ISSN: 2320-8694

Journal of Experimental Biology and Agricultural Sciences

Volume 4 | Issue (Spl.4.EHIDZ)| Dec, 2016

Special Issue on: EQUINE HEALTH, INFECTIOUS DISEASES AND ZOONOSIS (EHIDZ)

Lead Guest Editor Sandip Kumar Khurana Principal Scientist, National Research Centre on Equines, India – 125001

Guest Editor Amarpal Principal Scientist, Indian Veterinary Research Institute, India - 243122 Yashpal S. Malik Principal Scientist, Indian Veterinary Research Institute, India - 243122 Kuldeep Dhama Principal Scientist, Indian Veterinary Research Institute, India - 243122 K. Karthik Assistant Professor, Tamil Nadu Veterinary & Animal Sciences University, India Minakshi Prasad Professor, College of Veterinary Sciences, India - 125001

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Volume No – 4 Issue No – Spl-4-EHIDZ December, 2016 Journal of Experimental Biology and Agricultural Sciences

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JEBAS Technical Editors

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Dr. B L Yadav Head – Botany, MLV Govt. College, Bhilwara, India E mail: [email protected]

Dr. Yashpal S. Malik ICAR-National Fellow, Indian Veterinary Research Institute (IVRI) Izatnagar 243 122, Bareilly, Uttar Pradesh, India E mail: [email protected]; [email protected]

Dr. K L Meena Lecturer – Botany, MLV Govt. College, Bhilwara, India E mail: [email protected]

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Dr. Neeraj Associate Professor and Head, Department of Botany Feroze Gandhi College, RaeBareli, UP, India

Dr.Md.Moin Ansari Associate Professor-cum-Senior Scientist, Division of Surgery and Radiology Faculty of Veterinary Sciences and Animal Husbandry Shuhama, Srinagar-190006, J&K, India

JEBAS Associate Editors

Dr Biswanath Maity Carver College of Medicine, Department of Pharmacology University of Iowa – Iowa, City, USA Email: [email protected]

Wu Yao Senior Manager, China Development Bank, ChaoYang District Beijing, China Email: [email protected]

Auguste Emmanuel ISSALI Forestry Engineer, Head - Coconut Breeding Department at Marc Delorme Coconut Research Station, Port Bouet, Côte d’Ivoire, Regional Coordinator -COGENT Email: [email protected]

Dr. Omoanghe S. Isikhuemhen Department of Natural Resources & Environmental Design, North Carolina Agricultural & Technical State University, Greensboro, NC 27411, USA Email: [email protected]

Dr. Vincenzo Tufarelli Department of Emergency and Organ Transplantation (DETO), Section of Veterinary Science and Animal Production, University of Bari ‘Aldo Moro’, s.p. Casamassima km 3, 70010 Valenzano, Italy Email: [email protected]

Dr. Sunil K. Joshi Laboratory Head, Cellular Immunology Investigator, Frank Reidy Research Center of Bioelectrics, College of Health Sciences, Old Dominion University, 4211 Monarch Way, IRP-2, Suite # 300, Norfolk, VA 23508 USA Email: [email protected]

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Dr. Bilal Ahmad Mir Department of Genetics, University of Pretoria, South Africa-0002 E mail: [email protected]; [email protected]

Dr. Amit Kumar Jaiswal School of Food Science and Environmental Health, College of Sciences and Health Dublin Institute of Technology, Dublin 1, Ireland E mail: [email protected]

Dr. Gurudayal Ram Assistant Professor Jacob School of Biotechnology and Bioengineering (JSBB), Sam Higginbottom Institute of Agriculture, Technology and Sciences(SHIATS), Allahabad, Uttar Pradesh – 211007

Rajveer Singh Chauhan Division of Phycology, Department of Botany, University of Lucknow, Lucknow, INDIA E-mail: [email protected]

Y. Norma-Rashid (Norma Yusoff) Professor Institute of Biological Sciences – Faculty of Science, University of Malaya, 50603 Kuala Lumpur MALAYSIA E-mail: [email protected]

Dr.Peiman Zandi Department of Agronomy, I.A.University, Takestan branch,Takestan,Iran E-mail: [email protected]

Dr. Oadi Najim Ismail Matny Assistant Professor – Plant pathology, Department of Plant Protection College Of Agriculture Science, University Of Baghdad, Iraq E-mail: [email protected], [email protected]

Dr. Girijesh K. Patel Post Doc Fellow, 1660 Springhill Avenue, Mitchell Cancer Institute University of South Alabama, USA E-mail: [email protected]

Dr Anurag Aggarwal MD, DA, PDCC (Neuroanesthesia and Intensive Care), India E-mail: [email protected]

Dr Ayman EL Sabagh Assistant professor, agronomy department, faculty of agriculture kafresheikh university, Egypt

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From the Desk of Guest Editors ______

Dear Authors,

Equines include horses, donkeys, mules, zebra, and their hybrids. The utility of equines exists for transportation, agricultural, riding, sports, racing, equestrian events, army, police services, brick kilns, companions and ceremonial purposes. Equines are very sensitive and vulnerable to infectious diseases and require very good managemental practices in comparison to other animal species. There are several existing and emerging equine pathogens having zoonotic potential, which are threat to public health. Latest information relating to equine health management and infectious diseases with emphasis on diseases prevalence, surveillance, monitoring and strategic approaches in diagnosis, prevention, control and treatment of equine infectious diseases and their zoonotic aspects, is essentially required. Novel molecular diagnostics techniques, development of improved therapeutics and immunomodulatory strategies, vaccines and package of practices for prevention, control and eradication of infectious diseases are relevant today for effective prevention and control of equine infectious diseases. This issue is aimed for the benefit of equine veterinarians, equine researchers, other equine health care specialists and all other professionals and persons related with animal health in general.

This special issue is published with nine articles including eight review articles and one research article. The review on ‚Leptospirosis in horses: special reference to equine recurrent uveitis‛ provides an insight in to Leptospirosis, a bacterial zoonotic disease affecting several domestic and wild animal species. Special emphasis is on ocular and systemic manifestations in horses. The ocular manifestation of leptospirosis in equines, the equine recurrent uveitis (ERU) has been dealt in detail.

Another review on ‚Emergence of equine herpes virus 1 myeloencephalopathy: A brief review‛ elaborates the nervous form of EHV1, equine herpes myeloencephalopathy (EHM). The review describes the genetics, epidemiology, pathogenesis, treatment, prevention and control of EHM along with host, agent and environmental factors which play a role in the development of EHM.

The review on ‚Equine ocular setariasis and its management‛ enriches the knowledge regarding etiology, diagnosis and management of ocular setariasis in equine species. The common post operative complications along with their management are also discussed.

The research article ‚Parasitological, biochemical and clinical observations in ponies experimentally infected with Trypanosoma evansi‛ describes the observations on experimentally infected ponies with Trypanosoma evansi. Serum urea, uric acid, triglyceride, cholesterol, bilirubin indirect (BID) and total bilirubin (BIT) contents increased, while albumin contents significantly decreased indicating impairment of liver and kidney functions.

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The review on ‚Equine brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control‛ describes equine brucellosis, its epidemiology, pathogenesis, clinical signs along with appropriate prevention and control strategies with special mention of two important conditions in equines namely poll evil and fistulous withers.

The review on ‚Biotechnological tools for diagnosis of equine infectious diseases‛ focuses on biotechnological tools available for equine diseases diagnosis and its applications which hold great promise for improving the speed and accuracy of diagnostics for equine infectious diseases needed to promote optimal clinical outcomes and general public health.

The review ‚Evolving views on enteric viral infections of equines: an appraisal of key pathogens‛ discusses the current status of key enteric viruses that cause diarrheic disorders in foals and horses and their public health aspects.

The review ‚Lyme borreliosis in the horse: A mini-review‛ discusses beautifully the Lyme borreliosis which is a multisystemic tick borne disease and converses with the disease etiology, pathobiology, disease management and public health aspects.

The review ‚An overview of ozone therapy in equine- an emerging healthcare solution‛ unfolds the significance of ozone therapy in equine medicine providing an insight into the mechanism of action of ozone therapy and various conditions where ozone therapy could be used in equines.

The information compiled in this special issue will be useful for equine researchers, equine veterinary professionals, equine industry persons, students/scholars, public health experts to equip them with latest armour to develop disease diagnostics and therapeutics for prevention, control of equine diseases including zoonotic aspect. This special issue will help in formulating various strategies related to equine health and also provide a solution on zoonotic aspects. The gain in knowledge on various aspects through this special issue will help in dealing equine health issues in a better way.

Thank you

Sandip Kumar Khurana Amarpal Yashpal S. Malik Kuldeep Dhama K. Karthik Minakshi Prasad

JEBAS Guest Editors

Lead Guest Editor

Sandip Kumar Khurana (MVSc, Ph.D) Principal Scientist National Research Centre on Equines (NRCE) Sirsa Road, Hisar – 125 001, Haryana, India Email: [email protected]

Guest Editors Amarpal (MVSc, PhD, FISACP, FNAVS, FISVS) Principal Scientist and Head, Division of Surgery Indian Veterinary Research Institute Izatnagar-243122 (UP), India Email: [email protected]

Yashpal S. Malik (MVSc., Ph D, Post-Doc USA) National Fellow, Principal Scientist Division of Biological Standardization Indian Veterinary Research Institute (IVRI), Izatnagar 243122, India E-mail: [email protected]

Kuldeep Dhama (MVSc, Ph.D) Principal Scientist & NAAS Associate Division of Pathology, ICAR-Indian Veterinary Research Institute (IVRI) Izatnagar-243 122, Bareilly, Uttar Pradesh, India Email: [email protected]

K. Karthik (MVSc) Assistant Professor, Central University Laboratory, Tamil Nadu Veterinary & Animal Sciences University, Madhavaram Milk Colony Chennai-51, Tamil Nadu, India E mail: [email protected]

Minakshi Prasad (PhD), Fellow NAAS, Fellow NAVS Professor and Head, Department of Animal Biotechnology College of Veterinary Sciences LALRUVAS, Hisar 125001, Haryana Email: [email protected]

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Welcome Message - Managing Editor (Dr Kamal Kishore Chaudhary, M.Sc, Ph.D) ______

Dear Authors,

It is with much joy and anticipation that we celebrate the launch of special issue - Spl-4-EHIDZ (Volume 4) of Journal of Experimental Biology and Agricultural Sciences (JEBAS). On behalf of the JEBAS Editorial Team, I would like to extend a very warm welcome to the readership of JEBAS. I take this opportunity to thank our authors, editors and anonymous reviewers, all of whom have volunteered to contribute to the success of the journal. I am also grateful to the staff at Horizon Publisher India [HPI] for making JEBAS a reality.

JEBAS is dedicated to the rapid dissemination of high quality research papers on how advances in Biotechnology, Agricultural sciences along with computational algorithm can help us meet the challenges of the 21st century, and to capitalize on the promises ahead. We welcome contributions that can demonstrate near-term practical usefulness, particularly contributions that take a multidisciplinary / convergent approach because many real world problems are complex in nature. JEBAS provides an ideal forum for exchange of information on all of the above topics and more, in various formats: full length and letter length research papers, survey papers, work-in-progress reports on promising developments, case studies and best practice articles written by industry experts.

Finally, we wish to encourage more contributions from the scientific community and industry practitioners to ensure a continued success of the journal. Authors, reviewers and guest editors are always welcome. We also welcome comments and suggestions that could improve the quality of the journal.

Thank you. We hope you will find JEBAS informative.

Dr. Kamal K Chaudhary Managing Editor - JEBAS December 2016

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INDEX ______

Leptospirosis in horses: special reference to equine recurrent uveitis doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S123.S131

Emergence of equine herpes virus 1 myeloencephalopathy: A brief review doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S132.S138

Equine ocular setariasis and its management doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S139.S143

Parasitological, biochemical and clinical observations in ponies experimentally infected with Trypanosoma evansi doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S144.S150

Equine brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S151.S160

Biotechnological tools for diagnosis of equine infectious diseases doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S161.S181

Evolving views on enteric viral infections of equines: an appraisal of key pathogens doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S182.S195

Lyme borreliosis in the horse: A mini-review doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S196.S202

An overview of ozone therapy in equine- an emerging healthcare solution doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S203.S210

Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

LEPTOSPIROSIS IN HORSES: SPECIAL REFERENCE TO EQUINE RECURRENT UVEITIS

Sandip Kumar Khurana1,*, Kuldeep Dhama2, Minakshi P3, Baldev Gulati1, Yashpal Singh Malik2 and Kumaragurubaran Karthik4

1NRCE, Hisar, Haryana, India 2Indian Veterinary Research Institute, Izatnagar, Barrielly, U.P., India 3Department of Animal Biotechnology, LUVAS, Hisar, Haryana, India 4Tamil Nadu University of Veterinary and Animal Sciences, Chennai, Tamil Nadu, India

Received – August 27, 2016; Revision – September 04, 2016; Accepted – October 03, 2016 Available Online – December 04, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S123.S131

KEYWORDS ABSTRACT Equine

Leptospirosis Leptospirosis is a bacterial zoonotic disease with worldwide distribution. The disease affects several domestic and wild animals. Leptospirosis has seasonal nature with high incidence in hot rainy season ERU especially in tropical regions. The disease in horses has ocular and systemic manifestations. However, stillbirths and neonatal mortality due to this disease is also common. The ocular manifestation is equine recurrent uveitis (ERU), also known as periodic ophthalmia or moon blindness, where autoimmune mechanisms also play an important role. Several diagnostic assays are employed and microscopic agglutination test has been commonly employed in several parts of the world though isolation is the gold standard test. Recent diagnostic advances like PCR, real time PCR, LAMP also aid in early diagnosis of the disease so that the spread of disease to other animals and human can be prevented.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Sandip Kumar Khurana) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

S124 Khurana et al

1 Introduction 2 Equine Leptospirosis

Leptospirosis is a major animal and human health problem 2.1 Disease occurrence worldwide. Leptospires belong to family Leptospiraceae, order spirochaetales. Leptospires are about 0.1 µm in diameter and The incidence of leptospirosis in horses remains uncertain as 6-20 µm in length. Their major antigenic component is systematic studies on leptospirosis in horses are scanty. Its lipopolysaccharide (LPS). The disease is prevalent in many importance and economic impact in equines is also not as parts of the world both in urban and rural settings (Vinetz, accurately distinguished as for other species of animals. Most 1997; Bharti et al., 2003; Verma et al., 2013; Khurana et al., epidemiological studies are based on serology with highly 2015). The leptospirosis primarily causes chronic kidney variable incidence in different geographical regions. There is infection in several domestic and wild animals. The leptospira also variability in the serovars. A sero-prevalence of only 1.5% colonize in renal tubules and are shed in urine. These bacteria was reported in central Italy, for serovars Icterohaemorrhagiae, survive for prolonged periods in moist conditions and thus Bratislava or Pomona (Ebani et al., 2012). However, a transmit the infection. Brazilian study of 119 race horses showed a much higher sero- positivity rate of 71% against serovar Copenhageni (Hamond Rats and other rodents are natural reservoirs of leptospira with et al., 2012b). All these horses were apparently healthy with no no apparent signs of disease. They clear infections from their clinical signs of leptospirosis. In another study by the same bodies except the kidney tubules. Other animals which are not group, seropositivity of 48% was reported and 35% of urine natural carriers of infection have mild to severe infection and samples were detected positive by PCR, however these were even death. The leptospires may cause reproductive problems culturally negative (Hamond et al., 2013). Recently a mainly abortions (Coghlan & Bain, 1969; Faine et al., 1984; prevalence study conducted in Brazil in 38 mares having Faine et al., 1999; Bharti et al., 2003; Hamond et al., 2015; reproductive problems examined for leptospira by examining Hamond et al., 2016). Mother to foetus transmission is also the serum, urine and vaginal fluid by isolation and PCR. common (Vinetz, 1997; Bharti et al., 2003; Alder & de la Pena Seventeen serum samples were positive (44.7%) for leptospira Moctezuma, 2010; Verma et al., 2013; Hamond et al., 2014). and of which sero groups Australis accounts for 76.4% and Pomona 23.6%. PCR results were positive for 17 vaginal fluid Animal handlers and waste water/ recycle workers are highly samples and 10 urine samples and the PCR products were susceptible (Campagnolo et al., 2000). Leptospira spp. are sequenced which showed that the samples belonged to L. endemic to several tropical and subtropical areas affecting interrogans (sv Bratislava and Pomona) and L. military personnel, aid workers, tourists and general public (Ko borgpertersenii. Thus this study implies the presence of et al., 1999; Bharti et al., 2003). Leptospirosis is less common leptospira in reproductive tract (Hamond et al., 2015). Sixty in temperate regions. There are different reservoirs of infection two cart horse samples were screened in Curitiba, southern in rural and urban areas, domestic and wild animals act as Brazil by microscopic agglutination test (MAT) and real time reservoirs in rural settings, dogs and rats are reservoirs in urban PCR of which 80.8% of the samples were positive for areas (Vinetz et al., 1996; Levett, 2001; Meites et al., 2004). Icterohaemorrhagiae serovar (Finger et al., 2014). Natural disasters, like floods may be followed by leptospirosis outbreaks (Fuortes & Nettleman, 1994). The leptospirosis has Sero-positivity of 25% in Korea with serovars Sejroe and been described as “an occupational disease of soldiers” as the Bratislava being prominent (Jung et al., 2010), 79% in The soldiers fighting in adverse terrains and conditions during wars Netherlands, serovars Copenhegi and Bratislava (Houwers et are at a greater risk of acquiring leptospirosis infection al., 2011) and 25% in Sweden (Baverud et al., 2009) show (Johnston et al., 1983). marked differences in prevalence and varied serovars according to geographical region. It was also indicated that the Symptoms in human beings may vary, but commonly include majority of equine infections were asymptomatic. A North fever, headache, muscular pain, uneasiness, vomiting American report in aborting mares revealed 20 out of 21 as due conjunctivitis, uveitis, meningitis and jaundice. About 5 and to serovar Pomona subtype Kennewicki (Timoney et al., 10% of patients progress to icteric phase. Fatality rates at this 2011), which is correlated to the presence of this serovar and stage may be more than 20%. Mortality is primarily due to subtype in local wildlife mainly raccoons. Serovar Bratislava acute renal failure, pulmonary haemorrhages, intracerebral has been implicated in horses in Northern Ireland, based on haemorhage and multisystem organ failure (Vinetz, 1997; culture and serology (Ellis et al., 1983). Pikalo et al. (2016) Faine et al., 1999; Ko et al., 1999; Levett, 2001; Bharti et al., examined the presence of leptospiral antibodies in horses in 2003). middle Germany by MAT. 54 out of 314 (17.2%) horses were positive for one or more of eight leptospiral serovars analysed. Leptospirosis in horses has been considered a relatively Icterohaemorrhagiae (11.1%) was most prevalent followed by uncommon infection. Most of the infections are asymptomatic. Bratislava (9.6%) and Grippotyphosa (1.9%). Dorrego-Keiter A specific outcome of equine leptospirosis is recurrent uveitis et al. (2016) detected 57.5% (127/221) horses with ERU which appears to be mediated by autoimmune mechanisms. having antibodies against leptospira by MAT in Germany.

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Leptospirosis in horses: special reference to equine recurrent uveitis. S125

Figure 1Transmission and clinical signs of equine leptospirosis.

The most frequent antibodies were against Grippotyphosa apparently healthy animals suggest either a subclinical form of (79/127), followed by Icterohaemorrhagiae (34/127) and the disease in these animals or previous infection. Region-wise Bratislava (29/127). Tsegay et al. (2016) detected significant sero-prevalence of leptospiral antibodies showed that studs in antibody titres in 184 of 418 carthorses to at least one of 16 southern and western part of India country had higher sero- serovars of Leptospira species in central and southern Ethiopia. positivity irrespective of the group. Serovar Bratislava (34.5%) was found to be most prevalent. 2.2 Clinical signs, symptoms and disease manifestations National Reference Centre for Leptospirosis (NRCL) of Italy along with other units evaluated the occurrence and Equine leptospirosis is accompanied with mild fever. Loss of distribution of leptospira in Italy. Analysis of the data for the appetite and lethargy are common in mild form of disease. one year (2010-2011) revealed that Australis was common Jaundice, haemorrhages on the mucosa and depression are among horses in Italy (Tagliabue et al., 2016). predominant signs in the severe form. Renal failure is more common in foals in comparison to old horses. Classic icteric Khurana et al. (2003) studied the sero-prevalence of leptospira leptospirosis occurs mainly in foals and is comparatively rare in 436 equines in India by ELISA for detection of antibodies in adult horses. Leptospirosis may cause placentitis, abortions against six leptospiral serovars including Leptospira and stillbirths in pregnant mares (Figure 1) (Timoney et al., interrogans serovars Canicola, Pomona, Australis, Autumnalis, 2011). The letospires could be seen in foetal and maternal Icterohaemorrhagiae and Grippotyphosa. These samples tissues, microscopically (Poonacha et al., 1993). Leptospiral included 379 serum samples from apparently healthy equines, abortions in the late stage of gestation, with no apparent 12 from cases of abortion and 45 from equines in contact with clinical signs are common. Weak and icteric foals are also born aborted animals. Out of the 379 apparently healthy horses, 64 (Donahue et al., 1991; Donahue et al., 1995). Infected mares (16.89%) harboured antibodies against Leptospira spp. They shed leptospires in the urine for prolonged periods and transmit further reported the mares that came into contact with aborted the infection (Donahue & Williums, 2000; Newman & animals, 66.7% had positive titres indicative of leptospiral Donahue, 2007). infection. Animals in contact with aborted animals had also showed higher sero-prevalence of 64.4%. Antibody titres in

