM outbreak at a riding school reported antibody titers ranging from 1/16 to 1/256 31 . Since antibody titers are known to decline rapidly over time, the lower titers observed in our study may be attributed to the delayed collection of samples. qPCR PCR especially real time PCR is considered as a test of choice due to high sensitivity, specificity and abilty to detect small quantities of DNA 13.39 . In our study, qPCR results were positive only in tissue and blood samples of dead animal. Studies by other workers have reported a decrease in sensitivity of qPCR from 25% to 14% over the period of 22 days 9.26 . Virus Isolation and Characterization In our study, we could isolate the virus from the tissues of deceased animals in second outbreak. However, contrary to our expectations, viral isolation was not achieved from nasopharyngeal swabs or blood samples from animals that exhibited clinical signs and were positive in VNT. Isolation and identification of virus are the gold standard for the diagnosis of EHM 40 . Virus isolation is typically most successful in nasal secretions and blood during the early stages of EHV-1 infection 17.32 . In our study, however, nasal swabs and blood samples were collected after the onset of neurological signs, at which point viremia and nasal shedding might had already decreased. This could explain the failure to isolate the EHV-1 virus from our samples. Post-mortem findings Post-mortem examination, including histopathological analysis, is crucial for confirming the diagnosis of EHV-1 myeloencephalopathy during an outbreak 31 . In this study, all histopathological lesions in the brain and other organs exhibited primary vascular injury and secondary ischemic degeneration of the nervous tissue. Similar findings have been reported by other researchers 41.42 . Thrombosis and secondary ischemic degeneration were also observed as a result of EHV-1-induced myeloencephalitis, which manifested as endotheliotropism rather than specific neurotropism, distinguishing it from several other alpha herpesviruses 43 . Limitations The study had a limited sample size, and due to delayed reporting by the owner, the timeline of clinical sign development was not recorded. Conclusion This study describes two sporadic outbreaks of Equine Herpesvirus Myeloencephalopathy (EHM) in India, which were limited to a small number of horses. Serological, molecular, virus isolation, and histopathological analyses confirmed the presence of EHV-1. Additionally, latent infections were identified in clinically healthy animals, as positive samples were detected in asymptomatic individuals. EHV-1 infection is recognized as an emerging pathogen, and the findings of this study are significant in identifying risk factors and providing evidence-based recommendations for controlling the disease, including vaccination strategies, in the region. Credit Author contribution statement Harnoor Kaur Dawra – Clinical investigation and Sample collection, Ashwani Kumar Sharma – Conceptulization, development of methodology of work done and final editing of the draft Baldev Raj Gulati ,- Laboratory diagnosis and review of manuscript Kuldip Gupta –Histopathological analysis of tissue samples and review of manuscript and Adil Majid Bha t -Writing and reviewing of manuscript. References 1. Hussey GS, Goehring LS, Lunn DP, Hussey SB, Huang T, Osterrieder N, Powell C, Hand H, Holz C, Slater J. Experimental infection with equine herpesvirus type 1 (EHV-1) induces chorioretinal lesions. Vet Res. 2013; 44 :118. 2. 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Western Plain Ferozepur 22 3 (VNT positive) 1/8 1/8 1/12 Lip paralysis and facial nerve deficit (rotation of nostrils) Posterior paralysis, facial nerve deficit and prolapse of penis Fever, swelling of limbs and episodic seizures Undulating Plain Chandigarh 5 2 (1 PCR and 1 VNT) 1/8 Fever ataxia and paresis followed by death after 2 days Second horse had no clinical signs Mohali 11 1 (VNT positive) 1/8 No clinical signs Central Plain Jalandhar 34 1 (VNT positive) 1/16 No clinical signs Sub Mountain Undulating Hoshiarpur 17 1 (VNT positive) 1/8 No clinical signs Western Zone Barnala 11 - - Table 1: Summary of collected samples, clinical signs, and VNT titers of horses from various regions of Punjab, screened for EHV-1. Table 2 .Hematological findings of horses in EHV-1positive and negative cases. 1. Hb (g/dL) Positive 4 10.6±0.3 10.1-11.2 Negative 96 11.8±0.1 7.8-16.1 2. PCV (%) Positive 3 32.9±1.1 31.3-35.1 Negative 95 36.6±0.4 24.4-49.5 3. TEC (×10 6 /µL) Positive 3 7.0±0.3 6.3-7.3 Negative 95 7.3±0.1 4.2-9.7 4. Thrombocytes (×10 3 /µL) Positive 4 178±21 134-234 Negative 96 150±5 20-243 5. TLC (cells/µL) Positive 4 7598±1172 5990-11010 Negative 96 8934±205 4570-16790 6. Absolute neutrophil count (cells/µL) Positive 4 4890±640 3684-6606 Negative 96 6306±247 1923-15111 7. Absolute lymphocyte count (cells/µL) Positive 4 2653±528 1797-4184 Negative 96 2625±107 643-5164 8. Absolute eosinophil count (cells/µL) Positive 1 2.0±0.1 0-2 Negative 1 2.0±0.1 0.2 Information & Authors Information Version history V1 Version 1 17 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords ehm equine herpes virus myeloencephalopathy posterior paresis Authors Affiliations Harnoor Kaur Dawra Guru Angad Dev Veterinary and Animal Sciences University View all articles by this author Ashwani Sharma 0000-0002-9540-0503 [email protected] Guru Angad Dev Veterinary and Animal Sciences University View all articles by this author Baldev Raj Gulati National Research Centre on Equines View all articles by this author Kuldip Gupta Guru Angad Dev Veterinary and Animal Sciences University View all articles by this author Adil Majid Bhat Guru Angad Dev Veterinary and Animal Sciences University View all articles by this author Metrics & Citations Metrics Article Usage 220 views 170 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Harnoor Kaur Dawra, Ashwani Sharma, Baldev Raj Gulati, et al. Sporadic Outbreaks of Equine Herpes Myeloencephalopathy in Punjab, India: A virological, serological and molecular investigation. Authorea . 17 April 2025. 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