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S126 Khurana et al

Microscopically placental lesions include vasculities, 2.4 Prevention and control thrombosis, inflammatory cells in the stroma and villi, cystic adenomatous hyperplasia of allantoic epithelium. Foetal liver Treatment regimens for horses have mostly been derived by and kidneys are enlarged. Microscopic lesions in foetus include extrapolation from other species, due to non availability of suppurative and nonsuppurative nephritis, leukocytic specific information for horses. Streptomycin and penicillin are infiltration of the portal triads, giant cell hepatopathy, most common antibiotics of choice. Tetracyclines are used as pulmonary haemorrhages, pneumonia and myocarditis (Wilkie an alternative. The penicillin dose is related to titre of et al., 1988; Poonacha et al., 1993). Histopathological findings leptospiral antibodies. The streptomycin is has severe toxic in young horses are marked with petechiae and lymphoytic effects in horses (Bernard 1993; Newman & Donahue, 2007). infiltration in renal proximal tubules and glomeruli (Bernard, 1993; Faine et al., 1999). Equine recurrent uveitis is a common Till now there were no leptospirosis vaccine for horses. Cattle sequel in equines, which is dealt separately in this review. vaccines were being occasionally used in horses, which is not advisable. In leptospiral uveitis molecular mimicry occurs The pulmonary haemorrhage is not common in equines (Bharti between leptospiral proteins and ocular tissues, in this situation et al., 2003). Recently this syndrome is being reported more vaccination with whole-cell bacterin may prime equines with commonly than previous information (Broux et al., 2012) and cross-reacting antigens resulting in stronger immunological endoscopy revealing pulmonary haemorrhage in 35% of sero- responses and development of eye inflammation in subsequent positive adult horses (Hamond et al., 2012a). exposures. Ideally, proposed leptospirosis vaccine should be free of cross-reacting antigens. Several leptospiral antigens 2.3 Diagnosis of leptospirosis in equines have been tested for protective efficacy (Alder & de la Pena Moctezuma, 2010; Murray et al., 2013). No antigen has been The diagnosis of leptospirosis in horses is similar to that for tested in horses. Recently, Zoetis has introduced a licensed other species. The gold standard is the culture and equine leptospiral vaccine for prevention of leptospirosis identification of leptospira. PCR is a more convenient and caused by Leptospira Pomona. Prevention must therefore rapid (Alder & de la Pena Moctezuma, 2010). Real time PCR revolve around normal husbandry and hygiene practices, assay was recently compared with fluorescent antibody test vaccination of other animals on the farm, minimizing contact (FAT) and microscopic agglutination test (MAT) for effective with rodents and other wildlife carriers and other infected diagnosis of equine leptospirosis from foetal specimens like horses. placenta, kidney, liver and heart blood. Out of the 21 confirmed cases of equine abortion real time PCR could detect 3 Equine Recurrent Uveitis (ERU) all the positives correctly while MAT and FAT detected only 19 and 18 samples respectively. Thus qPCR is a better assay A major consequence of leptospirosis in horses is uveitis or compared to MAT and FAT for diagnosis of leptopsiral moon blindness also called periodic ophthalmia (Verma et al., abortion in equines (Erol et al., 2015). Silver staining and FAT 2013; Malalana et al., 2015). The uvea consists of three may be used to demonstrate leptospires in the placenta or components, the iris, ciliary body (anterior uvea) and choroid foetal kidney. FAT is more sensitive than silver staining and (posterior uvea). The uveal tract is highly vascular, usually more specific than MAT (Donahue & Williams, 2000; Szeredi pigmented (Samuelson, 2007; Gilger & Deeg, 2011: & Haake, 2006; Newman & Donahue, 2007). Enzyme-linked Hollingsworth, 2011). Direct proximity to the peripheral immunosorbent assay (ELISA) has also been developed using vasculature, makes the uveal tract vulnerable to any disease of different proteins of leptospira and its efficacy has been the systemic circulation (Hughes, 2010; Leiva et al., 2010; assessed time to time. Recently a cocktail of recombinant Gilger & Deeg, 2011). A blood-ocular barrier exists between proteins namely rLipL21, rLoa22, rLipL32, and rLigACon4-8 the peripheral vasculature and the inner structures of the eye, of Leptospira interrogans were analyzed for its potential as a divided into the blood-aqueous barrier (iris and ciliary body) diagnostic marker through ELISA. The assay was tested with and blood-retinal barrier (choroid). These barriers make the 130 serum samples and the results were compared with MAT eye a protected or immune-privileged site. Disruption of this and it was found that ELISA was sensitive and specific barrier allows the leakage of blood products and cells into the yielding similar results with MAT assay (Ye et al., 2014). eye and the activation of several immune responses. Leptospira-associated uveitis forms an important part of ERU MAT is the test of choice for serological diagnosis. In an cases (Halliwell et al., 1985; Hartskeerl et al., 2004; Witkowski endemic area the value of a single positive specimen is limited. et al., 2016). ERU is inflammation of uvea which occurs The four-fold rise in titre in paired sera is important for recurring episodes (Cook & Harling, 1983). It is reported to accurate diagnosis. In cases of leptospiral abortion, MAT on have a worldwide prevalence of around 10% and thought to be foetal fluids and maternal serum gives a very high titre a major cause of blindness in horses (Schwink, 1992; indicative of positive diagnosis (Donahue & Williams, 2000). Hartskeerl et al., 2004). Serological tests in different areas should include prevalent serovars of that area as test antigens. Commercial enzyme Pathogenesis of ERU is not exactly elucidated though several immunoassays incorporating locally prevalent serovars are possible ways has been reported. Eye of horses affected with available. ERU shows infiltration of macrophages, lymphocytes and

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Leptospirosis in horses: special reference to equine recurrent uveitis. S127 plasma cells into the ciliary body and also the iris. This shows treatment. A period of low inflammation follows the acute that immunologically privileged sites wall has been breached phase (Cook & Harling, 1983). Secondary cataract, anterior or by the organism. There is huge flow of CD4+ T lymphocytes in posterior attachment of iris, lens luxation, vitreous exudates the anterior uveal tract (Romeike et al., 1998). In these affected and retinal detachment are also witnessed due to severe horses T cell response is mainly of Th1 based (Gilger et al., inflammatory reaction (Rebhun, 1979; Cook & Harling, 1983; 1999). Kalsow et al. (1994) reported that T- and B-cells are Gilger et al., 2000; Gilger & Michau, 2004). A pathognomonic highly organized in the germinal centres in the horses affected sign of ERU is thick hyaline membrane near posterior aspect with ERU. This highly organized structure shows the antibody of iris and eosinophilic linear cytoplasmic inclusion bodies in response towards leptospira antigen in the anterior uvea nonpigmented ciliary epithelial cells (Cooley et al., 1990; (Kalsow et al., 1994). Leptospira can directly cause damage to Dubielzig et al., 1997). the eye leading to ERU but mainly it is caused by the autoimmune response due to the antigen (Verma et al., 2005). ERU has been associated with sulphonamides administration Two leptopsiral proteins namely LruA and LruB were or vaccination (Matthews & Handscombe, 1983; Whitcup, suggested to play a major role in ERU since IgG and IgA 2010). Higher prevalence in geldings compared to mares and specific for these proteins has been identified in the fluid from stallions has been reported (Szemes & Gerhards, 2000). No the eye (Verma et al., 2005). Hence these proteins can also be particular sex related differences in prevalence have been used as a diagnostic marker for detection of leptospiral reported (Gilger & Deeg, 2011; Kulbrock et al., 2013). Age of infection in equines. An evidence of a antigenic relationship presentation has been reported to vary in different studies between Leptospira and equine eye is established further cross (Dwyer et al., 1995; Szemes & Gerhards, 2000). reactivity as a mechanism of disease progression has been proposed (Parma et al., 1985; Parma et al., 1987; Parma et al., Diagnosis of Leptospira associated ERU is based on the 1997). Verma et al. (2005) described that intraocular presence of classical signs of uveitis, history of recurrence and expression of two leptospiral proteins, LruA and LruB seropositivity by MAT. No specific test is available for the antibodies was significantly higher than in the sera, indicating diagnosis of leptospiral uveitis. Negative MAT titres are not local production and antibodies in uveitic eyes. The lens always indicative of absence of leptospiral infection. proteins cross-reacting with LruA antiserum were identified as crystalline B and vimentin, and cross reacting retinal protein Reducing inflammation is of primary concern in ERU therapy. was identified as crystalline B2 (Verma et al., 2010). Therefore An intraocular device containing cyclosporine A is found cross reactivity between leptospiral and ocular proteins may be effective in treatment of leptospiral ERU (Werry & Gerhards, responsible for immunopathogenesis of ERU of leptospirai 1991; Gilger & Michau, 2004). However, usefulness of origin. antibiotics in treating ERU has not been fully explored. A recent review conducted in the North Carolina State University ERU has three distinct clinical forms: classic, insidious and Veterinary Health Complex with the medical records of ERU posterior (Gilger & Michau, 2004; Gilger & Deeg, 2011; from 1999 to 2014 showed that most cases had blindness, eye Malalana et al., 2015). Classic ERU is characterised by active globe loss and loss of eye function. Several owners opted for intraocular inflammation followed by quiet periods, where euthanasia and some opted to sell the animals due to recurrent subsequent inflammatory phases show increased severity. eye problem (Gerding & Gilger, 2016). Thus this problem of Insidious ERU is characterised by low grade persistent ERU has caused great financial loss to the horse owners. inflammation. The Vitreous, choroid and retina are primarily affected in posterior uveitis. Thus leptospiral infections result in reproductive and respiratory problems in equines. However, the most important The signs associated with an acute episode of anterior uveitis manifestation of leptospiral infection in equines is ERU, which are varied including ocular pain, blepharospasm, lacrimation, affects equine population by causing blindness, thus rendering chemosis, photohobia, oedema of the eyelid, swollen them useless. Therefore more researches are needed to explore conjunctiva and corneal oedema, aqueous flare, hypopyon, the pathogenesis and mechanism of occurrence of ERU with an hyphaema, miosis, iris colour changes and low intraocular aim of its prevention and control. pressure (Cook et al., 1983: Wada, 2006; Gilger & Deeg, 2011). Posterior uveitis is characterised by vititis with Conflict of interest liquefaction of the vitreous and retinal changes. Authors would hereby like to declare that there is no conflict of Changes associated with previous episodes of uveitis in an interests that could possibly arise. otherwise quiescent eye may give clues to previous episodes. Some of these changes may include depigmentation, corneal References scarring, atrophy and fibrosis of iris, abnormalities of the iris margin, cataract, glaucoma and fundic changes etc. (Williams Adler B, de la Pena Moctezuma A (2010) Leptospira and et al., 1971) but are not specifically pathognomonic (Cook et leptospirosis. Veterinary Microbiology 140: 287-296. doi: al., 1983; Barnett, 1987; Spiess, 2010; Mathes et al., 2012). 10.1016/j.vetmic.2009.03.012. The prognosis is dependent on an early diagnosis and

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Barnett KC (1987) Equine periodic opthalmia: A continuing Dorrego-Keiter E, Toth J, Dikker L, Sielhorst J, Schusser GF aetiological riddle. Equine Veterinary Journal 19: 90-91. (2016) Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous Baverud V, Gunnarsson A, Engvall EO, Franzen P, Egenvall A humor and serum of 225 horses with equine recurrent uveitis. (2009) Leptospira seroprevalence and associations between Berl Munch Tierarztl Wochenschr. 129 : 209-215. seropositivity, clinical disease and host factors in horses. Acta Veterinaria Scandinavica 51:15. doi: 10.1186/1751-0147-51- Dubielzig RR, Render JA, Morreale RJ (1997) Distinctive 15 morphologic features of the ciliary body in equine recurrent uveitis. Veterinary and Comparative Opthalmology 7: 163- Bernard WV (1993) Leptospirosis. The Veterinary Clinics of 167. North America, Equine Practice 9: 435-444. Dwyer AE, Crockett RS, Kalsow CM (1995) Association of Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, leptospiral seroreactivity and breed with uveitis and blindness Lovett MA, Levett PN, Gilman RH, Wiling MR, Gotuzzo E, in horses-372 cases (1986-1993). Journal of the American Vinetz JM (2003) Leptospirosis: a zoonotic disease of global Veterinary Medical Association 207: 1327-1331. importance. The Lancet, Infectious Diseases 3: 757-771. Ebani VV, Bertelloni F, Pinzauti P, Cerri D (2012) Broux B, Torfs S, Wegge B, Deprez P, van Loon G, (2012) Seroprevalence of Leptospira spp. and Borrelia burgdorferi Acute respiratory failure caused by Leptospira spp. in 5 foals. sensu lato in Italian horses. Annals of Agricultural and Journal of Veterinary Internal Medicine 26: 684-687. Environmental Medicine 19: 237-240.

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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EMERGENCE OF EQUINE HERPES VIRUS 1 MYELOENCEPHALOPATHY: A BRIEF REVIEW

Baldev Raj Gulati1,*, Gayathri Anagha2, Thachamvally Riyesh1 and Sandip Kumar Khurana1

1ICAR-National Research Centre on Equines, Hisar, Haryana-125001, India 2ICAR- Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243122, India

Received – October 15, 2016; Revision – October 26, 2016; Accepted – November 20, 2016 Available Online – December 04, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S132.S138

KEYWORDS ABSTRACT

Equine herpesvirus 1 Equine herpesvirus 1 (EHV1) is an economically important viral pathogen of equines and causes respiratory disease, neonatal foal mortality, late-term abortion and sporadic encephalomyelitis aka Equine herpesvirus equine herpes myeloencephalopathy (EHM) in affected horses. The nervous form of EHV1 (EHM) has myeloencephalopathy been recognized as early as 1950s in horse population; however, many aspects of this disease remained poorly understood. In recent years, there has been much progress in our understanding of genetics, Neuropathogenicity epidemiology and pathogenesis of EHM through close monitoring of field outbreaks in different parts of the world. Various host, agent and environmental factors have been found to a play a role in the ORF30 development of EHM, the most significant being the identification of a single nucleotide polymorphism in DNA polymerase gene (A2254 to G2254), which imparts neuropathogenic potential to the virus. EHM affects horses of all ages, including un-weaned foals and produces clinical symptoms that are indistinguishable from other viral encephalitis/ central nervous system (CNS) disorders. EHM treatment includes supportive therapy, and reducing inflammation of CNS. Diagnosis of affected horses and monitoring of in-contact animals is the best measures to prevent EHM outbreaks. This review in brief discusses about progress made in epidemiology, pathogenesis, treatment, prevention and control of EHM.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Baldev R. Gulati) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

S133 Gulati et al

1 Introduction internal repeat regions. The EHV-1 genome encodes for 76 open reading frames (ORFs). EHV1 isolates have special Equine herpesvirus 1 (EHV1) is a highly contagious virulence markers, which are thought to induce EHM. respiratory pathogen associated with a variety of disease conditions in horses. It is estimated that 80 to 90% of horses 3 Equine herpesvirus myeloencephalopathy have been exposed to EHV1 infections by two years of age (Allen, 2008). EHV1 infection causes upper respiratory tract EHM was present in equine population as early as 1950s, infection in young horses, abortion in pregnant mares, neonatal however, its importance came to limelight in the last decade foal mortality and neurological disorders. Abortion is the most after large outbreaks of EHM occurred in Europe and America economically crippling outcome of EHV1 infection with 95% (Perkins et al., 2009; Vissani et al., 2009; Pronost et al., 2010; of EHV1 associated abortions occurring in the last four months Smith et al., 2010; Fritsche & Borchers 2011; Pusterla et al., of pregnancy. Respiratory disease associated with EHV1 is 2012; Damiani et al., 2014; Stasiak et al., 2015). Neurological most commonly seen in young animals at the time of weaning disease can affect horses of all ages, including un-weaned (Allen, 2008). Neurological disease associated with EHV1 is foals, and often requires euthanasia of affected animal Horses called equine herpesvirus myeloencephalopathy (EHM). exhibiting neurologic diseases can shed the virus in their nasal Although clinical form of EHM is less frequently observed, it secretions and transmit the disease to in-contact animals can cause serious economic losses in breeding horses and has (Henninger et al., 2007).) very negative impact on equine industry (Friday et al., 2000; van Mannen et al., 2001; Henninger et al., 2007; Pronost et al., The ORF30 spanning the nucleotide region 51522-55184 2010). (3662 nt) in EHV1 genome encodes for a protein referred to as Pol, the putative DNA polymerase catalytic subunit which During past decade, incidence of abortion and possesses DNA synthesis activity. This gene is highly rhinopneumonitis due to EHV1 has been declining, possibly conserved throughout its length. Recently, a single nucleotide due to widespread vaccination practices. At the same time, polymorphism (SNP) of guanine (G) for adenine (A) at 2254 there has been rise in incidence of EHM in many parts of nucleotide position of the ORF30 region resulting in an amino world viz., Europe, North America, South America, Africa and acid variation, from asparagine to aspartic acid (N/D752) have Oceania (Perkins et al., 2009; Vissani et al., 2009; Pronost et been proven to be associated with the neuropathogenic al., 2010; Smith et al., 2010; Fritsche & Borchers 2011; potential of the EHV1 strain (Nugent et al., 2006). This DNA Tsujimura et al., 2011, Cuxson et al., 2014; Negussie et al., polymerase enzyme of EHV1 has two sets of identical protein 2015; McFadden et al., 2016). The neuropathogenic strains of subunits each of which contains two catalytic pockets (Liu et EHV1 (causing EHM) have also been reported from Asian al., 2006), serving as site for polymerase activity and the site countries such as Japan (Tsujimura et al., 2011) and India for 3’- 5’ exonuclease activity. In EHV1, neuropathogenic (Unpublished data). This article discusses the aetiology, strains, the point mutation results in a switch from no charge to pathogenesis, epidemiology of EHM and gives an overview of a negative charge and induces a conformational change within prevention, control and treatment of EHM. the viral polymerase structure and thereby increases the replicative capacity of the virus and produce significantly 2 Etiology higher viral loads (Nugent et al., 2006; Liu et al., 2006).

EHV1 is an enveloped, double-stranded DNA virus belonging 4 Prevalence of neuropathogenicty to the genus Varicellovirus of the subfamily Alphaherpesvirinae within family Herpesviridae (Davison et Increased numbers of EHM cases have been reported from al., 2009). As many as nine equine herpesviruses (EHV 1-9) various parts of the world during the last decade with majority species have been known to infect equines. Among these only of them from Europe and North America. Europian countries five (viz., EHV1, 2, 3, 4 and 5) have the ability to produce viz., France (Pronost et al., 2010; van Galen et al., 2015), diseases in horses. EHV3 is responsible for equine coital Germany (Fritsche & Borchers, 2011; Damiani et al., 2014), exanthema while EHV1 and 4 are the economically important Belgium (van der Meulen et al., 2003; Gryspeerdt et al., 2011), viruses affecting the horses globally (Davison et al., 2009) with Poland (Stasiak et al., 2015), Netherlands (Goehring et al., EHV1 capable of even causing abortion and neurological 2006) and Croatia (Barbic et al., 2012); North American disorders as compared to EHV4 (Patel & Heldens, 2005; Lunn countries viz., Canada (Burgess et al., 2012) and U.S.A et al., 2009). EHV2 and 5 do not cause any specific diseases (Nugent et al., 2006; Henninger et al., 2007; Perkins et al., but remain associated with upper respiratory tract diseases, 2009; Smith et al., 2010; Pusterla et al., 2012); South immunosupression, general malaise and poor performance American countries viz., Brazil (Mori et al., 2011) and (Thein 1978; Belak et al., 1980). Argentina (Vissani et al., 2009); Asian countries viz., Turkey (Yilmaz et al., 2012); Japan (Tsujimura et al., 2011) and India EHV1 genome is 150 kbp linear double-stranded DNA (Unpublished data); Islands viz ., Australia (Cuxson et al., composed of a unique long region and unique short region 2014) and Newzealand (McFadden et al., 2016); African flanked by inverted repeat regions, the terminal repeat and the countries viz., Ethiopia (Negussie et al., 2015) experienced

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Emergence of equine herpes virus 1 myeloencephalopathy: A brief review. S134 outbreaks of EHV1 infection by neuropathogenic strains of recovery, although some horses may be left with permanent EHV1. The incidence of neuropathogenic genotype from cases neurologic sequalae. of neurological illness reported from different countries varies between 20% and 86% (Perkins et al., 2009; Vissani et al., 7 Factors affecting EHM 2009; Pronost et al., 2010; Fritsche & Borchers, 2011; Cuxson et al., 2014). The prevalence of neuropathogenic strains in Mechanism behind EHM is poorly understood. Studies on the abortion outbreaks varies between 1.5% and 25.8%. The evaluation of the risk factors associated with the development percentage prevalence was highest (25.8%) in France (Pronost of EHM have been performed in Europe and in North America. et al., 2010) followed by 19.4% in U.S.A (Smith et al., 2010), Various factors viz., season, age, breed, sex, immunological 10.6% in Germany (Fritsche & Borchers, 2011), 7% in status and latency have been found to be associated with EHM. Argentina (Vissani et al., 2009), 3.1% in Poland (Stasiak et al., A study in the Netherlands revealed a strong association 2015), 2.7% in Japan (Tsujimura et al., 2011) and 1.5% in between season and outbreaks of EHV neurological disease Australia (Cuxson et al., 2014). with all outbreaks occurring between mid-November and mid- May. However, this season specificity has not been observed in 5 Pathogenesis of EHM all countries. Paillot et al. (2008) reported that neurological signs due to EHM were seen at an increased frequency in Upon entry into the animal body, virus multiplies in the standard breeds, Hispanic breeds and draught breeds, with no epithelial cells of upper respiratory tract. Following initial cases of EHV-induced myeloencephalopathy in archetypical replication, the virus spreads to the cells of lamina propria and ponies, Haflinger, Fjord and Icelandic horses. underlying tissues within 12-24 h, after crossing the basement membrane. By 1-2 days post-infection (dpi), the virus reaches Experimental infection proved that older horses are more in the local lymph nodes draining the respiratory tract where predisposed to the development of neurological disease as further replication and infection of leukocytes occurs. compared to young to young/middle aged horses. Adult horses Leukocytes harbouring the virus are released to the blood may develop viremia 100 times higher than young horses and stream (Leukocyte-associated viremia) between 4-10 dpi they are 8 times more likely to develop the disease (Allen, which enables the virus to reach internal organs including CNS 2008). Latency by alpha herpes viruses is an important (Kydd et al., 1994; Gryspeerdt et al., 2011). Secondary epidemiological strategy ensuring survival and spread within replication occurs in endothelial cells of CNS-associated the natural host population (Whitley & Gnann, 1993). EHV1 arterioles (in particular the vessels of the spinal cord), which latency has been demonstrated in lymphoid as well as in neural may result in nervous system disorders 9-13 dpi. As a tissues (Baxi et al., 1995; Borchers et al., 1999). Following consequence, vasculitis, thrombosis, perivascular cuffing of reactivation, latently infected carriers may shed the virus in lymphocytes at sites of endothelial infection occurs, probably their nasal secretion and also may result in EHM following caused by direct interaction of the host's immune system and invasion of nervous system (Allen & Timoney, 2007). infectious agent (Edington et al., 1986). This vascular damage leads to ischaemia and re-perfusion injury of the CNS. 8 Laboratory Diagnoses Neuropathogenic strains are capable of exhibiting longer and higher level viremia. This high level viremia, interfere the Laboratory diagnosis of EHM is currently based on at least one blood flow to CNS and resulting in development of of the following criteria: clinical symptoms, cerebrospinal fluid neurological diseases (Fritsche & Borchers, 2011). The exact examination, serological testing, virus isolation, molecular mechanism by which leukocyte-associated viremia leads to detection methods and post-mortem examination. Differential myeloencephalopathy is not known. diagnosis should also be made from other viral cause of encephalitis, rabies, protozoal myeloencephalitis and non 6 Clinical Signs of EHM infectious conditions like neuritis of the cauda equina, central nervous system (CNS) trauma and different plant/chemical Onset of clinical signs of EHM usually occur 6-10 dpi intoxications (Pusterla et al., 2009; Pusterla & Hussey, 2014). following the onset of viremia. Clinical signs depend on Horses presented with clinical symptoms as explained number and size of affected sites, as well as relevance and elsewhere may be suspected for EHM. An increased protein location of affected nervous tissue (caudal spinal cord is most concentration and albumin quotient may be noticed in CSF of affected). Clinical signs usually include fever, ataxia, affected horses. paresis/paralysis of hind limbs, bladder dysfunction, urinary incontinence and sensory deficit in the perineal area. In Serological examination suggesting a 4-fold or greater increase addition ventral oedema, scrotal or preputial oedema in male in serum antibody titer between acute and convalescent horses, and limb oedema are also noticed. In severe cases of samples in the clinically affected horses, along with antibodies EHM, paralysis may advance to tetraplegia and death of in CSF, is strongly suggestive of EHM (Friday et al., 2000; van animal is observed (van Mannen, 2002; Pusterla & Hussey, Maanen et al., 2001). However, many horses with EHM do not 2014). EHM affected horse that remains in standing posture exhibit a 4-fold rise in SN titer, since the antibody titers rise may have good prognosis. However, horses with severe rapidly and may have peaked by the time neurological signs neurologic disease may take more than a year for complete appear (Friday et al., 2000; van Maanen et al., 2001). Virus

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S135 Gulati et al isolation from nasal or nasopharyngeal swabs or buffy coat reducing the likelihood of introduction and dissemination of samples is considered as the ‘gold standard’ test for a EHV1 infection can prevent EHM in herd. The control laboratory diagnosis of EHV1 infection. However, EHM cases measures are mainly focused around quarantine and might not yield virus isolation, as virus shedding may stop by vaccination (Lunn et al., 2009; Pusterla et al., 2009). Affected the time neurological signs appear (Pusterla et al., 2009). or suspected horses must be removed from the stable immediately and placed in strict isolation. Once EHV1 Many of the published conventional PCR detection protocols infection is confirmed, horses should remain in strict (Ballagi- Pordany et al., 1990; Sharma et al., 1992; Wagner et quarantine until they are fully recovered and are asymptomatic al., 1992; Borchers & Slater, 1993; Kirisawa et al., 1993; for 21 days. Horses from farms experiencing EHM infection Lawrence et al., 1994., Wang et al., 2007) are unable to should be maintained in their existing stable and segregated differentiate between neuropathogenic and non- from other horses. There should be total movement restriction neuropathogenic viruses. Hence, PCR assays based on ORF30 of animals from such farms (Pusterla et al., 2009; Pusterla & followed by sequence analysis can be used to differentiate Hussey, 2014). The currently used EHV1 vaccines are not able neuropathogenic and non-neuropathogenic EHV1 isolates to provide protection against EHM. However, regular use of (Nugent et al., 2006; Allen, 2007; Leutenegger et al., 2008; commercially available EHV1 vaccines enhances herd Pusterla & Hussey, 2014). Use of novel PCR platforms, such immunity, reduce viral shedding at the event of exposure and as real-time PCR assays based on ORF30 enable the hence reduce EHM risk (Pusterla & Hussey, 2014). differentiation of neuropathogenic and non-neuropathogenic viruses (Allen, 2007; Leutenegger et al., 2008). Single 11 Conclusions and future perspectives nucleotide polymorphism (SNP)-real-time PCR (Smith et al., 2012) and primer-probe energy transfer method (Malik et al., The development of neurological disease due to EHV1 2010) have been used for diagnosis of EHM. A SNP-based infection is likely to be multi-factorial. Potential horse-specific real-time PCR has been developed in our laboratory that is able risk factors for EHM include advanced age, breed, post- to differentiate neuropathogenic and non-neuropathogenic exposure viraemic load, low cytotoxic-T lymphocyte EHV1 strains. Using this assay, we observed circulation of precursors and environmental factors. Antemortem diagnosis neuropathogenic EHV1 among Indian equine population of EHM relies mainly on real time-PCR detection of EHV1 in (unpublished data). nasal secretions and blood. Although several vaccines are commercially available to prevent respiratory and abortigenic 9 Treatment of EHM form of EHV1 infections, they do not provide protection from neurologic form of the disease. Even though there is a strong

There is no specific treatment for EHM and the line of association between EHM and the G2254 mutation, this treatment is aimed at supportive medication to reduce CNS nucleotide substitution is not the only determinant of inflammation. Antiviral drugs for reducing viremia, non- neurological disease. EHV1 isolates with A2254 genotypes have steroidal anti-inflammatory drugs (NSAID) for countering been associated with a number of cases of neurological inflammation and anti-thrombotic drugs for preventing clot disease. On the other hand, G2254 genotype EHV1 isolates have formation are commonly used for treatment (Lunn et al., 2009; been recovered from horses with no evidence of neurological Pusterla & Hussey, 2014). Treatment with corticosteroids, such symptoms. as prednisolone acetate or dexamethasone for 2 to 3 days, is frequently recommended for severely affected animals as their One of the possible reasons for this observation could be the use could aid in reducing the incidence of vasculitis, fact that besides A2254→G2254 substitution, other non- thrombosis, and the resultant neural injury. Flunixin synonymous nucleotide substitutions in ORF30 region can also meglumine (nonsteroidal anti-inflammatory drug), which is have an effect on the production of neurological disease by commonly used for the treatment of CNS vasculitis can be either enhancing/attenuating the capability of viral replication used as these drugs suppress cellular interactions between rates in vivo. Furthermore, DNA polymerase is only one out of infected lymphocytes and endothelial cells (Pusterla et al., six proteins involved in ‘elongation complex’ of DNA 2009). Drugs like dimethyl sulfoxide, acetylsalicylic acid and replication machinery Substitutions occurring in the ORF of pentoxifylline have also been used for thromboembolic events any one of these proteins could have a considerable impact on associated with vasculitis. Antiviral drugs such as acyclovir viral replication rates, which will in turn have an effect on have been found effective in in-vitro studies, however, limited neuropathogenicity. data is available on the in vivo efficacy of acyclovir. Administration of broad-spectrum antimicrobials is also found This is an area of research that needs further investigation. effective to combat the risk of development of cystitis in Comparative whole genome sequencing of neuropathogenic affected horses (Pusterla & Hussey, 2014). EHV1 strains from different geographical location might decipher other markers related to neuropathogenicity. There is 10 Control of EHM also need to understand the role of host factors in the pathogenesis of EHM, including host immunopathological There is no specific method for prevention and control of mechanisms in response to EHV1 infection and latency. EHM. However, routine management practices aimed at

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Emergence of equine herpes virus 1 myeloencephalopathy: A brief review. S136

Conflict of interest Cuxson JL, Hartley CA, Ficorilli NP, Symes SJ, Devlin JM, Gilkerson JR (2014) Comparing the genetic diversity of Authors would hereby like to declare that there is no conflict of ORF30 of Australian isolates of 3 equid alphaherpes viruses. interests that could possibly arise. Veterinary Microbiology 169:50-57.

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McFadden AM, Hanlon D, McKenzie RK, Gibson I, Bueno Sharma PC, Cullinane AA, Onions DE, Nicolson L (1992) IM, Pulford DJ, Orr D, Dunowska M, Stanislawek WL, Spence Diagnosis of equid herpesviruses -1 and -4 by polymerase RP, McDonald WL, Munro G, Mayhew IG (2016) The first chain reaction. Equine Veterinary Journal 24:20-25. reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand. New Zealand Veterinary Journal 64:125-134. Smith KL, Allen GP, Branscum AJ, Frank Cook R, Vickers ML, Timoney PJ, Balasuriya UB (2010) The increased Mori E, Borges AS, Delfiol DJ, Oliveira Filho JP, Gonçalves prevalence of neuropathogenic strains of EHV-1 in equine RC, Cagnini DQ, Lara MC, Cunha EM, Villalobos EM, Nassar abortions. Veterinary Microbiology 141:5-11. AF, Castro AM, Brandao PE, Richtzenhain LJ (2011) First detection of the equine herpesvirus 1 neuropathogenic variant Smith KL, Li Y, Breheny P, Cook RF, Henney PJ, Sells S, in Brazil. Revue scientifique et technique 30:949-954. Pronost S, Lu Z, Crossley BM, Timoney PJ, Balasuriya UB (2012) New real-time PCR assay using allelic discrimination Negussie H, Gizaw D, Tessema TS, Nauwynck HJ (2015) for detection and differentiation of equine herpesvirus-1 strains Equine Herpesvirus-1 Myeloencephalopathy, an Emerging with A2254 and G2254 polymorphisms. Journal of Clinical Threat of Working Equids in Ethiopia. Transboundary and Microbiology 50:1981-1988. Emerging Diseases doi: 10.1111/tbed.12377. Stasiak K, Rola J, Ploszay G, Socha W, Zmudzinski JF (2015) Nugent J, Birch-Machin I, Smith KC, Mumford JA, Swann Z, Detection of the neuropathogenic variant of equine herpesvirus Newton JR, Bowden RJ, Allen GP, Davis-Poynter N (2006) 1 associated with abortions in mares in Poland. BMC Analysis of equid herpesvirus 1 strain variation reveals a point Veterinary Research 11:102. mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks. Thein P (1978) The association of EHV–2 infection with Journal of Virology 80:4047-4060. keratitis and research on the occurrence of equine coital exanthema (EHV–3) of horses in Germany. In: Bryans JT, Paillot R, Case R, Ross J, Newton R, Nugent J (2008) Equine Gerber H (Eds) Equine infectious disease IV. Princeton New Herpes Virus-1: Virus, Immunity and Vaccines. The Open Jersey Veterinary Publications: 33-41. Veterinary Science Journal 2: 68-91. Tsujimura K, Oyama T, Katayama Y, Muranaka M, Bannai H, Patel JR, Heldens J (2005) Equine herpesviruses 1 (EHV-1) Nemoto M, Yamanaka T, Kondo T, Kato M, Matsumura T and 4 (EHV-4)-epidemiology, disease and (2011) Prevalence of equine herpesvirus type 1 strains of immunoprophylaxis: a brief review. Veterinary Journal 170: neuropathogenic genotype in a major breeding area of Japan. 14-23. Journal of Veterinary Medical Science 73:1663-1667.

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Emergence of equine herpes virus 1 myeloencephalopathy: A brief review. S138 van der Meulen K, Vercauteren G, Nauwynck H, Pensaert M Wagner WN, Bogdan J, Haines D, Townsend HG, Misra V (2003) A local epidemic of equine herpes virus-1 induced (1992) Detection of equine herpesvirus and differentiation of neurological disorders in Belgium. Vlaams Diergeneeskundig equine herpesvirus type 1 from type 4 by the polymerase chain Tijdschrift 72: 366-372. reaction. Canadian Journal of Microbiology 38:1193-1196. van Galen G, Leblond A, Tritz P, Martinelle L, Pronost S, Wang L, Raidal SL, Pizzirani A, Wilcox GE (2007) Detection Saegerman C (2015) A retrospective study on equine of respiratory herpesviruses in foals and adult horses herpesvirus type-1 associated myeloencephalopathy in France determined by nested multiplex PCR. Veterinary Microbiology (2008-2011). Veterinary Microbiology 179:304-309. 121:18-28. van Maanen C (2002) Equine herpesvirus 1 and 4 infections: Whitley RJ, Gnann JW (1993) The epidemiology and clinical an update. Veterinary Quarterly 24: 58-78. manifestations of herpes simplex virus infections. In Roizman B, Whitley RJ, Lopez C (Eds.) The Human Herpesviruses, van Maanen C, Sloet van Oldruitenborgh-Oosterbaan MM, New York: Raven Press: pp. 69-105. Damen EA, Derksen AG (2001) Neurological disease associated with EHV-1-infection in a riding school: clinical Yilmaz H, Altan E, Turan N, Gurel A, Haktanir D, Sonmez K, and virological characteristics. Equine Veterinary Journal Deniz S, Gulcubuk A, Gur E, Sonmez G, Richt AJ (2012) First 33:191-196. Report on the Frequency and Molecular Detection of Neuropathogenic EHV-1 in Turkey Journal of Equine Vissani MA, Becerra ML, Olguín Perglione C, Tordoya MS, Veterinary Science 32: 525-530. Miño S, Barrandeguy M (2009) Neuropathogenic and non- neuropathogenic genotypes of Equid Herpesvirus type 1 in Argentina. Veterinary Microbiology 139:361-364.

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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EQUINE OCULAR SETARIASIS AND ITS MANAGEMENT

Malik Abu Rafee* and Amarpal

Division of Surgery and Radiology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P India-243122

Received – October 15, 2016; Revision – November 02, 2016; Accepted – November 21, 2016 Available Online – December 04, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S139.S143

KEYWORDS ABSTRACT

Ocular setariasis Ocular setariasis is a common vision threatening ophthalmic condition in equine resulting from ectopic parasitism by Setaria digitata, Setaria equina and Thelazia lacrymalis. The disease occurs mostly in Eye worm summer and autumn seasons and it displays signs of lacrimation, photophobia, corneal opacity, conjunctivitis and loss of vision. Close inspection of the eye reveals a moving worm in the anterior Equine chamber of the eye. B-mode (brightness mode) ultrasonography helps in the diagnosis in case of Surgery complete opacity. The best treatment is the surgical removal of the parasite under regional/ general anesthesia. Needle paracentesis at 3 O’ clock and nick incision at 12 O’ clock position are most commonly used surgical procedure. Both the techniques give good results. A slightly modified technique of using a 21 gauge needle attached with the syringe to aspirate the worm into the syringe also gives satisfactory results. In medicinal therapy ivermectin is the most advocated drug for ocular equine setariasis, but long term tying of medicinal should be avoided and surgery should be advocated. Corneal opacity is the most common post operative complication reported. Post surgical use of placentrex has also been advocated to enhance healing and to resolve corneal opacity. The present review is aimed at etiology, diagnosis and management of ocular setariasis in equine species.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Malik Abu Rafee) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

S140 Rafee and Amarpal

1 Introduction 2 Diagnosis

Among the most common surgical conditions of equine ocular Lacrimation, photophobia, blepharospasm and corneal opacity setariasis is a vision threatening disease of equine resulting are the common signs seen in horses with eye worm. Keen from ectopic parasitism caused by Setaria spp, a genus of inspection of the eye usually reveals moving worm in the filaroid worms (Gangwar et al., 2008; Radwan et al., 2016). In anterior chamber of the eye. The affected eye reacts to bright India equine ocular setariosis, an important cause of corneal flash stimulus and fluorescein staining test is usually negative, opacity is commonly caused by Setaria digitata, Setaria whereas slit-lamp biomicroscopic examination reveals corneal equina and Thelazia lacrymalis (Sathu, 1974; Ladoucer & edema (Tuntivanich et al., 2011). In eyes with complete Kazacos, 1981; Parrah et al., 2004; Sellon & Long, 2013). S. corneal opacity B-mode ultrasonography (12 MHz, corneal digitata is a parasite of cattle and hoofed animals and is found contact technique) can be performed to visualize the anterior mainly in Asia. S. equina infects horses and other equids chamber and other intraocular structures (Patil et al., 2012). worldwide. The usual predilection site of adult Setaria worms Though CBC (complete blood count) does not show major is the peritoneal cavity. Occasionally they can get into the changes but a decrease in erythrocyte count, haemoglobin and central nervous system or the eyes (Yadav et al., 2006). haematocrit , together with leucocytosis and an accelerated Microfilariae (immature larvae) are found in the blood. The erythrocyte sedimentation rate (ESR) has been reported in parasite is transmitted by mosquitoes (Anopheles previous studies (Muhammad & Saquib, 2007). Microscopic peditaneniatus and Culex nilgiricus) through the blood stream. examination of wet blood films is also recommended as it Adult female worms release microfilariae in sometimes reveals motile microfilariae. Knott’s test (a the abdominal cavity of their hosts. These microfilariae get technique for the detection of microfilariae by haemolysis and into the blood stream and reach the capillaries in the skin. concentration of blood samples) can be performed to detect the Mosquitoes become infected with microfilariae when they feed microfilariae of the Setaria species (Slim & Fouad, 1965). blood of infected hosts that contains microfilariae. These microfilariae develop to infective larvae inside the mosquitoes 3 Surgical treatments in 2 to 3 weeks. The infected mosquitoes then transmit these infective larvae to other susceptible hosts during their blood Although both medical and surgical treatments have been meals. advocated for the equine ocular filariasis (Muhammad & Saquib, 2007), the best treatment is the surgical removal of the The ocular setariasis spreads mostly in summer and autumn parasite (Tuntivanich et al., 2011) that can be performed under when the mosquito vectors are most prevalent (Mritunjay et al., general anesthesia or regional nerve blocks with or without 2011; Al-Azawi et al., 2012). The parasite exhibits migratory sedation. Regional nerve blocks like supraorbital, behavior in unusual hosts such as horses, donkeys or human auriculopalpebral and retrobulbar can be performed using 2% beings and can be found in various organs such as heart, lung, lidocaine as per the standard methods described in literature spleen, kidney, uterus, oviduct, ovary, and urinary bladder (Lumb & Jones, 2001). Akinesis of the eyelids can further be (Varma et al., 1971). All equines are generally more prone for achieved by blockade of the ventral and dorsal branches of the ocular worm (Pratap, et al., 2005; Jayakumar et al., 2012; palpebral nerve (Facial VII) (Skarda, 1996). Radwan et al., 2016).The immature worm can also invade eye (Sreedevi et al., 2002; Tuntivanich et al., 2011) through the The supraorbital nerve is desensitised as it emerges from the vascular system (Townsend, 2013). The eye infection occurs supraorbital foramen, which is easily palpated 1 cm caudal to when the adult worm meanders through intraocular tissue, thus the upper orbital rim, 5–7 cm dorsal to the medial canthus. By it is also called as eye worm. using a 23–25 gauge needle, 1–3 ml lidocaine can be injected subcutaneously and into the foramen. This desensitises the The infected animals usually display signs of photophobia and forehead and the middle two-thirds of the upper eyelid. Motor lacrimation (Basak et al., 2007). The serrated cuticle of the paralysis of the auriculo-palpabral nerve (VII) is achieved by worm and lashing movements within the anterior chamber of perineural administration of local anesthetics to this nerve at the eye caused severe trauma and inflammation to the cornea the most dorsal point of the zygomatic arch or just caudal to which then results into corneal opacity, which eventually the vertical ramus of the mandible, just ventral to the results into blindness (Jaiswal et al., 2006). Basak et al. (2007) zygomatic arch. The retrobulbar block may be achieved using has reported corneal edema caused by dead filarial worm a 19 gauge 80 mm long spinal needle passed over the attachment to the endothelium in the anterior chamber. The zygomatic arch in a ventro-medial direction until it encounters dead worm possibly liberates toxins into the anterior chamber, the medial wall of the bony orbit (Fletcher, 2004; Labelle & which may be lethal to the endothelium and resulting into Clark-Price, 2013). The cornea and sclera may be desensitised corneal edema. It may lead to devastating sequel like synechia, most effectively spraying topical application of 1% solution of cataract, and retinal detachment (Paglia et al., 2004). Though, amethocaine (Durham et al., 1992) or 1% tropicamide the involvement of the eye is commonly unilateral but bilateral (McMullen et al., 2014). occurrence has also been reported (Shin et al., 2002; Buchoo et al., 2005).

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Equine Ocular Setariasis and Its Management S141 Surgical interventions used for the treatment of ocular Placentrex facilitate post surgical healing at the insertion site in setariasis include needle paracentesis at 3 O’ clock (Sreedevi et equine ocular setariasis (Mritunjay et al., 2011). al., 2002; Vadalia, 2013) and nick incision at 12 O’ clock (Buchoo et al., 2005). Prior to surgery, it is better that horses 4 Medicinal therapies should receive topical non-steroidal anti-inflammatory agent (0.3% flurbiprofen) along with systemic non-steroidal anti- Taking in consideration complications of surgical treatment inflammatory agents (flunixin meglumine or ketoprofen) and like phthisis bulbi, corneal oedema, and scarring and prolapse antibiotics. For preparation of the eye for surgery topical of the iris (Lavach, 1990), various medicinal therapies have antiseptic (like 0.5% betadine) can be used (Patil et al., 2012). been advocated. However, Medical treatment has not been The head is held in still position with a twitch. Eye lids are considered suitable because of the slow absorption of dead retracted with the Castroviejo eye speculum and a stab incision parasites and the attendant antigenicity (Moore et al., 1983; is made at 12 O’ clock with BP blade No. 11 (Buchoo et al., Lavach, 1990). The standard antifilarial drug, 2005). The parasite usually gets ejected along with the aqueous diethylcarbamazine citrate (DEC) has given inconsistent humor; however, sometimes the parasite gets stuck in the results (Perumal & Seneviratna, 1954; Ahmad & Gupta, 1965). incision. In such cases the worm is removed with the help of Also, an inconveniently large number of repeat treatments (for forceps. example, 32 treatments over 45 days) (Razig, 1989) has precluded DEC as a practical chemotherapeutic agent for The incision is left unsutured. Dorsal and lateral approaches equine setariasis. Muhammad & Saquib (2007) have advocated allow monitoring of the incision postoperatively and at the a medicinal therapy for ocular equine microfilariasis using same time does not create the potential for possible suture ivermectin and death of the parasite in the eye took 15 days trauma associated with excursions of the nictitating membrane after administration of ivermectin. These suggested that in (Kalpravidh et al., 1992). However, when additional protection situations in which surgical intervention is difficult, the off- of wound by nictitating membrane is required a stab incision at label use of ivermectin would be appropriate to treat ocular the ventral margin of limbus is preferred (Patil et al., 2012). equine setariasis. The use of viscoelastic substance like hypromellose is injected into the anterior chamber to decelerate the vigorous movement Conclusion of the worm to facilitate the removal of the worm (Patil et al., 2012). Ocular setariasis commonly known as eye worm is a common surgical condition of equine eye affecting horse, donkey and In the second method, a 16 gauge needle is inserted into the pony equally. The condition can be easily diagnosed on the anterior chamber of the eye at 3 O’ clock position (Sreedavi et basis of clinical symptoms like lacrimation, photophobia, al., 2002) or at 6-8 O’ clock (Gopinathan et al., 2013) position blepharospasm, corneal opacity and visible worm in the of the cornea, approximately 1 mm away from the limbus, as anterior chamber of the eye. Surgical treatment under regional/ soon as the worm appear near this site. Due to the aqueous general anesthesia is an effective treatment of the condition. humor pressure, the eye worm usually escapes through the hub Though, medicinal therapy with ivermectin is advocated, of the needle or it appears at the puncture site thereby however relying on the medicinal treatment for too long should facilitating removal. Aqueous humor leakage is minimal as the be avoided. Ophthalmic ointments decreasing inflammation needle puncture hole is very small (Gopinathan et al., 2013). In and chances of infection and/or enhancing the healing can be a slightly modified needle technique a 21 gauge needle used to reduce the chances of postsurgical complication. attached with the syringe is inserted through the conjunctiva into the anterior chamber and directed carefully towards the Conflict of interest worm to aspirate the worm into the syringe (Yang et al., 2014). The puncture site is left without suturing. Needle stabbing Authors would hereby like to declare that there is no conflict of technique, is economical, time saving and recommended for interests that could possibly arise. the removal of parasite (Singh et al., 1976). Postoperatively sub-conjunctival injection of dexamethasone (2 mg)- References gentamicin (20 mg) may be given. Topical application of ofloxacin or other eye ointment is considered. Ahmad SA, Gupta BN (1965) Filaria oculi in equines: a therapeutic trial with the filaricidal drug Hetrazan Corneal opacity at the site of stab incision is the most common (diethylcarbamazine citrate) Lederle. Indian Veterinary Journal postoperative complication reported (Sharma et al., 2005). 42:140-142. Sometimes it diffuses to involve the whole upper quadrant (Patil et al., 2012). This takes days to 3 to 8 weeks to get Al-Azawi AK, Fadhl AR, Fadhl SR (2012) Epidemiological resolved (Buchoo et al., 2005; Jaiswal et al., 2006; Patil et al., study of Setaria equina infection in donkeys. Iraq Veterinary 2012). Human placenta extract has anti-inflammatory and Journal 36 : 93-97. analgesic effects and enhance wound healing (Piyali & Debasish, 2012; Changole et al., 2015; Shukla et al., 2016).

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Basak SK, Hazra TK, Bhattacharya D (2007) Persistent McMullen RJ Jr1, Davidson MG, Gilger BC (2014) The effect corneal edema secondary to presumed dead adult filarial worm of 1% tropicamide-induced mydriasis and cycloplegia on in the anteriorchamber. Indian Journal of Ophthalmology 55: spherical refraction of the adult horse. Veterinary 67-69. DOI: 10.4103/0301-4738.29501. Ophthalmology 17 : 120–125. doi: 10.1111/vop.12055.

Buchoo BA, Pandit BA, Shahardar RA, Parrah JD, Darzi, MM Moore CP, Sarazan RD, Whitley RD, Jackson WF (1983) (2005) Surgical management and prevalence of ocular filariasis Equine ocular parasites: a review. Equine Veterinary Journal in equines. Indian Veterinary journal 82: 81-82. Supplement 2:76-85. DOI: 10.1111/j.2042- 3306.1983.tb04565.x. Changole S, Gupta B, Nandagawali V, Palyekar A, Chipde H (2015) Comparative Study of Efficacy of Human Placental Mritunjay K, Monsang SW, Pawde AM, Singh SK, Madhu Extract Over Beta Glucan Collagen Sheets in Partial Thickness DN, Zama MMS (2011) Post surgical healing effect of Burn Patients. Bombay Hospital Journal 57 : 279-284. placentrex in equine (Equus cabalus) ocular setariasis: A review of 22 cases. The Indian Journal of Field Veterinarians Durham RA, Sawyer DC, Keller WF, Wheeler CA (1992) 6: 71-73. Topical ocular anesthetics in ocular irritancy testing: a review. Laboratory animal Sciences 42: 535-541. Muhammad G, Saqib M (2007) Successful treatment of ocular equine microfilariasis (Setaria species) with ivermectin. Fletcher BW (2004) How to perform effective equine dental Veterinary Record 160: 25-26. nerve blocks. Proceeding of American Association of equine Practioners 50: 233-239. Paglia DT, Miller PE, Dubielzig RR (2004) James Wardrop and equine recurrent uveitis. Archives of ophthalmology Gangwar AK, Devi S, Singh HN, Singh A (2008) Ocular 122:1218–1223. doi:10.1001/archopht.122.8.1218. filariasis in equines. Indian Veterinary Journal 85: 547-548. Parrah JD, Buchoo BA, Moulvi BA (2004) Ocular filariasis in Gopinathan A, Singh K, Saxena AC, Khurana KL, Amarpal equines. A study of 9 cases. Centaur 4: 70-71. (2013) Evaluation of two techniques for management of ocular Setariasis in horses. Research Opinion in Animal and Patil DB, Parikh PV, Nisha J, Jhala SK, Din DMU,Tiwari DK Veterinary Sciences 3 : 407-411. (2012) Equine eye worm: a review of 50 cases. Indian Journal of Veterinary Surgery 33: 61-62. Jaiswal S, Singh SU, Singh B, Singh HN (2006) Ocular setariosis in a horse. Intas Polivet 7: 67-68. Perumal PC, Seneviratna P (1954) Kumri (Syn: Kamri) in horses associated with ocular setariasis with a short note on Jayakumar K, Dharmaceelan S, Rajendran N, Senthilkumar S, attempted treatment. Ceylon Veterinary Journal 2:92-94. Kathirvel S, Nagarajan L, Kumaresan A (2012) Ocular Setariasis in a Pony. Indian Veterinary Journal 89 : 64 – 66. Piyali DC, Debasish B (2012) Aqueous Extract of Human Placenta. In: Zheng J (Ed.), Recent Advances in Research on Kalpravidh M, Bramasa A, Kalpravidth C (1992) Surgical the Human Placenta, InTech Publisher ISBN: 978-953-51- removals of intraocular parasites from the anterior chambers of 0194-9. the horse eyes. Thailand Journal of Veterinary Medicine 22: 13-20. Pratap K, Amarpal A, Aithal HP, Pawde AM (2005) Survey of eye disorders in domestic animals. The Indian Journal of Labelle AL, Clark-Price SC (2013) Anesthesia for Ophthalmic Animal Science 75:33–34. Procedures in the Standing Horse. Veterinary Clinics of North America: Equine Practice 29 : 179–191. doi: Radwan AM, Ahmed NE, Elakabawy LM, Ramadan MY, 10.1016/j.cveq.2012.12.001. Elmadawy RS (2016) Prevalence and pathogenesis of some filarial nematodes infecting donkeys in Egypt. Veterinary Ladoucer CA, Kazacos KR (1981) Thelazia lacrimalis in World 9 : 888-892. doi: 10.14202/vetworld.2016.888-892. horses in India. Journal of American Veterinary Medical Association 178: 301-302. Razig SA (1989) A preliminary clinical trial on the use of diethylcarbamazine citrate for the treatment of equine Lavach JD (1990) Parasitic diseases. In Large Animal filariasis. Acta Veterinaria (Belgrade) 38 : 145-151. Ophthalmology. Vol 1. Philadelphia, Mosby pp 260-263. Sathu S (1974) Intraocular parasites in horses. A report of five Lumb WV, Jones EW (2001) Local and regional anesthetic cases. Indian Veterianary Journal 5: 225. and analgesic techniques. In: Veterinary anesthesia, 3rd ed. Lea and Febiger, Philahelphia pp 449. Sellon CD, Long M (2013) Equine Infectious Diseases. 2nd ed. Science Direct publication, St. Louis, MI.

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Sharma R, Shrivastava HK, Chauhan S, Kumar R (2005) Townsend WM (2013) Food and fiber-producing animal Equine keratouveitis by Setaria spp. microfilaria and its ophthalmology. In: Gelatt KN (Ed). Essentials of veterinary management- A review of 14 cases. Intas Polivet 6: 260-261. ophthalmology, 2nd ed., Wiley-Blackwell publication, UK, pp: 532. DOI: 10.1002/9781118910337 Shin S, Cho K, Wee SH (2002) Ocular infection of cattle with Setaria digitata. Journal of Veterinary Medical Science 64: 7- Tuntivanich N, Tiawsirisup S, Tuntivanich P (2011) Success 10. of Anterior Chamber Paracentesis as a treatment for Ocular Setariasis in Equine Eye: Case Report. Journal of Equine Shukla AD, Kamath AT, Kudva A, Pai D, Patel N (2016) Our Veterinary Science 31: 8-12. DOI: Experience in the Management of Traumatic Wound Myiasis: http://dx.doi.org/10.1016/j.jevs.2010.11.017. Report of 3 Cases and Review of the Literature. Case Reports in Dentistry. DOI:10.1155/2016/7030925. Vadalia JV (2013) Surgical Treatment of Ocular Setariosis in a Stallion. The Indian Journal of Veterinary Science 13:11. Singh H, Chaudhuri PC, Kumar A (1976) Paracentesis oculi: a preferred technique for removal of intra-ocular parasites in Varma AK, Sahai BN, Singh SP, Lakra P, Shrivastava VK horses. Indian Veterinary Journal 53: 467-468. (1971) On Setaria digitata, its specific characters, incidence and development in Aedes vittatus and Armigeres obturbans in Skarda S (1996) Regional anaesthetic techniques In: Thurnon India with a note on its ectopic occurrence. Zeitschrift für JC, Tranquilli WJ, Benson JG (Eds) Lumb and Jones’ Parasitenkunde 36: 62-72. DOI: 10.1007/BF00328975. Veterinary Anesthesia, 3rd edn., Lea and Febiger, Baltimore. pp 448-477. Yadav A, Kumar A, Bhadwal MS, Khajuria JK, Gupta A (2006) Ocular setariosis in horses: A case study. Journal of Slim MK, Fouad KA (1965) Incidence of equine filariasis in Veterinary Parasitology 20 : 1-10. Egypt. Veterinary Medical Journal 10:113-118. Yang YJ, Cho YJ, Choi SK, Cho GJ (2014) Modified Needle Sreedavi C, Sudhakar K, Murthy PR, Prasad V (2002) Clinical Aspiration Technique for Extracting Live Eye Worm in a microfilariasis in a horse:a case report. Indian Veterinary Thoroughbred Horse. Journal of Animal and Veterinary Journal 79: 487-488. Advances 13: 998-1001. DOI: 10.3923/javaa.2014.998.1001.

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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

PARASITOLOGICAL, BIOCHEMICAL AND CLINICAL OBSERVATIONS IN PONIES EXPERIMENTALLY INFECTED WITH Trypanosoma evansi

Yadav SC1,*, Jaideep Kumar2, Gupta AK1, Jerome A4, Prabhat Kumar3, Rajender Kumar1, Kanika Tehri3 and Ritesh Kumar3

1ICAR-National Research Centre on Equines, Sirsa Road, Hisar, India 2Research Scholar, Guru Jambheshwar University of Science and Technology, Hisar, India 3Research Associate, cJunior Research Fellow, ICAR-National Research Centre on Equines, Sirsa Road, Hisar, India 4ICAR-Central Institute for Research on Buffaloes, Sirsa Road, Hisar, India

Received – October 25, 2016; Revision – November 08, 2016; Accepted – November 25, 2016 Available Online – December 04, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S144.S150

KEYWORDS ABSTRACT

Trypanosoma evansi The present investigation aimed to study the parasitological, biochemical and clinical alterations in Ponies ponies during the course of Trypanosoma evansi experimental infection. Six female ponies were experimentally infected sub-cutaneously with mice adapted 2x106 T. evansi parasites, isolated from Surra naturally infected horse, while two ponies were maintained as uninfected healthy controls. All six ponies became parasitologically positive between 5-7 days post infection (DPI) tested by standard Biochemical changes parasitological detection method (SPDM) by blood smear examination showing varying degree of parasitaemia and two prominent peaks during the course of infection. The main clinical signs observed Clinical signs were intermittent fever, weakness, emaciation, anaemia, anorexia and incoordination in hind quarters leading to significant weight loss at terminal stage of infection. All the infected ponies developed sub- Haematology acute to acute disease within 56 days and reached to recumbency stage. Of them, four ponies died at different stages of infection and few of them showing neurological signs at terminal stage of infection. Parasitaemia The present investigation also revealed that horse ponies are more susceptible than donkeys in experimental infection of T. evansi. Haematological studies showed a gradual fall in the levels of haemoglobin (Hb), hematocrit (HCT) and red blood cell (RBC) count from 10.57 to 4.83 (g/dl), 32.81 to 16.33 (%) and 8.53 to 3.33 (x1012 cells/l) respectively, in infected animals over the study period. Serum

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: yadavsc@rediffmail com (S. C. Yadav) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

S145 Yadav et al

urea, uric acid, triglyceride, cholesterol, bilirubin indirect (BID) and total bilirubin (BIT) contents

increased, while albumin contents significantly decreased in T. evansi infected ponies at different

stages indicating impairment of liver and kidney functions. However, no changes in parasitological and

biochemical responses were observed in the healthy controls.

1 Introduction of the present study was to record the characteristic clinical course of the disease as well as parasitological, haematological Animal trypanosomosis is caused by different species of and biochemical aspects in ponies experimentally infected with trypanosomes, including Trypanosoma vivax, T. evansi, T. Indian isolate of T. evansi. congolense, T. simiae and T. equiperdum in different parts of the world. Of them, T. evansi is the most widely spread 2 Material and Methods organism with the greatest range of hosts (Hoare, 1972), thus making it one of the most significant animal health problems in 2.1 Source and maintenance of T. evansi the world. T. evansi is transmitted mechanically and non- cyclically by haematophagus flies such as horseflies (Tabanus) T. evansi was isolated at National Research Centre on Equines and stable flies (Stomoxys) which act both as vector and host at Hisar, Haryana during 2009 from infected horse and was reservoir. This parasite is responsible for the disease 'surra' in maintained in mice by in vivo propagation. Briefly, the blood domestic as well as wild animals, causing severe constraints to was collected from the parasitological positive horse in agriculture development and leading to significant impact on ethylene diamine tetra acetic acid (EDTA) and injected livestock in Asia, sub-Saharan Africa, and Latin America. In intraperitoneally (i/p) in mice. The level of parasitaemia was India, trypanosomosis caused by T. evansi is enzootic, since it checked microscopically by collecting blood daily from tail of affects many species of domestic and wild animals. Several the mice. After attaining parasitaemic peak, T. evansi isolate sporadic outbreaks of equine trypanosomosis have been was maintained in the laboratory by inoculating the 104 reported from different states (Yadav et al., 2012; Kumar et al., parasites in naive mice, through i/p route. The parasites were 2013). The course of the disease lasts from one week to six also kept in cryopreserved form in liquid nitrogen for further months (Woo, 1977) and usually results in emaciation and use, as and when required. death. The pre-patent period varies from 4 to 13 days and parasitaemia displays an undulating course (Ramirez et al., 2.2 Experimental infection in ponies 1979). Eight female ponies, aged 9-12 months, procured from local Anaemia is commonly found in horses, donkeys, dogs and market, were divided in two groups (infected and healthy coatis experimentally infected with T. evansi (Soodan et al., groups), comprising of six (P1 to P6) and two ponies (P7 & 1996; Marques et al., 2000; Aquino et al., 2002; Herrera et al., P8), respectively. All the animals were examined for the 2002). The disease manifests itself in different forms: acute, presence of helminth ova/oocyst and treated with sub-acute, chronic and in-apparent. Clinical signs are only albendazol@10 mg/kg body weight six weeks prior to indicative of surra, which include progressive weakness, experiment. Further these animals were also confirmed for emaciation, fever, anaemia and death of affected animal. More abesence of T. evansi and Theilaria equi antibodies prior to recently rising trends of neurological cases due to T. evansi infection by antibody ELISA. These animals were housed in have also been reported, showing marked ataxia, hyper- fly proof stable and maintained throughout the experiment excitability, circling, depression, gradual onset of paralysis of under intensive system of management. They were fed on hind quarters (Rodrigues et al., 2009; Berlin et al., 2009; balanced diet consisting of water and green fodder ad libitum, Ranjith kumar et al., 2013). The disease progresses in two during the experiment. phases; an acute phase, characterized by high levels of parasitaemia and noticeable clinical symptoms, and a chronic The experimental infection was set up in six ponies by phase, characterized by low parasitaemia, which can either inoculating 2×106 parasites sub-cutaneously (s/c) / pony. lead to emaciation or become clinically unapparent with Another group of two ponies was kept as uninfected control undetectable changes in variables such as body temperature during the course of experiment. The serum/blood samples and haematocrit count (Fernández et al., 2009). The chronic were collected initially, at short intervals i.e. on day 0, 3, 5, 7, form is most common and is likely to present an association 10 and 14 from both groups. Thereafter, weekly blood samples with secondary infection due to immune-suppression caused by were collected for parasitological, biochemical and clinical T. evansi infection (Ahmed, 2008). observations till 56 days post-infection. During the period, three ponies which became terminally ill reached in recumbent Some alterations in blood biochemistry, including decrease in stage and were euthanized. The remaining three ponies at the blood albumin and increase in globulin levels, hypoglycemia end of experiment were treated with quinapyrimine sulphate @ and increase in icterus index, have been reported in donkeys 3 mg/kg body weight and monitored clinically after treatment. and horses (Soodan et al., 1996; Marques et al., 2000). The aim

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Parasitological, Biochemical and Clinical observations in ponies experimentally infected with Trypanosoma evansi. S146 The animal experimentation was carried out according to the 2.5 Statistical Analysis rules and regulations set forth by Committee for the Purpose of Control and Supervision of Experiments on Animals Statistical analysis was performed using SPSS (version 16), (CPCSEA) Animal Welfare Division, Ministry of using repeated measures ANOVA. Data represented as mean I Environment, Government of India. The research protocol for S E and considered significant at p < 0.05 the experimentation was duly approved by the Institute Animal Ethics Committee of the National Research Centre on Equines, 3 Results Hisar, Haryana. 3.1 Parasitological observations 2.3 Parasitological observations All six ponies inoculated sub-cutaneously with 2x106 T. evansi During the course of experiment, the parasites were observed became parasitologically positive by 5-7 DPI as tested by by wet blood smear examination and further counted in 50 standard parasitological detection method (SPDM) using wet/ microscopic fields at 400x magnification in thin blood smears thin blood smear examination. Parasitaemia was consistent in stained with Giemsa (Cadioli et al., 2006). all ponies (low to moderate) and parasites were regularly observed in blood examination during the course of experiment 2.4 Haematological and biochemical indices except 10th and 35th DPI (only P3 showed high count). The first peak of parasitaemia occurred on day 7 with an average of Erythrocyte count, packed cell volume and haemoglobin 416.17 parasites followed by next peak by 28 DPI with an content were obtained from HM5 Vet Scan haematology average parasite count of 324.67 (Figure. 1). It was interesting analyzer (Abaxis, Pvt. Ltd, USA) as per standard procedure to note that all the individual ponies on day 10 had nil parasite using the Vet Scan haematology kit, while biochemical count except pony P-3 which got high parasite count at 35 DPI. metabolites related to liver and kidney functions mainly [Urea, Thereafter, all the ponies showed relapsing or undulating Triglyceride (Tgl), Cholesterol (Chol), Albumin (Alb), Total parasitaemia ranging from only few trypanosomes in blood to Serum Protein (Prot), Bilirubin indirect (BID) and Total maximum densities 1-6 x107 parasites / ml. Body temperature bilirubin (BIT), Creatinine (Cre), Uric acid (UA), Calcium increased up to 102-107°F but was intermittent in nature (Cal)] were evaluated at different intervals in serum samples of throughout the experiment. The first rise in temperature both healthy and infected animals using XL system packs for occurred on 5th day after the appearance of trypanosomes in the each metabolite in a Clinical Chemistry Analyzer (ERBA- blood, followed by a succession of peaks. EM200).

Figure 1 Parasite count in ponies infected with T. evansi at different intervals during infection. *Only pony P3 was recorded with high parasite count of 439* parasites at 35 DPI, while all other five infected ponies had nil count

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S147 Yadav et al

with unsteady and irregular steps. Ponies subjected to physical exertion readily fell down and were unable to support weight on the hind limbs by 56 DPI.

The experiment was terminated at 61 DPI, as three ponies (P1, P2 & P5) died/ euthanized during 58-61 DPI while rest three of the debilitated ponies, which showed sub-acute disease (unable to stand, walk and anorexic) were treated with quinapyrimine sulphate (3 mg/kg body weight). After treatment, ponies were monitored for parasitaemia and found negative by 48 hrs using SPDM methods. These animals recovered well at 10 weeks post-treatment, showing normal haematological indices and maintained good health, except for pony- 4 that showed acute neurological signs, tilting of head, circling motion, hyper-excitability and was finally recumbent and euthanized.

3.3 Haematological observations

Haematological studies showed that there was a continuous and sharp fall (p < 0.05) in the levels of haemoglobin (Hb), hematocrit (HCT) and red blood cell (RBC) count from 10.57 to 4.83 (g/dl), 32.81 to 16.33 (%) and 8.53 to 3.33 (x1012cells/l), respectively in infected animals (Figure. 2) while in healthy ponies, no significant change in these parameters was observed. Moreover, no clinical, parasitological and biochemical changes were detected in two uninfected control ponies.

3.4 Changes in biochemical indices

Blood urea contents ranged from 20.52 to 57.37 mg/dl and 14.65 to 22.05 mg/dl, while uric acid content ranged from 0.84 to 1.96 and 0.66 to 0.89 mg/dl in infected and healthy ponies, respectively (Figure 3). In infected ponies, urea content Figure 2 Changes in haemoglobin, RBC and hematocrit values increased significantly 7 DPI onward and remained high in T. Evansi infected and uninfected ponies. throughout the experiment duration as compared to healthy ponies and zero days of infected ponies. Further a very sharp 3.2 Clinical signs increase in this index was observed after 56 DPI in three ponies (P1, P2 and P5) which died on 61DPI. In rest of the Apart from increased body temperature, there were few more three infected ponies (P3, P4 and P6), which survived on 61 symptoms of disease during the first 4 weeks of infection. The DPI, urea content either remained static or decreased slowly. main clinical signs observed in most of ponies were Uric acid content also showed a significant increase after 21 intermittent fever, fast breathing, lacrimation from eyes and DPI and followed similar pattern between 56 to 61 DPI. No became anorexic by 6 DPI. All the infected ponies displayed significant increase was observed in creatinine and calcium progressive emaciation, jaundice, mucosal pallor and in few contents of infected ponies as compared to healthy ones. cases sub-mandibular oedema also. Besides this, motor Triglyceride content showed a continuous increase in infected incoordination of hind limbs was observed (pony 4 and 5), ponies 7 DPI. Further cholesterol, protein BID and BIT reduced appetite and lateral recumbency (pony 1, 2 & 5) while contents increased significantly about one to two weeks before ponies 3 & 4 showed oedema in brisket, abdominal regions, death of infected ponies (P1, P2 and P5) while albumin content staggering gait and incoordination in hind quarters were also decreased sharply in later one as compared to healthy ponies. observed during the course of infection. At terminal stage of Among infected ponies, three ponies which remained alive, disease, all the ponies were reluctant to move, revealed changes in levels of various metabolites were appreciably high pronounced hind quarters weakness with ataxia and between 7 to 35 DPI but during later stages (56 DPI onward), incoordination of the hind limbs, demonstrating staggering gait no significant increase was observed.

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Parasitological, Biochemical and Clinical observations in ponies experimentally infected with Trypanosoma evansi. S148

Figure 3 Changes in the level of various metabolites in T. evansi infected (I) and healthy (H) ponies during the study period.

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S149 Yadav et al

Conclusion and Discussion Among biochemical indices, both liver and kidney functions were observed to be affected in T evansi infected ponies as In the present study, six healthy ponies inoculated their serum urea, uric acid, triglyceride, cholesterol, albumin, subcutaneously with T. evansi, became positive within 5-7 DPI BID, BIT contents varied and increased significantly at as detected by wet blood smear examination, however, pre- different stages during experimental period. Similar patent period has been reported to vary from 4 to 13 days and observations mainly in terms of serum albumin and globulin parasitaemia displays an undulating course during the infection have also been reported in donkeys (Cadioli et al., 2006). (Ramirez et al., 1979). In present investigation parasitaemia was consistent, throughout the experiment and all ponies Acknowledgements developed acute disease within 56 days post-infection. Three ponies died exhibiting symptoms of acute trypanosomosis and The authors are thankful to Director, National Research Centre fourth died due to neurological signs which appeared 10 weeks on Equines, Hisar for providing facilities to conduct the work. post-treatment with quinapyramine sulphate. Contrary to above The authors also wish to acknowledge Department of observations, the experimentally infected donkeys showed Biotechnology (DBT), Government of India for partial funding comparatively low parasitaemia and the disease persisted years through research Grant no. BT/PRi4499/ADV/57/107/2010 in together in sub-clinical stage without mortality (Kumar et al., terms of manpower support. The authors also wish to 2013). acknowledge and thank Mr. R.K. Dayal for technical support during the investigation. These observations clearly indicated that ponies are susceptible of T. evansi infection, while donkeys are resistant with a long Conflict of interest statement course of disease. The parasite count, body temperature and blood indices of the infected ponies underwent significant All authors disclose that they have no financial and personal changes. The high temperature was observed in all infected relationships with other people or organization that could ponies 8-15 DPI and was intermittent throughout the inappropriately influence (bias) their work, including experimental period and there was no definitive correlation employment, consultancies, stock ownership, honoraria, paid with peak parasitaemia. The increase in the temperature of expert testimony, patent applications/registration, and grants or infected animals is a characteristic that has been previously other funding. described and related to the waves of parasitaemia in experimental infections with T. evansi (Oshiro et al., 1989; References Uche & Jones 1992; Aquino et al., 1999; Marques et al., 2000; Dargantes et al., 2005). Thereafter, similar observations of Ahmed A (2008) Epidemiological studies (parasitological, intermittent fever, weakness, emaciation, anaemia, anorexia serological and molecular techniques) of T. evansi infection in and incoordination in hind quarters were recorded as reported camels in Egypt. Veterinary World Journal 1: 325-328. in experimental infection in equines (Marques et al., 2000; Wernery et al., 2001). Motorial disturbance, a frequent Aquino LPCT, Machado RZ, Alessi AC, Marques LC, de symptom of equine Surra, usually reported to affect mainly the Castro MB, Malheiros EB (1999) Clinical, parasitological and hind legs (Curasson, 1943; Horchner et al., 1983) was also immunological aspects of experimental infection with observed during this investigation. Trypanosoma evansi in dogs. Memorias do Instituto Oswaldo Cruz 94: 255-260. The alterations in the haematological indices observed during the course of infection are consistent with the findings of Aquino LPCT, Machado RZ, Alessi AC, Santana AE, Castro previous workers in horses infected with T. evansi, wherein fall MB, Marques LC, Malheiros EB (2002) Hematological, in haematocrit, erythrocyte counts and haemoglobin content biochemical and anatomopathological aspects of experimental was described. Anaemia has been a consistent finding in the infection with Trypanosoma evansi in dogs. Arquivo Brasileiro infected animals throughout the study period. Marques et al. de Medicina Veterinária e Zootecnia 54: 8-18. (2000) reported about 35% decrease in red cells count, packed cell volume and haemoglobin concentration in the Berlin D, Loeb E, Baneth G (2009) Disseminated central experimentally infected horses in three weeks of infection and nervous system disease caused by Trypanosoma evansi in a thereafter small variations were observed. However, the horse. Veterinary Parasitology 161: 316-319. animals remained anaemic until the end of observation period. Despite being a significant feature of trypanosomosis, the Cadioli FA, Marques LC, Machado RZ, Alessi AC, Aquino origin of anaemia in surra is not completely elucidated. LPCT, Barnabé PA (2006) Experimental Trypanosoma evansi Evidences suggest that its aetiology is multifactorial and infection in donkeys: hematological, biochemical and haemolysis, haemodilution or/ and noncompensatory histopathological changes. Arquivo Brasileiro de Medicina erythropoiesis are some of the mechanisms proposed (Jenkins Veterinária e Zootecnia 58: 749-756. & Facer 1985). In conformity with our findings, death of T. evansi infected animals without clear previous indications has been also reported earlier (Horchner et al., 1983).

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Parasitological, Biochemical and Clinical observations in ponies experimentally infected with Trypanosoma evansi. S150 Curasson G (1943) Trypanosoma vivax et variétés. In Traité de Oshiro ET, Rodrigues M, Nunes VLB, Ribeiro OC (1989). protozoologie vétérinaire et comparée Tome 1 Trypanosomes. Trypanosoma (Trypanozoon) evansi (Steel, 1885) Balbiani, Paris: Vigot Frères : 270-278. 1888, infecco experimental emequino com amostraisolada de capivara, Hydrochaeris hidrochaeris Linnaeus, 1766 Dargantes AP, Reid SA, Copeman DB (2005) Experimental (Rodentia: hydrochacridae). Semina 10: 51-55 Trypanosoma evansi Infection in the Goat. I. Clinical Signs and Clinical Pathology. Journal of Comparative Pathology Ramirez LE, Wells EA, Betancourt A (1979) La 133: 261-266. Tripanosomiases en los Animales Domisticos en Clolumbia. Centro Internacional de Agricultura Tropical pp 71. Fernández D, González-Baradat B, Eleizalde M, González- Marcano E, Perrone T, Mendoza M (2009) Trypanosoma Ranjithkumar M, Saravanan BC, Yadav SC, Kumar R, Singh evansi: A comparison of PCR and parasitological diagnostic R, Malik TA, Dey S (2013) Neurological trypanosomiasis in tests in experimentally infected mice. Experimental quinapyramine sulfate-treated horses – A breach of the blood- Parasitology 121: 1-7. brain barrier? Tropical Animal Health and Production 46: 371- 377. Herrera HM, Alessi AC, Marques LC, Santana AE, Aquino LP, Menezes RF, Moraes MA, Machado RZ (2002) Rodrigues A, Fighera A, Souza TM, Schild AL, Barros CSL Trypanosoma evansi experimental infection in the South (2009) Neuropathology of Naturally Occurring Trypanosoma American coati (Nausa nausa): hematological, biochemical and evansi Infection of Horses. Veterinary Pathology 46: 251-258. histopatological changes. Acta Tropica 81: 203-210. Soodan JS, Sood NK, Khahra SS (1996) Clinic-pathological Hoare CA (1972) The Trypanosomes of Mammals: A studies in donkeys experimentally infected with Trypanosoma Zoological Monograph. Blackwell Scientific Publications. evansi. Indian Journal of Animal Science 66: 443-448. Oxford, UK. Uche UE, Jones TW (1992) Pathology of experimental Horchner F, Schonefeld A, Wust B (1983) Experimental Trypanosoma evansi infection in rabbits. Journal of infection of horses with Trypanosoma evansi I. Parasitological Comparative Pathology 106: 299-309. and clinical results. Annales de la Societe Belge de Medecine Tropicale 63: 127-135. Wernery U, Zachariah R, Mumford JA, Luckins T (2001) Preliminary evaluation of diagnostic tests using horses Jenkins GC, Facer CA (1985) Hematology of African experimentally infected with Trypanosoma evansi. The trypanosomes. In Tizard, I. Immunology and Pathogenesis of Veterinary Journal 161: 287–300. Trypanosomiasis. CRC Press, Boca Raton pp13-44. Woo PTK (1977) 7-Salivarian trypanosomes producing disease Kumar R, Kumar S, Khurana SK, Yadav SC (2013) in livestock outside of sub-saharan Africa. In: Julius Kreir (Ed) Development of an antibody-ELISA for seroprevalence of Taxonomy Kinetoplastids and Flagellates of Fish. Academic Trypanosoma evansi in equids of North and North-western Press, New York, pp 269-296. regions of India. Veterinary Parasitology 196: 251-257. Yadav SC, Kumar R, Manuja A, Goyal L, Gupta AK (2012) Marques LC, Machado RZ, Alessi AC, Aquino LPCT, Pereira Early detection of Trypanosoma evansi infection and GT (2000) Experimental infection with Trypanosoma evansi in monitoring of antibody levels by ELISA following treatment. horses: clinical and haematological observations. Brazil Journal of Parasitic Disease 38 : 124-127. Journal of Veterinary Parasitology 9: 11-15.

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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EQUINE BRUCELLOSIS: REVIEW ON EPIDEMIOLOGY, PATHOGENESIS, CLINICAL SIGNS, PREVENTION AND CONTROL

Kumaragurubaran Karthik1,*, Govinthasamy Prabakar2, Ramasamy Bharathi1, Sandip Kumar Khurana3 and Kuldeep Dhama2

1Tamil Nadu Veterinary and Animal Sciences University, Chennai- 51, India 2Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India 3NRCE, Hisar, Haryana, India

Received – November 03, 2016; Revision – November 25, 2016; Accepted – December 02, 2016 Available Online – December 04, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S151.S160

KEYWORDS ABSTRACT

Brucellosis Brucellosis is one of the major zoonotic diseases that affect several domestic animals, wild animals and also marine mammals. Though there is no specific Brucella sp. that can affect horses, B. abortus and B. Equine suis can affect horses naturally and B. canis experimental infection has also been reported in equines. Brucellosis in equines is characterized by two conditions namely Poll evil and fistulous withers. B. abortus Organism has its predilection for joints, ligaments and tendons in case of equines and causes inflammatory conditions leading to formation of fistula. Equine brucellosis has been documented from Poll evil several parts of the world and prevalence has been reported time to time mostly based on serological diagnosis. Diagnosis of brucellosis mainly depends on serological methods though isolation of the Fistulous withers organism is the gold standard. Due to hazardous nature of the pathogen, tests like Rose Bengal plate agglutination test, Standard tube agglutination test and other serological assays are commonly LAMP employed. Isothermal amplification assay like LAMP are gaining momentum these years due to swiftness in diagnosis of the pathogen. LAMP with high specificity and sensitivity for detection of Brucella spp. and also B. abortus has been developed in the recent years. Prevention and control of brucellosis is of utmost important to halt the spread of the organism to other animals and human. Trauma is a major reason for predisposition of poll evil and fistulous withers hence proper fitting of saddle will help to prevent the disease. Housing and feeding the horses separately can prevent spread of disease from cattle. The present review discusses equine brucellosis, its epidemiology, pathogenesis, clinical signs along with appropriate prevention and control strategies to be adapted.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Kumaragurubaran Karthik) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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1 Introduction countries like Australia, Canada, Japan, Israel and New Zealand have controlled the disease (Nicoletti, 2007). Reports Brucellosis is a major zoonotic disease caused by the shows that age, breed and sex specificity does not exist for bacterium of the genus Brucella of which several species has brucellosis in equine though more cases are documented in been listed to cause disease in domestic and wild animals. horses above 3 years of age (Nicoletti, 2007). History shows that the disease was first identified in Malta in the year 1887 by Sir David Bruce who isolated Brucella 2 Epidemiology melitensis, the then Micrococcus melitensis from a soldier died due to Maltese fever (Godfroid et al., 2005). Recent years has Three species of Brucella namely B. abortus, B. suis and B. seen this bacterium as a pathogen causing disease in sea canis have been incriminated to infect horses, of which B. mammals thus showing the long journey of this pathogen from abortus and B. suis have been reported with natural infection Malta to Marine. There are eleven Brucella species namely B. and B. canis infection has been reported with experimental abortus (cattle), B. melitensis (sheep and goats), B. ovis infection (Hagler et al., 1982). (sheep), B. suis (hogs), B. canis (dogs), B. neotomae (wood rats), B. microti, B. maris, B. ceti and B. pinnipedialis (marine Brucella antibodies have been reported from several parts of mammals) and B. inopinata (isolated recently from women the world at various time frames showing that horses get breast implant) (Verger et al., 1987; Foster et al., 2007; Scholz infected from other animals. A study in Brazil was conducted et al., 2008). where 123 crossbred cart horse serum samples were collected from a period April 2005 to June 2006. These samples were These eleven Brucella spp. do not follow the strict rule towards subjected to Rose Bengal Plate agglutination Test (RBPT) host specificity as they can infect other animals and also which showed eight animals (6.5%) to be positive for brucella human beings hence this pathogen is considered as an antibodies. This study did not report isolation of the pathogen important zoonotic organism. B. abortus and B. suis affects and the seropositive animals did not show any clinical wild animals and reports regarding B. melitensis infection in manifestation of the disease (Antunes et al., 2013). A wild animals are less (Rhyan, 2000; Godfroid & Kasbohrer, multispecies farm in Nigeria comprising of different livestock 2002). There is no specific Brucella sp. that can cause disease species like cattle, horse, goat and sheep was investigated for particularly in horses. All the documented reports of B. abortus infection status. This study revealed that all the brucellosis in horse are mainly by the cattle pathogen, B. seven horses under the study were found positive by RBPT. abortus and to a milder extent B. suis biovar 3 (Lucero et al., Vaginal samples from 6 horses were subjected for isolation of 2008; Colavita et al., 2016). B. abortus which showed that two samples were positive for B. abortus. This study is the first report of isolation of the Members of the genus Brucella are facultative intracellular pathogen from non clinical cases from horse (Bertu et al., gram negative cocco-bacilli or short rods measuring around 0.6 2015). The report of this study warrants attention as disease to 1.5 μm long and 0.5 to 0.7 μm width. Brucella genus neither free horses can also play role in transmission of the pathogen form capsules nor spores nor motile though carry the genes for to other animals and also to human. Several other studies have motility (except the chemotactic system that helps in been conducted earlier in Nigeria to investigate the presence of assembling a functional flagellum) (Fretin et al., 2005). brucellosis in horses which documented 8.4% (14 positive of

Brucella genus is classified phylogenetically within the α2 166 animals) and 14.7% (11 samples out of 75 horses) which subdivision of Proteobacteria which comprises of were lesser compared to the recent survey documenting 100% Agrobacterium, Bartonella, Ochrobactrum, Rhizobium, positivity (Bale & Kwanashie, 1984; Ehizibolo et al., 2011). Rhodobacter, and Rickettsia (Moreno et al., 1990). Genome of Multispecies housing can increase the possibility of disease B. abortus has two circular chromosomes of 2.1 Mb and 1.5 spread. Already there are established reports that horse to horse Mb size without plasmids (Michaux et al., 1993; Michaux- or other animals is a less likely event and these animals do not Charachon et al., 1997). Recent years has seen the completion excrete the organism (Corbel & Henry, 1983; Macmillan & of genome of B. abortus (Sanchez et al., 2001), B. melitensis Cockrem, 1985). Sadiq et al. (2013) reported 5.5% sero- (GenBank NC 003317 and NC 003318) (DelVecchio et al., positivity of brucellosis by RBPT and Microtiter Serum 2002) and B. suis (GenBank NC 002969) thus increasing the Agglutination Test in donkeys from Borno and Yobe states of opportunity for understanding the pathogenicity of Brucella. Nigeria.

Brucellosis mainly causes reproductive diseases in various A very recent study conducted on the Mambilla plateau of animals, and in horses the clinical manifestation is termed as Taraba state, Nigeria showed 16% prevalence of brucellosis "poll-evil" or "fistulous withers" due to the inflammation of and adult horses were affected more that young animals (Ardo supraspinous bursa and connective tissue, leading to abscess & Abubakar, 2016). Another study conducted in the same formation and fistulation in the affected region and Taraba state of Jalingo region showed 7 animals out of 90 adult occasionally abortions and other reproductive problems are horses screened by RBPT (Ardo et al., 2016). also reported (Denny, 1972). B. abortus has been reported worldwide causing infection in domestic animals though some

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Equine Brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control. S153 Studies conducted in Iran also reported isolation of B. abortus few hours kills the bacteria and they are also readily killed by from infected horses and serological diagnosis by RBPT, commonly used disinfectants (Glynn & Lynn 2008). standard tube agglutination test (STAT) and 2-mercapto ethanol (2ME) showed 2.5% to 12% prevalence (Tahamtan et 3 Pathogenesis al., 2010). Another study in the Hamadan region of Iran showed 0.5% prevalence by RBPT and STAT (Ghobadi & The major route of entry of the pathogen into the host Salehi, 2013). Positive animal in this study was also in close (equines) is through the consumption of feed contaminated contact with other animals hence there was a higher chance of with B. abortus and contact infection from cattle has been infection. Earlier study in the Mashhad region of Iran also reported by several workers (Denny 1973; O’Sullivan 1981; showed 2.5% positive cases (Tahamtan et al., 2010). Ocholi et al., 2004). There prevails mystery regarding the role played by horse in the transmission of infection to other horses An epidemiological survey was conducted in Peshawar district or to other animals/ humans. Hence there is no clear evidence of Pakistan employing 500 serum samples from horses (n= that supports that horse’s act as a ware house/ reservoir for 196), donkeys (n= 267) and mules (n= 37) collected during a transmission of brucellosis in endemic areas (Acosta-Gonzalez time period of January to December, 2012. Higher prevalence et al., 2006). (71.93%) was reported in horses than donkeys (63.67%) and mules (5.4%). Study also reported females and animals of 5-11 Being a facultative intracellular pathogen, Brucella survives, years were affected most (Safirullah et al., 2014). Younger multiplies and evades host immune mechanism simultaneously animals tend to clear the pathogen faster though there is found developing inside phagocytic cells (Gorvel & Moreno, possibility of latent infection which may be the reason that the 2006). Ecological niche inside the phagosomal compartment of study had higher infection rate among older animals (Quinn et host macrophages is conducive for the survival of brucellae al., 2004). Sex hormone and erythritol tend to increase as the and maintaining chronic infections depends upon the ability of age advances and this may be another reason brucella infection surviving and replicating within these phagocytic cells (Roop is seen in older animals after sexual maturity (Radostits et al., et al., 2004; Neta et al., 2010) (Figure 1). Brucella infection 2000). occurs through ingestion and the organism enters the oral and pharyngeal cavities (Brinley et al., 1990). The bacteria are Serum samples of 227 equines (178 donkeys, 43 horses and 6 transported following penetration of the mucosal epithelium mules) collected in the Mossoró, Rio Grande do Norte, Brazil either free or within phagocytic cells to the regional lymph were subjected to RBPT, STAT and 2 ME showed 1.76% nodes through macrophages leading to spread and prevalence of brucellosis in horse serum (Dorneles et al., multiplication of organism in lymph nodes, spleen, liver, bone 2013). A massive survey was conducted recently employing marrow, reproductive organs, tendon sheath and bursae occur serum samples of 6,439 animals comprising samples from (Canning et al., 1986; Riley et al., 1984; Memish et al., 2000; 5,292 horses, 110 donkeys and 1,037 mules from 1936 herds Adams, 2002). The capacity of Brucella to hide inside the of Minas Gerais State, Brazil over a period of September 2003 macrophages makes it difficult to diagnose the disease and also and March 2004 (Junqueira et al., 2015). RBPT and STAT was hinder in the treatment of the disease (Glynn & Lynn 2008). performed which showed that 70 horses out of 5,292 animals to be positive (1.32%), 1 out of 110 donkeys (0.91%) and 14 4 Clinical signs out of 1,037 mules (1.35%). This study documented that males (52 out of 4106 males) had higher sero-positivity compared to In equines, the clinical signs due to brucellosis are mostly female (33 out of 2333 females) (Junqueira et al., 2015). noticed in the musculoskeletal system mainly as the organism localise in the bursae (causing septic bursitis), joints (causing A study in Western Sudan employing serum samples from 346 septic arthritis) and tendon sheaths (causing septic horses and 28 donkeys showed 4.9% and 3.6% prevalence of tenosynovitis) (Denny, 1972, Denny,1973; Carrigan et al., brucellosis in horses and donkey (Musa, 2004). A total of 1954 1987; Ocholi et al., 2004). Few reports regarding abortion, serum samples (horses 782 and donkeys 1172) were collected vertebral osteomyelitis and infertility in male horses have also from Sanliurfa and Diyarbakir provinces of South-East Turkey been documented (Collins et al., 1971; Denny 1973). Most and were subjected to RBPT. Prevalence was higher in horses classical clinical signs observed in horses due to brucellosis are (13.68%) than donkeys (6.05%) (Tel et al., 2011). Several poll evil (septic supra-atlantal bursitis) and fistulous withers other workers have earlier studied the prevalence of brucellosis (septic supraspinatous bursitis). Draining sinuses are seen as in horse in various regions of Turkey. Solmaz et al. (2004) these problems are chronic in case of equines (Crawford et al., reported 60.59% prevalence of brucellosis by RBPT in horses 1990). An experimental infection of B. abortus instillation into in the Van province of Turkey. Göz et al. (2007) reported 9.5% the conjunctival sac was carried out in a horse to know the horses to be positive by STAT in Hakkari region of Turkey. clinical signs. Serum antibody level was evident after 7 to 12 days of infection and intermittent bacteraemia was observed Brucella organism is to an extent resilient which has been for 2 months (MacMillan et al., 1982). recovered from manure and fetal samples even after 2 months when kept in cool weather condition. Exposure to sunlight for

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S154 Karthik et al

Figure 1 Pathogenesis and clinical signs of equine brucellosis.

Lesions are of granulomatous type were observed in lung, colony to appear. At times it may even take longer; hence liver, testes and metatarsophalangeal synovial membranes culturing work is laborious and time consuming. The major (Megid et al., 2010). No apparent clinical signs were noted and setback in culturing is that Brucellosis is zoonotic and the female animals bred normally yielding negative results to handlers are always at the risk of infection (Karthik et al., isolation of organism (MacMillan & Cockrem, 1986). B. 2014a). Hence it needs level 3 biocontainment facilities and abortus induced abortion in equines are not common though highly skilled technical personnel for handling live culture some documented reports state that mid to late term abortion (Alton et al., 1988). may occur (McNutt & Murray 1924; McCaughey & Kerr 1967; Shortridge, 1967; Robertson et al., 1973; Hinton et al., Serological tests like Rose Bengal Plate Test (RBPT), Standard 1977). Organism in the vaginal excretions does not last longer Tube Agglutination Test (STAT) and Enzyme Linked as compared to cattle. Immunosorbent Assay (ELISA) are commonly employed (Nicoletti, 2007). Other serological tests include complement Some authors reported the isolation of B. suis from aborted fixation test (CFTs), 2-mercaptoethanol (2ME), buffered equine fetuses (McNutt & Murray, 1924), horses affected with Brucella antigen tests (BBAT), Milk ring test (MRT), etc. septic bursitis (Portugal et al., 1971) and also from the (Acha & Szyfres, 2003; Godfroid et al., 2010). These tests are reproductive organs of mares without apparent clinical signs inexpensive, fast and sensitive but not necessarily highly (Cvetnic et al., 2005). specific, antibodies may cross react with Yersinia enterocolitica serotype O:9, Escherichia coli O: 157, 5 Diagnosis Francisella tularensis, Salmonella urbana O: 30, Vibrio cholerae, and others (Radostits et al., 2000). The gold standard test for diagnosis of Brucellosis is isolation and identification of the organism which needs 5-10% carbon Molecular techniques like PCR has been employed with dioxide for its growth. Even with all the conditions conducive various samples like blood, serum tissues from aborted foetus, for the growth of Brucella, it will take around 3-5 days for a semen and milk for diagnosis of brucellosis (Fekete et al.,

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Equine Brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control. S155 1992; Leal-Klevezas et al., 1995; Queipo-Ortuno et al., 1997; material gets discharged through one or more opening. These Amin et al., 2001; Kanani, 2007). More recently, real-time fistulas may heal slowly and there are always chances that they PCR has been used for detection of Brucella, offering reappear (Cohen et al., 1992). Few years back researchers have improvement in detection times and specificity (Queipo- found an Iron Age horse cranium from Tuva Republic, Central Ortuno et al., 2005). Real-time PCR is the latest method used Asia and the cranium had occipital lesions, cavities around the in which hybridization probes are used to increase specificity nuchal ligament attachment site was noted in the skull which (Bricker, 2002; Probert et al., 2004; Elfaki et al., 2005). may be due to inflammation followed by necrosis (Bendrey et al., 2011). The researchers concluded that condition is poll evil Isothermal amplification assay has the advantage of employing originating due to bacterial cause. Authors discussed that this a set temperature for amplification of DNA, reducing the time case may be due to B. abortus the common cause of poll evil in for amplification and also there is no need for post horses. Thus this study shows that the pathogen existed long amplification protocol for result visualization (Dhama et al., back causing poll evil in horses (Bendrey et al., 2011). 2013). LAMP for the Brucella was developed against Brucella cell surface protein (bcsp)31 gene and omp25 gene and Drainage of the infected tissue (poll evil and fistulous withers) sensitivity of both LAMP assay was higher than PCR (Ohtsuki and treatment with systemic antibiotics can be employed to et al., 2008; Lin et al., 2011). LAMP with visual detection treat brucellosis in horse. Chloramphenicol, tetracyclines, based on calcein has also been developed targeting the same streptomycin and some sulphonamides are commonly used for omp25 (Pan et al., 2011). Reports regarding development of treatment of Brucellosis but these antimicrobials cannot LAMP targeting IS711 genes and real time quantitative LAMP penetrate the infected tissues (Nicoletti, 2007). Clofazimine are available (Pérez-Sancho et al., 2013). Visual LAMP has been reported to have good effect in the treatment of targeting omp25 gene including loop primers was developed brucellosis in equines (Knottenbelt et al., 1989). B. abortus recently that can detect all Brucella spp. Developed LAMP S19 vaccine has also be used with good effect for treatment of assay with loop primers was 10 fold more sensitive than brucellosis with the regimen ranging from one dose to three commonly employed PCR (Karthik et al., 2016). LAMP assay doses at 10 days interval (Denny, 1973; Gardner et al., 1983; has the intrinsic property of product carry over contamination Cohen et al., 1992; Nicoletti, 2007). Use of this vaccine for hence a novel closed tube LAMP assay was also developed for treatment also involves local and systemic reactions and death detection of Brucella spp. (Karthik et al., 2014b). LAMP assay has been reported in a horse that received intra venous for specific diagnosis of B. abortus was also developed which injection (Denny, 1973; Cohen et al., 1992). Periodical was 100 fold more sensitive than the commonly employed drainage, cleaning the region with antiseptics and dimethyl PCR (Karthik et al., 2014c). Severity of bone damage in cases sulphoxide will aid to control further complications of poll evil of poll evil and fistulous withers can be identified by and fistulous withers (Cohen et al., 1992). radiography. 7 Prevention and control 6 Poll evil and fistulous withers a. In most of the instances horse gets brucellosis when Poll evil and fistulous withers are chronic inflammatory they are housed or allowed for grazing together with conditions affecting supra-atlantal bursa and supraspinatus cattle. Hence horse should be housed or allowed for bursa and its associated tissues respectively (Gaughan et al., grazing away from cattle suspected for brucellosis 1988; Rashmir-Raven et al., 1990; Cohen et al., 1992). The (Cramlet & Bernhanu, 1979). term fistula refers to draining wound from a normally closed b. Trauma is a major cause of predisposition of fistulous structure, through the skin and fistulous withers is an infection withers in horses hence proper fitting of saddle has to of the bursa overlying the spines of the withers by usually be taken care. caused by Brucella sp. in horse. Though B. abortus has been c. Parasitic problem Onchocerca spp. can also cause incriminated as the major cause for this condition, there are fistulous withers hence minimizing the parasitic load other pathogens/ wound which also plays role in causing this by proper hygienic measures needs to be practiced condition in horses. Other pathogens like Streptococcus (Cramlet & Bernhanu, 1979). zooepidemicus, Streptococcus equi, Staphylococcus aureus, d. Many of the countries follow testing and quarantine Staphylococcus epidermidis, Corynebacterium spp. the horses or euthanize the horse since brucellosis is Actinomyces bovis, Bacteroides fragilis, Proteus mirablis, zoonotic. Hence measures needs to be followed for Escherichia coli and Pasteurella spp. have also been isolated early diagnosis of the disease so that horses can be from horses suffering from fistulous withers (Cohen et al., segregated or culled to prevent further spread of the 1992; Hawkins & Fessler, 2000). Signs of fistulous withers disease. include single or multiple draining tracts and in some case disseminated swelling in the wither region without drainage. 8 Conclusion Walls of the bursa gets thickened carrying clear, thick fluid which is of straw coloured (Megid et al., 2010). Initial signs Brucellosis, an age old disease is one of the important zoonotic include pain, swelling, heat at the bursal region leading to disease that can infect several domestic animals, wild animals stiffness of the neck. Later stages the bursa ruptures and pus and also marine mammals. Eleven brucella species have been

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S156 Karthik et al described till date of which many are host restricted though it Megid J (2013) Serology for Brucella abortus in cart horses can infect other animals and also human. There is no single from an urban area in Brazil. Arquivo Brasileiro de Medicina species of brucella that can cause brucellosis in equines. B. Veterinária e Zootecnia 65 : 619-621. DOI: S0102- abortus and B. suis have been implicated to cause natural 09352013000200044 disease in equines. Mostly brucellosis in domestic animals are oriented to reproductive tract infection but in equines it causes Ardo MB, Abubakar DM (2016) Seroprevalence of horse mainly two important conditions affecting the musculoskeletal (Equus caballus) brucellosis on the Mambilla plateau of Taraba system termed as Poll evil and Fistulous withers. Abortions are State, Nigeria. Journal of Equine Veterinary Science 27 : 1–6. also noticed in some cases but shedding of bacteria through the DOI: 10.1294/jes.27.1 vaginal discharge is not documented yet. Though the disease has been reported for several years in horses there are still Ardo MB, Abubakar DM, Adamu Z (2016) Prevalence of several questions that need to be clarified like the pathogenesis Brucella antibodies in horses (Equus Caballus) in Jalingo, of bacterium in horse, predilection of bacterium towards Taraba State, Nigeria. Journal of Public Health and musculoskeletal system in horse while mainly reproductive Epidemiology 8 : 111- 114. DOI: 10.5897/JPHE2016.0811 tract in other domestic animals. Diagnosis of brucellosis at the early stage is important to identify the diseased animals so that Bale JO, Kwanashie GG (1984) Seroprevalence of bruellosis prior segregation of the animals can be done to minimize its among horses in Northern Nigeria. Journal of Animal spread to other animals. Advances in the field of diagnostics Production Research 4:161-164. have made it possible to identify the agent early though proper sampling procedures needs to be practiced to achieve the same. Bendrey R, Cassidy JP, Bokovenko N, Lepetz S, Zaitseva GI Similarly treatment and vaccination aspects needs to be (2011) A Possible Case of ‘Poll-Evil’ in an Early Scythian strengthened in order to control and eliminate the disease from Horse Skull from Arzhan 1, Tuva Republic, Central Asia. the animal population so that its spread to humans can also be International Journal of Osteoarchaeology 21: 111–118. prevented. Bertu WJ, Ocholi RA, Gusi AM, Abdullahi S, Zwandor NJ, Conflict of interest Durbi IAA, Opara J, Okewole PA (2015) Brucella abortus infection in a multispecies livestock farm in Nigeria. Authors would hereby like to declare that there is no conflict of International Journal of Biotechnology and Food Science 3: interests that could possibly arise. 36-40.

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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

BIOTECHNOLOGICAL TOOLS FOR DIAGNOSIS OF EQUINE INFECTIOUS DISEASES

Minakshi Prasad1,*, Basanti Brar1, Ikbal1, Koushlesh Ranjan2, Upendra Lalmbe1, J. Manimegalai1, 1 4 3 Bhavya Vashisht , Sandip Kumar Khurana and Gaya Prasad

1Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India, 125004 2Department of Veterinary Physiology and Biochemistry, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, India, 250110 3Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, Uttar Pradesh, India, 250110 4NRCE, Hisar, Haryana, India, 125001

Received – November 05, 2016; Revision – November 20, 2016; Accepted – December 04, 2016 Available Online – December 04, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S161.S181

KEYWORDS ABSTRACT

Biotechnology Rapid diagnosis of infectious diseases and appropriate treatment with in time are important steps that Immunoassay promote optimal clinical outcomes and general public health. Today there is large number of new technologies such as nanotechnology, biosensors, and microarray techniques, are being developed and Equine used as diagnostic tools for equine infectious diseases. Nucleic acid based techniques such as polymerase chain reaction (PCR) have become conventional tools in veterinary research and plays an Infectious disease important role in specific typing determinations as well as for rapid screening of ample numbers of samples at the time of equine disease outbreaks. Other biotechnological techniques are populous to be used in the coming times as they can enhance diagnostic efficacy in less time and cost as compared to conventional techniques. This review focuses on biotechnological tools available for equine diseases diagnosis and its applications hold great promise for improving the speed and accuracy of diagnostics for equine infectious diseases.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Minakshi Prasad) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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1 Introduction 2 Serological assays

Modern molecular biology provides us newer technology to Protein based assays are based on antibody and antigen diagnose and control several equine diseases. It is also used for interaction. These types of several assays such as enzyme the development of novel diagnostic tools for equine infectious linked immunosorbent assay (ELISA), falcon assay screening disease control (Pusterla et al., 2006; Amaya, 2014). Several test –ELISA, indirect or direct immunofluroscencent antibody diagnostic tools such as nucleic acid probes, monoclonal tests, immunoblotting dot-ELISA, peptide based-ELISA, antibodies, restriction fragment length polymorphisms, real complement fixation test, agar gel immunodiffusion and time PCR, proteomics, biosensors and nanotechnology have neutralization test are used for equine infectious disease increased the livestock productivity. These methods have been diagnosis. These serological assays are highly sensitive and commonly used for equine disease diagnosis and control (Yeh specific than other techniques like microscopy and it allow et al., 2010; Johnson et al., 2010; Rakhshandehroo et al., clearance of post-therapeutic pathogen. 2014). Several viral and bacterial pathogens such as Japanese encephalitis virus, West Nile virus, Hendra virus, borna virus, 2.1 Enzyme-linked immunosorbant assay (ELISA) equine rabies, Rhodococcus equi, Bacillus anthracis etc., are causes several serious diseases in equines and induce economic Components of immune system used for detection of immune to human population and these are zoonotic in nature (Yeh et response against infection in ELISA test. For detection of al., 2010; Booth et al., 2010; Priestnall et al., 2011; Khurana, specific immune response, ELISA assay involves antigen, 2015). antibody and enzymes. The antigens are adhered to surface of microtitre plate and antibody specific to the antigen is applied On various occasion equines are used for various purposes over the surface for binding. It was followed by the such as ceremonies, riding, sports, draught racing, transport conjugation of antibody with an enzyme-Horseradish and antitoxin/antibody production, throughout the world peroxidise. Further, substrate was added to the plate for (Burnouf et al., 2004). There is possibility of disease producing visible colour change in a reaction mixture. Based transmission and spread at the time of equines movement from on use or not of a secondary antibody, the ELISA test may be one country to another. Therefore, OIE (World Organisation either direct or indirect (Figure 1). This test is successfully for Animal Health) has enlisted several diagnostic tests for used for diagnosis of various diseases in equines. Singha et al. international movement of equines (Table 1) (OIE, 2016). (2014) have reported an indirect ELISA using truncated TssB Biotechnology may play an important role in prevention of protein for serodiagnosis of glanders. disease caused by these pathogens. A sensitive antigen capture ELISA was developed for the The correct knowledge of molecular biology of infectious detection of secreted NS1 from infected equines with West agents and their hosts is very important for controlling the Nile virus (Macdonald et al., 2005; Chung & Diamond, 2008). disease (Tavares et al., 2011). Biotechnological and protein Similarly, ELISA has been developed for the detection of based assays can play a main role in equine disease control due EHV-1, EHV-4 (Yasunaga et al., 2000), equine rhinitis virus A to its everlasting developments with the use of developed anti (ERAV) (Kriegshauser et al., 2009) and equine rhinitis virus B pathogenic drugs and diagnostic chemicals. Even though (ERBV) (Kriegshauser et al., 2008). In the recent studies, conventional techniques are still used commonly, recent ELISA targeting antibodies to the spike (S) of equine corona biotechnological assays have widened the scope of equine virus was developed and validated to detect antibodies to diseases detection and give us powerful new techniques for EqCoV in infected horses (Kooijman et al., 2016). quick and specific identification of equine diseases. This manuscript reviews the current and potential uses of biotechnology tools for equine infectious disease diagnostics.

Table 1 Prescribed test for equine diseases according to OIE, 2016.

Disease name OIE prescribed tests African horse sickness CF, ELISA Contagious equine metritis Agent identification. Dourine CF Equine infectious anaemia AGID Equine piroplasmosis ELISA, IFA Equine viral arteritis Agent identification (semen only), Virus Neutralization Glanders Complement Fixation

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Figure 1 Principle of ELISA test a) Direct ELISA b) Indirect ELISA

2.2 Dot-ELISA The overall specificity and accuracy of IFAT was shown to be better than that of the western blot and modified western blot, Dot-ELISA works on the basis of attachment of small amount which showed its potential to use as a diagnostic assay for of antigen on to a nitrocellulose membrane. Specific antibody detection of EPM caused by Sarcocystis neurona (Duarte et al., is incubated with antigen containing dotted membrane 2003). The FAT and immune-histo-chemistry (IHC) assay followed by adding of enzyme conjugated anti-antibody. A confirmed the presence of Australian bat lyssa virus (ABLV) substrate is added in the last which causes precipitation of a antigen in horse brain tissues (Shinwari et al., 2014). A FAT detectable coloured dot on the membrane (Svobodova et al., assay has been used for the direct identification of bacterial 2013). It was reported that the dot-ELISA is simple, quick, Helicobacter on the equine gastric mucosa (Perkins et al., specific, sensitive, low cost field test that detects minute levels 2012). This technique has been used to describe the spatial of antibodies much faster than complement fixation test and distribution of Helicobacter species in the stomach of healthy indirect hemagglutination antibody test (Verma & Misra, 1989; horses to demonstrate the microbiota of normal appearing Verma et al., 1990). Dot-ELISA has been used for the sero- squamous and glandular mucosa (Burton et al., 2007). diagnosis of glanders (John et al., 2010). By the use of nitrocellulose membrane in this test makes it applicable in the 2.4 Complement Fixation Test field. This assay is quick and specific in detection of various diseases. It gives us low background as compared to ELISA Complement fixation test (CFT) is an immunological test used assay that can easily differentiate between the positive and for detection of presence of either antigen or antibody in the negative samples. serum sample. It was generally used for microorganisms which are not easily cultured in research laboratory (Figure 3). 2.3 Fluorescent Antibody Test (FAT) Although, several studies have revealed its low specificity and sensitivity for virus detection, it is still used for many equine In Fat assay, antibody is labelled with fluorescent dye, is used viral disease diagnoses. CFT is the OIE recommended test for in visualization of antigen in a clinical specimens. The glanders. Due to low prevalence of glanders in equine antibody conjugated with fluorescent dye and antigen-antibody population it is important to use test with high specificity and complex gives a visible glow sign when examined under a sensitivity. CFT was found reproducible and reliable assay for fluorescent microscope. The fluorescent dye can be tagged clinical investigation and detection of latent infection of directly with primary antibody which is known as direct Equine herpes virus 1 (EHV1) (Hartley et al., 2005). A CFT fluorescent antibody test or with a secondary anti-antibody assay has some limitations such as laborious, time consuming known as Indirect Fluorescent Antibody Test (Figure 2). The and often cross reactivity in nature. The non-specific hemolysis

FAT is used in diagnosis of several equine diseases. This assay of RBC can be prevented using Potassium Periodate (KIO4). was recently investigated for diagnosis of equine leptospiral The KIO4 treatment to horse sera prevented the non-specific abortion in mare (Erol et al., 2015). The sarcocystis neurona hemolysis which helped in determination of precise titers causes a dreadful disease, equine protozoal myeloencephalitis during CF test for EHV-1 diagnosis (Bannai et al., 2013). The (EPM) in equines. The IFAT was successfully validated for CFT and virus neutralisation assays were used for CSF testing for confirmation of EPM in equines (Duarte et al., determination of sero-conversion of EHV1 and EHV4 during 2006; Johnson et al., 2013). obtaining acute and convalescent serum samples (Hartley et al., 2005).

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Figure 2 Principle of Direct Fluorescent antibody test (FAT).

Figure 3 Complement fixation test.

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Figure 4 Principle of lateral flow test

2.5 Lateral Flow Test (LFT) infection of cells when added to the plate. At the time of very high concentrations of antibody to the virus in target are This test is also known as a lateral flow present in the test sample, virus neutralization will be occur at immunochromatographic strip assay. LFT is used as diagnostic high serum dilutions. Whenever, where some or no antibody to techniques in medical and veterinary applications. It is an easy the virus is present in the serum sample, it will be able to and fast assay used for identification of interest target in neutralize the infectious virus at the first dilution used in the sample without using any special equipment (Figure 4). This test. The result of the test is the target at which the serum technique can be used for qualitative or specific semi- sample has been diluted such that it no further neutralizes the quantitative identification of many interest targets such as entire virus in the test. This dilution indicates the titre of the antibodies, antigens and nucleic acid products. The assay serum tested. The sero-prevalence of EHV-1 and EHV-9 indicates a procedural control line which shows that the assay infections was reported by serum neutralization test (Borchers was performed properly and validates the test result. Therefore, et al., 2005; Taniguchi et al., 2000). presence of two lines gives positive result, while indication of only control line shows negative test in experiment. However, 2.7 Agar Gel Immunodiffusion (AGID) the appearance of no lines or only test line shows invalid result and test must be repeated. This test has been successfully used AGID technique is used for the detection, identification and in diagnosis and detection of various disease associated with quantification of antibodies and antigens present in biological equines from biological samples. The recombinant viral capsid samples. In this technique, a gel plate is cut to form a series of protein p26 conjugated to colloidal gold based simple wells in agar gel. A sample aliquot of interest target is placed immunochromatographic lateral flow (ICLF) test was validated in one well, and antibodies are placed in nearby well and the for specific detection of Equine infectious plate was incubated for 48 hours. During incubation time the anemia virus (EIAV) antibodies in equine sera (Alvarez et al., antigens in the target sample and the antibodies each diffuse 2010). Similarly, LFT was also used for detection of vesicular out from their corresponding wells. At the point where the two stomatitis virus in cattle and horse clinical samples (Ferris et diffusion lines intersect, if any of the antibodies is specific to al., 2012). any of the antigens then they will bind to each other and form a complex. This antigen-antibody complex precipitated gives a 2.6 Virus neutralization test thin white line in the gel, helps in the visual identification of antigen recognition. AGID can be used to diagnose Equine In virus neutralization tests, serial dilutions of heat inactivated Infectious Anemia (EIA) (Beltrao et al., 2015). It detects test serum are prepared and poured in a 96 well plate and are antibodies against the main capsid viral protein (p26) in horse incubated with a defined amount (generally 100 TCID 50) of serum samples and this test is simple, inexpensive and specific infectious virus (antigen). After incubation time period, to identify EIAV-infected animals (Alvarez et al., 2010). susceptible virus cells are added to the virus-serum mixture and the final serum, virus and cell combination is kept for 2.8 Peptide based-ELISA period of 2-3 days. Depending on the virus, this may be done by microscopic examination of the plate for the indication of Petide based-ELISA, a plate based techniques for detecting and viral cytopathic effect (CPE). Serum containing antibodies quantifying peptides, proteins, hormones and antibodies. In specific to the virus in target are capable to neutralize the this technique, a synthetic peptide is to be mobilized onto a aliquot of virus used in the test line, hence preventing the solid support and complexes with an antibody, linked to an

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S166 Prasad et al enzyme. Detection is done by assessing the conjugated enzyme Soutullo et al. (2001) evaluated the performance of an equine activity after incubation with a substrate to produce a infectious an Aemia-ELISA designed with synthetic peptides. measurable end product (Figure 5). The very important step for This assay could be important to prove for large throughput the detection strategy is a specific antigen-antibody interaction. screening and early detection of equine infectious anaemia Peptide-ELISAs are performed in a 96-well microtiter plate (EIA), when the results of the traditional Coggins test are still with synthetic peptide in carbonate buffer followed by negative. Recently, a sensitive and specific peptide-based incubating the plate overnight at 4°C. Now, block the plate ELISA was developed to determine the sero-prevalance of with blocking buffer for 1 hour at 37°C followed by addition EHV-1 and EHV-9 (Abdelgawad et al., 2015). For of freshly prepared diluted primary antibody into each wells discrimination between serological responses to EHV 1 and and incubate the plate at 37°C for 1 hour. Subsequently, anti- EHV4 immunoglobulins-IgG based type specific ELISA was mouse IgG, diluted in 100 μl/well antibody dilution buffer is developed (Ma et al., 2013). This technique was also used to added with the incubation at 37°C for 30 minutes. In last discriminate between EHV-1 and EHV-4 glycoprotein E enhancement solution is added in the plate and incubated at peptides for EHV-1 and glycoprotein G (gG) for EHV-4 (Lang 37°C for 15 minutes. The plate was washed in between each et al., 2013; Yasunaga et al., 1998). Recently, a glycoprotein G step at least five times with 1X PBST. Read the absorbance at based peptide ELISA was developed for detection of equine appropriate wavelength with an appropriate time resolved plate herpesvirus type 4 (Bannai et al., 2016). reader.

Figure 5 Principle of peptide- ELISA

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2.9 Nucleic acid diagnostics conventional assays (Gasser, 2006). PCR can utilize almost all kind of biological samples such as meat, blood, urine, skin Nucleic acid-based detection methods including polymerase scrapings and faeces for parasitic infection study. When chain reaction, reverse transcriptase-PCR, nested-PCR, compared to the conventional method the detection limit of restriction fragment length polymorphism, amplified fragment PCR is higher. length polymorphism, random amplified polymorphic DNA, loop-mediated isothermal amplification, microarray, real-time Therefore, it is useful for detecting low amount of antigen in PCR are used for identification of several equine diseases suspected samples (Varrasso et al., 2001; Nugent et al., 2006). (Pusterla et al., 2007; Monego et al., 2009; Yeh et al., 2012; It can also be used for detection of many equine diseases Quereda et al., 2000; Larrasa et al., 2002; Eischeid, 2011). (Oldfield et al., 2004; Ocampo-Sosa et al., 2007; Pusterla et al., 2007; Letek et al., 2008; Monego et al., 2009). Polymerase 2.10 Polymerase Chain Reaction (PCR) chain reaction (PCR)–based diagnostic tests can allow rapid and sensitive detection of equine infectious pathogen (Paxson, PCR uses the enzyme DNA polymerase that amplifies a small 2008). Yeh et al. (2010) developed a duplex reverse length of targeted DNA using primers which are specific to the transcriptse PCR which is sensitive, specific and very rapid target. It will amplify the selected target sequence from a and is useful in both humans and as well as in horses for the mixture of genome. PCR acts as an important tool for the simultaneous and differential diagnosis of West Nile and identification of parasites due to the insufficient amount of Japanese encephalitis viruses. availability of antigen and antigen products by using

Table 2 PCR testing results for a variety of infectious equine pathogens.

Pathogen PCR test References West Nile Virus RT-nPCR Johnson et al. (2001) Strongylus edentatus, Strongylus equinus and Strongylus PCR-RLB Traversa et al. (2007) vulgaris Herpes viruses 4 and 1 PCR Brendan & Michael (1993) Equine herpesvirus 1 and 4 Differential multiplex PCR Carvalho et al. (2000) Distinguish between EHV-1 and EHV-4 PCR Wagner et al. (1992) EHV1, EHV4, EHV2 and EHV5 Multiplex PCR Wang et al. (2007) Rhodococcus equi PCR Khurana et al. (2015); Pal & Rahman, (2015) Rhodococcus equi Multiplex PCR Chhabra et al. (2015) Equine arteritis virus RT-PCR St-Laurent et al. (1994); Zhang et al. (2008) Theileria equi Nested PCR and Nested PCR with Wise et al. (2013) hybridisation Babesia caballi Nested PCR Battsetseg et al. (2001) Babesia equi Nested PCR Battsetseg et al. (2001) Streptococcus equi PCR Ijaz et al. (2012) Equine influenza virus RT-PCR OIE, (2016) Leptospira spp. PCR Faber et al. (2000) Salmonella spp. PCR Amavisit et al. (2001) Alternaria spp. PCR Dicken et al. (2010) Emmonsia crescens Single step PCR Pusterla et al. (2002) Lawsonia intracellularis Faecal PCR Lavoie et al. (2000) Corynebacterium pseudotuberculosis Real-time PCR/ RAPD-PCR Foley et al. (2004) Anaplasma phagocytophilum nPCR Lee et al. (2015); M'ghirbi et al. (2012) Streptococcus equi Species-specific PCR Javed et al. (2016) Equine encephalitis virus RT-PCR Linssen et al. (2000) Differentiation of B. mallei and B. pseudomallei Multiplex qPCR Janse et al. (2013) Streptococcus equi Triplex quantitative PCR Webb et al. (2013)

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Table 3 Examples of Real-time Polymerase chain reaction for the diagnosis of equine infectious pathogens.

Pathogen Target gene Reference Streptococcus equi eqbE and SEQ2190 Webb et al. (2013) Salmonella ompC gene of S. Heidelberg Amavisit et al. (2001) Salmonella spp, invA gene Pusterla et al. (2010) Anaplasma phagocytophilum 16S rRNA M'ghirbi et al. (2012) Streptococcus 16S rRNA Javed et al. (2016) Streptococcus equi SeM gene Javed et al. (2016) Streptococcus equi sodA gene Javed et al. (2016) Leptospira ligA and B Palaniappan et al. (2005) Corynebacterium pseudotuberculosis PLD exotoxin gene Spier et al. (2004) Equine arteritis virus EAV ORF7 gene Balasuriya et al. (2002) Equine herpes virus 1 Glycoprotein B Diallo et al. (2006) Equine herpes virus 4 Glycoprotein B Diallo et al. (2007) Equine herpes virus 1 gD and IR6 gene Goodman et al. (2007) Strongylus vulgaris rDNA Nielsen et al. (2008) Equine Influenza Virus Matrix and hemagglutinin gene Quinlivan et al. (2005) Pseudomonas syringae pv. aesculi Gyrase B Green et al. (2009) Lawsonia intracellularis Aspartate ammonia lyase gene Pusterla et al. (2008) Theileria equi 18S rRNA Kim et al. (2008) Equine infectious anemia virus gag gene Cook et al. (2002) African horse sickness NS1 Rodriguez-Sanchez et al. (2008) Babesia equi ema-1 gene Ueti et al. (2005) Corynebacterium pseudotuberculosis Phospholipase D gene Sharon et al. (2004) Burkholderia mallei ISBma2 Janse et al. (2013)

Further, Yeh et al. (2012) developed a diagnostic algorithm (McFadden et al., 2016). Various types of PCR are used for which serologically differentiates West Nile virus from testing a Variety of Infectious Equine Pathogens (Table 2). Japanese encephalitis virus infection and its validation in field surveillance of horses. Rakhshandehroo et al. (2014) had done 2.11 Random Amplified Polymorphic DNA (RAPD) an intra-specific variation study for Habronema muscae in horses using cytochrome c oxidase subunit 1 gene based It is a type of PCR reaction which amplifies segments of DNA identification by PCR technique. Helicobacter bacterium randomly. It uses short primers of nucleotide length varies infection was reported by Contreras et al. (2007) using the16S from 8-12 nucleotides and template DNA for PCR rRNA gene specific PCR in equines. amplification. By resolving the resulted amplified product on agarose gel electrophoresis a semi unique profile pattern can be PCR can also be combined with other molecular methods such visualized from a RAPD reaction. Although RAPD is as reverse transcriptase or nested PCR to genotype the comparatively easy to perform, but it is also a PCR dependent organisms. RT-PCR has also been developed for Western assay so it needs specific PCR protocol to give reproducible Equine Encephalitis Virus (WEEV) diagnosis (Linssen et al., result. Any kind of mismatch in template and primer results in 2000; Lambert et al., 2003). RT-PCR assay was successfully complete absence of PCR product and makes it difficult to used for identification of west nile and japanease encephalitis interpret the results of RAPD. This assay has the potential to virus (Lanciotti & Kerst, 2001). A multiplex PCR was play a useful role in genetic analyses of livestock species such designed to amplify herpes simplex virus types 1 and 2, as horses (Cushwa & Medrano, 1996). Larrasa et al. (2002) cytomegalovirus, and varicella-zoster virus DNA present in a described the development of quick and relevant DNA diverse range of clinical material (Druce et al., 2002). extraction and RAPD methods that can be used for genotyping Multiplex PCR assays for the simultaneous identification of Dermatophilus congolensis field isolates. Larrasa et al. (2004) varicella zoster virus (VZV), herpes simplex viruses (HSV), reported the molecular typing of D. congolensis from horse CMV, human herpesvirus 6, and Epstein-Barr virus in skin sample by RAPD and pulsed field gel electrophoresis cerebrospinal fluid (Quereda et al., 2000) and assays for HSV (PFGE) techniques. and VZV in mucocutaneous specimens (Jain et al., 2001; Nogueira et al., 2000) and CSF (Read & Kurtz, 1999) have 2.12 Real time PCR been reported, each with improved utility over existing methods in the diagnostic setting. In a recent study herpesvirus The real-time PCR assay gives us the quantification of several (EHV) type 1 was detected using PCR and neuropathogenic types of biological samples using verities of fluorescent genotype of EHV-1 was identified by DNA sequencing materials such as TaqMan probes, SYBR Green dye and

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Biotechnological tools for diagnosis of equine infectious diseases. S169 Scorpion primers (Nado, 2009). The pathogenic nucleic acids redistribution during melting. Eischeid analyzed and reported from various biological and environmental samples can be about the behaviour of other DNA dyes in Real-time PCR and quantified to give the information about the extent of infection. showed that EvaGreen and SYTO dyes out performed SYBR The SYBR Green dye based Real-time PCR assays have been Green in real-time PCR (Eischeid, 2011). validated for many equine diseases from several decades. Table 3 presents an overview of real-time PCR routinely used 2.13 Probe Hybridization for the detection of equine pathogens such as bacterial, viral and parasitic pathogens. Although Real-time PCR is excellent Fragments of DNA or RNA usually around 100-1000 bases in showing sensitive and specific results but it is still length used to detect the presence of nucleotide sequences that uncommon in routine laboratory diagnosis especially in rural are complementary to the probe sequence called hybridization endemic areas due to its sophistication. In the Real-time probe and this probe hybridizes to single-stranded nucleic acid amplification protocols, other procedures such as DNA sequence (Wetmur, 1991). Due to the nucleotide base extraction, choice of primers may cause heterogeneity in complementarily between the target and probe, the nucleotide results and causes difficulty in standardization of assay sequence of probe allows pairing of probe and the target (Bretagne & Costa, 2006). (Figure 6). The labelled probe is then hybridized to the target RNA (Northern blotting) or ssDNA (Southern blotting) A SYBR Green based assay was developed that could detect immobilized on a membrane or in situ. The probe is tagged 100% of the different WNV target region variants in their with a molecular marker of either radioactive (P32, I125 etc.) study, whereas a TaqMan assay failed to detect 47% of molecules or non-radioactive fluorescent molecules to detect possible single nucleotide variations in the probe-binding site the hybridization (Digoxigenin).The probe hybridization based (Papin et al., 2004). Johnson et al. (2010) designed a pan- assays have been used for diagnosis of equine infections such flavivirus RT-PCR using degenerate primers for the NS5 gene as equine arteritis virus (Balasuriya et al., 2002; Westcott et al., to allow the detection of a range of flaviviruses including 2003). The probe hybridization assay is relatively easy to WNV. This SYBR Green based RT-PCR was able to detect the perform. EHV-1virus strain was reported by means of WNV however the sensitivity was much lower compared to Southern blot and dot-blot hybridization (Morris & Field, WNV-specific TaqMan RT-PCR assays (Johnson et al., 2010). 1988). The probe hybridization assay was confirmed and the SYBR Green has been shown to inhibit the PCR reaction to sensitivity was inferior to classical techniques such as virus some extent and melt curve analysis is troublesome by dye isolation (Morris & Field, 1988).

Figure 6 Principle of probe hybridization

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2.14 Microarray cyclical step builds upon the product of the non-cyclical step which involves two outer primers along with the Bst DNA The nucleic acid microarray technique is a collection of polymerase. The loop primers might be involved in the cyclic microscopic nucleic acid spots attached to a solid glass surface. step when six primers are used (Nagamine et al., 2002). LAMP Picomoles of specific nucleic acid sequence are present on assay due to its unique properties has provided a powerful each spot called probe (Bumgarner, 2013). Probes are allowed diagnosis of various pathogens (Notomi et al., 2000). LAMP to hybridize labelled target nucleic acid (cDNA or cRNA/anti- technique amplifies nucleic acid at a very faster rate along with sense RNA). This probe-target hybridization can be detected maintaining high specificity, sensitivity and efficiency (Parida and also can be quantified by silver, fluorophore or et al., 2008). The most inventive feature of this technique is the chemiluminescence-labeled targets (Figure 7). This technique simplicity of its protocol (Figure 8), and the low cost of overall is also used to measure the expression levels of many amplification. Alhassan et al. (2007) developed a LAMP expressed genes of same or different species simultaneously. method for diagnosis of equine piroplasmosis. Microarray has also been used for diagnosis of equine disease detection. Equine-specific microarray has been used to Reverse transcription loop-mediated isothermal amplification estimate gene expression in laminitis (Noschka et al., 2009) (RT-LAMP) has been developed and it should be applicable to and articular cartilage repair (Mienaltowski et al., 2009). A detect the equine rotavirus infection in molecular laboratories recent study using microarray technology on placental tissues (Nemoto et al., 2010). Equine influenza virus was also reported identified a >900-fold upregulation of mRNA encoding the using LAMP assay (Nemoto et al., 2011; Nemoto et al., 2012). cytokine interleukin-22 in chorionic girdle, which is the first Novel LAMP methods was developed specific to the time IL-22 has been reported in any cells other than immune pathogenic bacteria found in equine secondary pneumonia, cells (Brosnahan et al., 2012). On the basis of whole genome namely, the Bacteroides–Prevotella group, Klebsiella single nucleotide polymorphism (SNP) analysis of all available pneumoniae, Stenotrophomonas maltophilia and Venezuelan equine encephalitis viruses (VEE) antigenic Staphylococcus aureus (Kinoshita et al., 2015). Two different complex genomes, verifies that a SNP-based phylogeny LAMP assays targeting Escherichia coli (Hill et al., 2008) accurately captured the features of the phylogenetic tree based or Pseudomonas aeruginosa (Goto et al., 2010) were used by on multiple sequence alignment, and reported a high resolution Kinoshita et al. (2015), on clinical respiratory specimens and a genome wide SNP (Gardener et al., 2016). high accordance was found between the results of the LAMP assays and bacterial culture. Use of these LAMP assays could 2.15 Loop-mediated isothermal amplification (LAMP) enable rapid detection of pathogenic bacteria and swift administration of the appropriate antimicrobials. In this way, it LAMP is a nucleic acid amplification procedure that works is possible to concurrently perform LAMP assays to detect under a unique amplification principle; involves two steps: both the primary and secondary causative pathogens of lower these are cyclic or non-cyclic phase (Ushikubo, 2004; Parida et respiratory bacterial infections in horses in only 60 min with al., 2008).The non-cyclical step precedes the cyclical phase of the naked eye; this will make it possible to institute appropriate amplification (Parida et al., 2008). It involves the four primers antimicrobial therapies more quickly in horses with secondary as well as the Bst DNA polymerase with strand displacement bacterial pneumonia (Kinoshita et al., 2015). activity, play a role in this first stage of LAMP reaction. The

Figure 7 Principle of microarray assay.

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Figure 8 Principle of LAMP.

2.16 Sequence analysis study especially B. fragilis and P. heparinolytica are dominant anaerobes in lower respiratory tract infection in horses. Whole genome sequencing is a process that gives the complete DNA sequence of an organism's genome at a single 3 Biosensors time. High-throughput genome sequencing technologies have largely been used as a research tool and are currently being Biosensor is an advanced technique for the detection of either introduced in the clinics (Van et al., 2013; Gilissen, 2014; the antigen or antibodies. This assay involves the use of a Nones et al., 2014). Genome sequencing of the domestic horse receptor (mostly an antibody), a disease specific antibody and and subsequent advancements in the field of equine genomics a transducer that converts a biological interaction into a have led to an explosion in the development of tools for measurable signal (Cruz et al., 2002). These biosensors are mapping traits and diseases and evaluating gene expression frequently coupled to sophisticated instrumentation to produce (Finno & Bannasch, 2014). In 2011, whole-genome highly-specific analytical tools, most of which are still in use sequencing of an individual American quarter horse mare was only for the research and development purpose due to the high performed using massively parallel paired-end sequencing cost of instrumentation, high cost of individual sample (Doan et al., 2012). Several single-gene disorders in quarter analysis, and the need for highly trained persons to oversee the horses, such as polysaccharide storage myopathy (McCue et testing. Fibre optic biosensors have the potential to do multi- al., 2008; Tryon et al., 2009), hyperkalemic periodic paralysis, analyte analyses in an automated format. Portable fibre optic glycogen branching enzyme deficiency (Rudolph et al.,1992), biosensors, has been reported to detect four different analytes and hereditary equine regional dermal asthenia (Ward et al., in one coupon (King et al., 2000). Biosenors can be used as 2004; Finno et al., 2009) has been reported due to whole- self-contained field devices for the detection of foreign animal genome sequencing of an individual American quarter horse disease agents. West nile virus was detected using biosensors mare. A high-quality draft assembly was constructed and and microfluidic systems, a linear, 15 amino acid fragment of additional sequence were provided by the inclusion of bacterial domain III of WNV was successfully used as an antigen on an artificial chromosome end sequences from a related male amperometric immunosensor (Ionescu et al., 2007). Neng et al. thorough bred horse (Leeb et al., 2006). Kinoshita et al., (2010) reported that, a surface enhanced Raman scattering (2014) reported the genera Bacteroides and Prevotella immunoassay was shown to be highly sensitive for the

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S172 Prasad et al detection of anti-WNV immunoglobulin. Hu et al. (2004) future. These instrumentations are designed to specifically developed a genetically biotinylated single chain fragment select, separate by molecular mass, and identify the complex variable antibody (scFv) against Venezuelan equine mixture of proteins in a sample, which can be compared to encephalitis virus (VEE). Patrick et al. (2014) studied the known samples for diagnostic purposes. evolution of equine influenza and the origin of canine influenza with the help of biosensor. In equine medicine, proteomics is been used in the diagnosis of different metabolic as well as orthopedic diseases which show 4 Nanotechnology some of the alteration in the expression levels of marker proteins (Amaya, 2014). In the proteomic marker analysis The systems or devices which are related to the features of conducted in biopsy samples of horse muscles, it was found nanometre scale are broadly defined as nanotechnology. This that three significantly increased proteins: alpha actin, scale of technology as it applies to diagnostics would include tropomyosin alpha chain and creatine kinase M chain (CKM). the detection of molecular interactions. The tiny dimensions of CKM was represented by multiple spots probably due to this technology led a basement to the use of nanoarrays and posttranslational modification, one of which appeared to be nanochips as test platforms (Jain, 2003). The potential use of unique for tying-up suggesting that altered energy distribution this technology is to analyse a sample for an array of infectious within muscle cells is part of the disease etiology (Freek et al., agents on a single chip. Many research groups are considering 2010a). In another study they have identified, 20 differential the use of chip assays that detect several agroerrorism agents in spots representing 16 different proteins. Evaluation of those each sample. Small, portable platforms are being designed to proteins complies with adaptation of the skeletal muscle after allow pen-side testing of animals for diseases of concern. normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain The use of nanoparticles to label antibodies is another facet of fatty acids, and to store energy in the form of glycogen. nanotechnology. These labelled antibodies can be used in various assays to identify specific pathogens, structures or Intensified training leads to additional changed spots. Alpha-1- molecules. The use of gold nanoparticles, nanobarcodes, antitrypsin was found increased after intensified training but quantum dots and nanoparticle probes are the examples of not after normal training. This protein may thus be considered nanotechnology (Yguerabide & Yguerabide, 2001). as a marker for overtraining in horses and also linked to Nanopores, nanosensors, resonance light scattering and overtraining in human athletes (Freek et al., 2010b). In an cantilever arraysare some of the additional nanotechnologies another study, which was conducted on the proteomics, study and it is anticipated that many of the specific nanotechnologies of cerebrospinal fluid, a total of 320 proteins were confidently will eventually be applied to the diagnosis of endemic identified across six healthy horses, and these proteins were veterinary diseases in the future. Klier et al. (2012) reported further characterized by gene ontology terms mapped in about an aerosol formulation of biodegradable, biocompatible UniProt, and normalized spectral abundance factors were and nontoxic gelatine nanoparticle-bound CpG-ODN2216, to calculated as a measure of relative abundance and these results treat equine recurrent airway obstruction in a clinical study. provide an optimized protocol for analysis of equine CSF and laid the basement for future studies involving the CSF study of 5 Proteomics equines in the context of pathogenic disease states (Carolyn et al., 2014). The analysis of osteoarthritis and osteochondrosis Proteomics is the new emerging field to isolate and conducted by Elisabetta et al. (2012) has identified some characterize the protein produced by various etiological agents. putative protein markers which can be further tried for the Different bacterial, viral as well as parasitic proteins can be definitive early diagnosis of osteoarthritis in the horses. A targeted with the help of this technology. Hence, proteomics highly sensitive proteomic comparison together with insightful has potential applications in veterinary diagnostics. The usual data mining enabled us to identify proteins and pathways approach of proteomic involves separation of the proteins with involved in early OA which could aid the development of early the help of two dimension gel electrophoresis and staining OA diagnostic markers and therapeutics (Peffers et al., 2012). them with appropriate protein marker. The protein ‘pattern’ is In case of a very unpredictable disease of equines, laminitis different in different species; hence it can be recognized as a identification and measurement of novel protein biomarkers fingerprint. It is then analyzed by performing image analysis present in blood that predict the onset and resolution of (Krah & Jungblut, 2004). Proteins that are up- or down- laminitis would both aid clinical management of at-risk equine regulated due to disease are compared and find by using patients and shed light on underlying mechanisms with the proteome maps. A protein of interest can be cut and taken out intent of developing novel preventive strategies and therapeutic from the gel and purified. This purified protein can be further approaches (Joseph et al., 2008). fully characterized using peptide-mass fingerprinting and/ or mass spectrometry methods. Veterinary diagnostics may make Conclusion use of proteomics to identify or look for known disease markers or patterns with biochip technology and A profound change has been occurred in recent years in instrumentation that combines mass spectrometry with other veterinary diagnostics with the introduction of new separation chromatography or molecular techniques in the biotechnological assays which completely changed the

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Biotechnological tools for diagnosis of equine infectious diseases. S173 scenario of time-tested, traditional diagnostic techniques of detection. Recently, advanced software tools and the veterinary disease diagnosis. These new biotechnological computing power for bioinformatics analysis of parasitic large methods, includes the production of more specific antigens by genome size data is a need of modern molecular diagnosis. The the use of recombination, expression vectors and synthetic major challenge regarding development of new technologies is peptides. When coupled with the use of monoclonal antibodies, to optimize and evaluate the tools for control and eradication the sensitivity and specificity of a number of traditional programs of parasitic diseases. So it will help in the diagnostic assays have been significantly improved. Various development of newer technologies to a level of analytical forms of PCR have become a routine diagnostic tool in sensitivity which will be appropriate for testing of clinical veterinary laboratories for rapid screening of large number of samples directly without previous processing. samples during disease outbreaks to develop prevention and control measures and also to make specific typing Conflict of interest determinations for research purpose. Authors would hereby like to declare that there is no conflict of Other technologies are likely to be widely adopted in the future interests that could possibly arise. as they demonstrate the ability to improve the diagnostic capabilities while reducing the time and, perhaps cost References associated with more conventional technologies. 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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

EVOLVING VIEWS ON ENTERIC VIRAL INFECTIONS OF EQUINES: AN APPRAISAL OF KEY PATHOGENS

Shubhankar Sircar1, Sharad Saurabh1, Jobin J. Kattoor1, Pallavi Deol1, Kuldeep Dhama1, Sandip K Khurana2 and Yashpal S. Malik1,*

1ICAR-Indian Veterinary Research Institute, Izatnagar 243 122, Uttar Pradesh, India 2ICAR-National Research Centre on Equines, Hisar - 125 001, Haryana, India

Received – November 08, 2016; Revision – November 22, 2016; Accepted – December 01, 2016 Available Online – December 04, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S182.S195

KEYWORDS ABSTRACT Equines Equines, the earliest known mammalian species, have been found highly susceptible to several enteric Enteric pathogens including viruses, fungi, parasites and bacteria. This review conserves with the key viral pathogens that affects foals and horses such as rotavirus, adenovirus, coronavirus, parvovirus, Virus picobirnavirus etc. With the advent of next generation sequencing approaches the list of new enteric viruses has expanded. Viruses like Cyclovirus, Kirkovirus and Anellovirus are the new members Epidemiology identified in equines recently. Close proximity of horses to human settlements and/or other domestic animals pretense the threat of infectious diseases spread to humans/animals and vice-versa. Therefore, Diagnosis horse diseases are not only of veterinary importance but also are among important factors for public health. Herein, we intend to appraise current status of key enteric viruses that cause diarrheic disorders Control in foals and horses.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Yashpal S. Malik) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

S183 Sircar et al

1 Introduction economic importance that cause diarrheic disorders in foals and horses. Furthermore, current updates in the rotavirus and Equines are among the earliest known mammalian species that coronavirus infection in equines are covered. have been incessantly domesticated by the human race since 4000 BC. Apart from being companion animals to humans, 2 Adenovirus they have also been used as amusing or sporting animal. During late 19th century, equine microbiology came under Adenoviruses belong to the Adenoviridae family, are un- existence with the discovery of Burkholderia mallei as the enveloped and have icosahedral nucleocapsid, with 90–100 nm feared pathogen of glanders and farcy of horse. Later on, a size, encapsulating a dsDNA genome of 26–45 Kbp (Davison filterable agent was identified capable of causing African horse et al., 2003). Adenoviruses are known to affect a wide sickness (Slater, 2013). With the help of classic virological vertebrate’s host range and are genetically/antigenically methods, numerous equine viruses have been added to this list heterogeneous (Harrach et al., 2011). Presently, this family such as equine influenza virus, West Nile virus, equid comprises of 5 genuses as Mastadenovirus, Atadenovirus, herpesviruses, equine encephalitis viruses, equine arteritis Aviadenovirus, Ichtadenovirus and Siadenovirus (Harrach et virus, equine infectious anemia virus, equine coronavirus, al., 2011). Hendra virus and vesicular stomatitis virus. Though, the study of equine infectious diseases has been an important part of Horses and foals are affected by adenoviruses of genera veterinary sciences since ages, a large number of the equine Mastadenovirus, have been primarily connected with diseases still remains unfamiliar. Close proximity of horses to respiratory and gastrointestinal tract infections (Reubel & human settlements and to other domestic animals pose the Studdert, 1997; Cavanagh et al., 2012). Equine adenoviruses threat of infectious diseases spread to humans/animals and are separated into 2 serotypes and consequent molecular and vice-versa. Zoonotic pathogens such as Alphaviruses, Hendra phylogenetic readings established them as distinct species virus, Influenza A virus, Rabies virus and West Nile virus are which are nowadays called as Equine adenovirus 1 (EAdV-1) reported to infect horses (Johnson, 2011; Cullinane & Newton, and Equine Adenovirus 2 (EAdV-2) (Reubel & Studdert, 1997; 2013; Onmaz et al., 2013; Slater, 2013). It is therefore Cavanagh et al., 2012). Out of these two, EAdV-1 is primarily noteworthy that horse diseases are not only of veterinary associated with infections in the respiratory tract of young importance but also pose potential threat of zoonosis/reverse foals and horses whereas EAdV-2 has been reported mainly zoonosis (Khurana et al., 2015; Mukarim et al., 2015) and are from horses having diarrheal illness (Studdert & Blackney, among important factors from human public health 1982). The first equine adenovirus was reported and isolated in perspective. USA (Todd, 1969) it was followed by isolation reports from Germany and Australia whereas physical characterization of Several enteric pathogens leading to multifactorial diseases the virus was published in 1973 of an adenovirus isolated from have been detected and isolated from diseased foals and the pneumonic lung tissue of an Arabian foal (Ardans et al., horses. The major enteric pathogens associated with neonatal 1973). foal diarrhea are rotavirus, coronavirus, Clostridium perfringens type A, Salmonella spp., Rhodococcusequiand Antibodies pertaining to both the Equine Adenovirus types Clostridium difficile (Fielding et al., 2015; Franco Ayala & were reported in New South Wales, Australia (Giles et al., Oliver Espinosa, 2015; Barr, 2016). Foals co-infecting with 2010). In a study during 1982 on the diarrheal outbreaks in number of infectious agents has also been documented in young foals, researchers isolated and identified an Equine diarrheic foals (Slovis et al., 2014). One of the prime causes of adenovirus strain which did not contain the hemagglutination- equine enteritis is the viral infections. A recent metagenomics inhibiting antibody to EAdV-1 (Studdert & Blackney, 1982). study highlighted several new viruses like Cyclovirus, Similar research findings were published in New Zealand Kirkovirus and Anellovirus along with known enteric viruses revealing two serologically different EAdV strains isolated such as parvovirus, adenoviruses, coronaviruses, rotaviruses from thoroughbred foals suffering from diarrhea and and picobirnaviruses in equines (Li et al., 2015). In last few respiratory disease (Horner & Hunter, 1982).These research years, several reviews appeared on various aspects of equine findings supported the assumption that adenoviruses found in health excluding enteric viral pathogens of equines, except a the fecal specimens should be considered as the prototype for few that highlighted coronavirus and rotavirus infections in EAdV-2. Compared to EAdV-1 adenovirus there has been less equines (Bailey et al., 2013; Papp et al., 2013; Dhama et al., studies done with respect to EAdV-2 adenovirus with the very 2014; Pusterla et al., 2015a). Viral etiological agents which are first sequence data appeared on the GenBank database for able to cause diarrheic outbreaks are comprehended in Table 1. EAdV-2 in the year 1997 (Reubel & Studdert, 1997) (GenBank In the event of emergence of several new viral infections L80007.1). The study by Ruebel and Studdert also established worldwide including Ebola and Zika (Dhama et al., 2015a; the first molecular evidence that EAdV-2 is distinct to EAdV- Singh et al., 2016) there is urgent need to understand the 1, not only in antigenic aspect but is quite different on burden of viral infections in different animal hosts. Here, we molecular level too. intend to review current status of major enteric viruses of

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Evolving Views on Enteric Viral Infections of Equines: An Appraisal of Key Pathogens. S184

Table 1 Enteric viruses reported in equines having relevance in causing enteritis highlighting their main features.

Sl Viruses Genome Genus Species/serotypes Relevance in References No. enteric diseases 1. Adenoviruses dsDNA Mastadenovirus Equine Adenovirus 1 No Cavanagh et al.(2012) Equine Adenovirus 2 Yes Giles et al. (2015) 2. Coronaviruses ssRNA Betacoronavirus Betacoronavirus-1 Yes Nemoto et al. (2015b) 3. Rotaviruses dsRNA Rotavirus A G3P[12], G14P[12], G3P[3], Yes Ghosh & G5P[7], G6P[1], G8P[1], Kobayashi (2014) G10P[1], G10P[11] and G13P[18] 4. Picobirnavirus dsRNA Picobirnaviruses Equine picobirnavirus Yes Li et al. (2015) 5. Parvovirus ssDNA Copiparvovirus Ungulate Copiparvovirus 3 No Li et al. (2015) 6. Anellovirus ssDNA Mutorquevirus Unclassified No Li et al. (2015) 7. Cyclovirus ssDNA Cyclovirus Unclassified No Li et al. (2015)

A whole genome characterization of an EAdV-2 strain was (Holmes, 2001). The different animals reported to have also reported recently in the year 2015 in Australia (Giles et Coronavirus mediated infections are swine, cattle, horse, dog, al., 2015) (GenBank Acc. No. KT160425). Interestingly, till cat and avian species like chicken and turkey (Saif et al., 1991; this review in September 2016 there were only 2 sequence White & Fenner, 1994; Studdert, 1996; Jamieson et al., 1998; report available for EAdV-2 in the GenBank database out of Guy et al., 2000; Lai & Holmes, 2001; McIntosh, 2002; which one is complete genome and another is 5.5 kbp in length Strauss & Strauss, 2002; Ksiazek et al., 2003; Van der Hoek et comprising the hexon and endopeptidase genes. al., 2004; Brian & Baric, 2005; Weiss & Navas-Martin, 2005; Decaro & Buonavoglia, 2008; Boileau & Kapil, 2010; Woo et Unlike EAdV-1, which can be easily isolated and grows al., 2012). fastidiously in primary tissue culture derived from equine fetal kidney (EFK) cells (Studdert, 1978), EAdV-2 responds very Further, based on the antigenic properties coronaviruses are poorly to cell culture and has been infrequently isolated divided into 3 major antigenic groups which infect several (Horner & Hunter, 1982; Studdert & Blackney, 1982). The animal hosts and humans (group 1 and 2) and also avian major technique used for the diagnosis and characterization of species (group 2). The group 1 comprise of human coronavirus adenovirus is PCR which is based on the hexon gene of (strain 229E), canine coronavirus, porcine transmissible adenovirus genome which contains highly conserved regions gastroenteritis virus and feline infectious peritonitis virus. The (Reubel & Studdert, 1997). group 2 is represented by human coronavirus strains (OC43 and HKU1), murine hepatitis virus, bovine coronavirus, With the available literature regarding the infection of porcine hemagglutinating encephalomyelitis virus, canine adenovirus in equines EAdV-1 has been studied widely in respiratory coronavirus (Erles et al., 2007), and bubaline comparison to EAdV-2. Despite the significant improvement coronavirus (Decaro et al., 2010). Viruses such as turkey in the development of diagnostic techniques there has been coronavirus and infectious bronchitis virus are included in scarcity of information regarding EAdV-2 which requires the group 3 (Resta et al., 1985; Studdert, 1996; Davis et al., 2000; attention of veterinarians and scientist working in the field of Guy et al., 2000; Lai & Holmes, 2001; McIntosh, 2002; equine infectious diseases. Strauss & Strauss, 2002; Van der Hoek et al., 2004; Smith & Denison, 2012; Woo et al., 2012; Smith et al., 2013). 3 Coronavirus The earliest known Equine Coronavirus (ECoV) recognized by Coronavirus is an enveloped, ssRNA, positive sense virus electron microscopy (EM) in the foals diarrheic fecal samples having a helical symmetry with a genome length of 26 to 32 and adult horses (Bass & Sharpee, 1975; Huang et al., 1983). Kb, which is largest among the RNA viruses. The Later, in 2000 Davis and colleagues developed an antigen Coronaviridae family is divided into 2 subfamilies capture ELISA for the detection ECoV in the feces of diarrheic (Torovirinae and Coronavirinae). The later subfamily foal along with immunohistochemistry of affected foal’s classified into four different genera viz. Alphacoronavirus, intestine suffering from neonatal enterocolitis (Davis et al., Betacoronavirus, Deltacoronavirus and Gammacoronavirus 2000). In the same year, an ECoV strain NC99 was isolated based on the serological and molecular differences (Woo et al., and its N protein gene was characterized using the previously 2012). These are mainly associated with respiratory, described BCoV primers (Guy et al., 2000). The ECoVs have neurologic, hepatic or gastrointestinal disorders in animals been found highly related with bovine coronaviruses (Imagawa

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S185 Sircar et al et al., 1990; Guy et al., 2000). Subsequently, the complete It has been usually observed that ECoV infection in horses genomic constellation of ECoV (strain NC99) was achieved spread when they are sheltered together, which has been which consists of 2 replicase polyproteins, 4 accessory proteins confirmed by the presence of BCoV and other related (ns2, p4.7, p12.7, and I) and 5 structural proteins viz. spike, coronavirus antibodies in the feces of horses (Imagawa et al., hemagglutinin esterase, membrane, envelope and nucleocapsid 1990; Anzai et al., 2001). Better management practices (Zhang et al., 2007). regarding the sheltering and transit of young foals and adult horses can arrest the infection from spreading to other places. Diagnosis of coronaviruses has been routinely done through EM in the earlier times (Reed et al., 1983; Biermann et al., 4 Rotaviruses 1991; Guy et al., 2000). Using BCV antibodies serum neutralization tests has also been employed to detect ECoV Rotaviruses are the foremost cause of diarrhea in neonates of infections in horses (Bass & Sharpee, 1975; Imagawa et al., humans, animals and avian (Estes & Kapikian, 2007; Dhama et 1990; Anzai et al., 2001). Molecular techniques like nested al., 2015b). Till date, nine different species have been PCR and quantitative PCR have been actively used in the identified in rotaviruses from (A-I) as per the antigenic diagnosis of ECoV (Guy et al., 2000; Slovis et al., 2010). differences in the major inner capsid protein and nucleotide During early stages of infection highly sensitive technique like sequence identities gene encoding the VP6 protein real-time RT-PCR has been used (Pusterla et al., 2013; (Matthijnssens et al., 2012a; Mihalov-Kovacs et al., 2015), Miszczak et al., 2014). In 2015, a RT-LAMP was also although recently reported species I needs further endorsement developed for the isothermal detection of ECoV which can be by the ICTV. Rotaviral genome is made up of eleven dsRNA economical in comparison to other diagnostic tests (Nemoto et segments that encode 6 structural proteins viz; VP1, VP2, VP3, al., 2015a). Recently a one-step real time RT-PCR was also VP4, VP6 & VP7 and 5/6 non-structural proteins i.e. NSP1– developed and demonstrated for the sensitive detection of NSP5/6). The major coat proteins (VP4 & VP7) induce the ECoV in respiratory and fecal samples (Miszczak et al., 2016). neutralizing antibodies production and are considered Improving the specificity of the detection one ELISA was important due to the genetic classification which is based on recently developed for the specific detection of ECoV in these genes. As per the latest update of Rotavirus naturally infected horses and record the seroprevalence in them Classification Work Group (RCWG) till 27th June, 2016 there (Kooijman et al., 2016). are 32 G-genotypes (VP7) and 47 P-genotypes (VP4) reported in various host species In earlier studies, ECoV was suspected to cause enteritis in (https://rega.kuleuven.be/cev/viralmetagenomics/virus- foals but their pathogenicity in young foals remained unproven classification/ rcwg). (Davis et al., 2000; Van der Hoek et al., 2004; Arguedas, 2007). Mainly the tissue tropism for ECoV infections have Among all the species of rotavirus, only group A rotaviruses been found to be inside the gastrointestinal tract of young foals (RVA) has been detected in the equines so far (Matthijnssens and horses (Miszczak et al., 2014; Fielding et al. 2015; Pusterla et al., 2012b). The first detection of equine RV has been et al., 2015b). Multiple ECoV outbreaks have occurred in the reported in the year 1975 from England (Flewett et al., 1975). ÜSA and Japan (Oue et al., 2011; Oue et al., 2013; Pusterla et Since from this, equine RVs have been known to be the major al., 2013). Besides the possible respiratory and mechanical causes of diarrhea in foals (Imagawa et al., 1991; Collins et al., transmission of coronaviruses in equines, infections spreads 2008; Frederick et al., 2009). Based on the serological reports through fecal-oral route (Studdert, 1996; Anzai et al., 2001). equine RVAs have been shown to be ubiquitous in the equine Although, signs of upper respiratory tract infections are populations (Pearson et al., 1982). predominant with ECoV infected foals but their infrequent detection in nasal secretions shows their lack of tropism to the Numbers of diagnostic techniques have been employed for upper respiratory tract of young horses. In an experimental detecting RVs in diarrheic foals and adult horses. Electron study on Japanese draught horses, the horses were microscopy was the first technique to be applied for the experimentally inoculated with ECoV strain NC99 and Obihiri identification of RVs in foals but unfortunately it requires 12-2 to confirm and investigate the clinical signs and virus costly instruments and expertise. Moreover the diagnostic shedding pattern of ECoV in horses. sensitivity of EM turns out to be very low as it can’t detect virus particles lower than 107per ml of stool samples Though, nasal secretions came positive in PCR assay, (McIntosh, 1996). Culturing the RV in cell line is again a experiments could not define whether this was due to nasal daunting task. Equine RV has been isolated in MA-104 cell replication of the virus and subsequent shedding or due to line in the year 1981 when field sample from a diarrheic foal environmental factors or both (Nemoto et al., 2014). has been successfully adapted in UK (Imagawa et al., 1981). Subsequently, in 2015 a study reported the whole genome RNA-PAGE has been widely applied for the detection of sequencing of three earlier isolated ECoV strains from Japan equine RVs due to the peculiarity of different RV groups to Obihiri 12-1, Obihiri 12-2 and Tokachi09. The study found the migrate differently on the RNA-PAGE. Certain studies has three strains genetically similar to NC99 strains of USA with reported the typical migration pattern being observed with minor exceptions in NS2, NSP3 and p4.7 genes (Nemoto et al., equine RVA isolates with the segment 3 & 4 migrating 2015b). together close to each other and unlike the other RVA isolates

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Evolving Views on Enteric Viral Infections of Equines: An Appraisal of Key Pathogens. S186 here the segment 7, 8 and 9 migrate as a triplet (Hardy et al., al., 2013). The G3 RVA strains have been further 1991). differentiated into 2 subtypes (G3A and G3B) as per reactivity with monoclonal antibodies (Browning et al., 1992). In the ELISA and other immunodiagnostic have been now used for year 1991, a highly unfamiliar equine RVA stain (L338) rapid detection of RV which are usually based on the group carrying G13P[18] genotype combination was reported from specific VP6 gene product (outer coat protein). Many test kits UK (Browning et al., 1991a). Using the RNA-RNA available for human RV detection has also been employed in hybridization technique, it was shown that the strain L338 the detection of equine rotaviruses, though the sensitivity of possess a distinctive genotype of G13 and P[18] and unusual ELISA has been found more in respect to other NSP1 genetic makeup which found distinct to other human and immunodiagnostic test (Nemoto et al., 2010a). The indication animal RVAs (Wu et al.,1993; Taniguchi et al., 1994; Kojima of widespread RVA infections in horses was evident by the et al., 1996). Certain bovine-like equine RVAs have also been finding of RVA specific antibodies in adult horses (Pearson et reported which are highly similar to bovine RV strains al., 1982; Eichhorn & Huan-Chun, 1987). It was also evident employing labeled probes and RNA-RNA hybridization by the detection of RVA in equine population from different techniques from UK and Japan, respectively (Imagawa et al., countries which includes the United Kingdom (Strickland et 1991; Imagawa et al., 1993; Isa et al., 1996), al., 1982), the United States of America (Kanitz, 1977), Australia (Studdert et al., 1978; Tzipori & Walker, 1978) Similarly, an unusual G6 and G10P6[1] rotavirus was detected Germany (Elschner et al., 2005), New Zealand (Durham et al., in equines between 2003 and 2005 in addition to G1 strains 1979;Schroeder et al., 1983), France (Puyalto-Moussu & from India, but later this report was taken back following the Taouji, 2002), Greece (Ntafis et al., 2010), Italy (Monini et al., wrong classification of G6 RVA strains as G16 (Matthijnssens 2011), the Netherlands (Van der Heide et al., 2005), Argentina et al., 2012b). In Argentina also an uncommon feline-like RVA (Barrandeguy et al., 1998), Venezuela (Ciarlet et al., 1994) and was also reported in diarrheic foal (Garaicoechea et al., 2011). India (Gulati et al., 2009). Certain unusual equine RVA genotype combinations has also been discovered such as G3P[3], G5P[7], G6P[1], G8P[1], RT-PCR assays are being used as test of choice nowadays for G10P[1], G10P[11] and G13P[18], (Gulati et al., 2007; the detection as well as for genotyping of equine RVA strains Garaicoechea et al., 2011). Till date only three inactivated (Gouvea et al., 1994; Tsunemitsu et al., 2001; Garaicoechea et equine RVA vaccine have developed by Argentina, Japan and al., 2011). Hybridizing probe based diagnostics and genotyping USA and are being used in several countries contains the has also been attempted in bovine rotavirus which can be opted common genotype of G3P[12] strain (Imagawa et al., 2005). for equine strains too (Minakshi et al., 2005). More recently, one isothermal technique RT-LAMP has been developed During three decades (1981-2010) there have been several targeting the VP4 with P[12] specificity (Nemoto et al., studies in Japan leading to the genetic analysis and 2010b). Moreover, in USA RT-PCR kit is commercially characterization of equine RVAs (Takagi et al., 1994; available for early and quick detection of RVs (Slovis et al., Tsunemitsu et al., 2001; Fukai et al., 2006; Nemoto et al., 2010). As submissions of molecular sequence of rotavirus have 2012;). Till 2013 only few reports for the whole genome been increased in public database, sequence based typing of characterization and analysis of equine RVA strains have been rotavirus groups got interest. Till now, there have been 6 G- documented (Ghosh et al., 2012; Matthijnssens et al., 2012b; genotypes (Imagawa et al., 1994; Isa et al., 1994; Isa et al., Mino et al., 2013). The strains characterized for their complete 1996) and 6 P-genotypes (Garaicoechea et al., 2011) are genomic constellations were three from Europe (Strain: L338; reported in equines (Table 2). 03V04954 and 04V2024), three from Argentina (Strain: E30; E403 and E4040), one from South Africa (Strain: EqRV-SA1) Table 2 G and P genotypes of equines rotaviruses reported till and four from Japan (Stain: B1; HH-22; CH-3 and OH-4) date. (Ghosh et al., 2012; Matthijnssens et al., 2012b; Ghosh et al., 2013; Mino et al., 2013). G-Type P-Type G3 P[1] In February 2015, a report appeared in which 23 equine RVA G5 P[3] strains from late 1990s and 2009-2010 along with the vaccine G8 P[7] strain HO-5 were characterized through the next generation sequencing (Nemoto et al., 2015c) taking the total count of G10 P[11] whole genome reports for equine RVA to 37 isolates till dates G13 P[12] (Table 2). In the whole genome and further phylogenetic G14 P[18] analysis of 37 isolates revealed that the G3 strains carried a uniform genetic constellation for their 11 gene segments and Among the widely reported and commonly found genotype were closely related to the HO-5 vaccine strain irrespective of combinations of equine RVA, G3P[12] and G14P[12] have time they were confirmed (Nemoto et al., 2015c). In disparity, been described several times worldwide (Tsunemitsu et al., the G14 strains showed some divergence in respect to VP7 and 2001; Elschner et al., 2005; Collins et al., 2008; Ntafis et al., NSP4 gene. 2010; Garaicoechea et al., 2011; Nemoto et al., 2011 Papp et

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S187 Sircar et al

Figure 1 Whole genome backbone consisting of 11 segments of equine group A rotavirus (RVA) strains with known genomic constellations.

SL. No Strain Name Year of Isolation VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5 Reference 1 RVA/Horse-wt/ARG/E403/2006/G14P[12] 2006 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E12 H7 Matthijnssens et al. (2012) 2 RVA/Horse-wt/ARG/E4040/2008/G14P[12] 2008 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E12 H7 Matthijnssens et al. (2012) 3 RVA/Horse-wt/ARG/E30/1993/G3P[12] 1993 G3 P[12] I6 R2 C2 M3 A10 N2 T3 E12 H7 Matthijnssens et al. (2012) 4 RVA/Horse-wt/IRL/03V04954/2003/G3P[12] 2003 G3 P[12] I6 R2 C2 M3 A10 N2 T3 E2 H7 Matthijnssens et al. (2012) 5 RVA/Horse-wt/IRL/04V2024/2004/G14P[12] 2004 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Matthijnssens et al. (2012) 6 RVA/Horse-wt/ZAF/EqRV- 2006 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Matthijnssens et al. (2012) 7 SA1/2006/G14P[12]RVA/Horse-tc/GBR/L338/1991/G13P[18] 1991 G13 P[18] I6 R9 C9 M6 A6 N9 T12 E14 H11 Matthijnssens et al. (2012) 8 RVA/Horse-wt/ARG/E3198/2008/G3P[3] 2008 G3 P[3] I3 R3 C3 M3 A9 N3 T3 E3 H6 Mino et al. (2013) 9 RVA/Horse-tc/GBR/H-1/1975/G5P9[7] 1975 G5 P[7] I5 R1 C1 M1 A8 N1 T1 E1 H1 Ghosh et al. (2013) 10 RVA/Horse -tc/JPN/OH -4/1982/G6P[5] 1982 G6 P[5] I2 R2 C2 M2 A13 N2 T6 E2 H3 Ghosh et al. (2013) 11 RVA/Horse -tc/JPN/BI/1981/G3P[12] 1981 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Ghosh et al. (2013) 12 RVA/Horse -tc/JPN/HH -22/1989/G 3P[12] 1989 G3 P[12] I6 R2 C2 M3 A10 N2 T3 E2 H7 Ghosh et al. (2013) 13 RVA/Horse -tc/JPN/CH -3/1987/G14P[12] 1987 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Ghosh et al. (2013) 14 RVA/Horse-tc/JPN/HO-5/1982/G3P[12] 1982 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 15 RVA/Horse-tc/JPN/JE29/1997/G3P[12] 1997 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 16 RVA/Horse-tc/JPN/JE75/1997/G3P[12] 1997 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 17 RVA/Horse-tc/JPN/JE76/1996-1997/G3P[12] 1996 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 18 RVA/Horse-tc/JPN/JE97/1996/G3P[12] 1996 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 19 RVA/Horse-tc/JPN/JE102/1997/G3P[12] 1997 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 20 RVA/Horse-tc/JPN/No.1/2010/G3P[12] 2010 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 21 RVA/Horse-tc/JPN/No.2/2010/G3P[12] 2010 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 22 RVA/Horse-tc/JPN/No.4/2010/G3P[12] 2010 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 23 RVA/Horse-tc/JPN/No.7/2010/G3P[12] 2010 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 24 RVA/Horse-tc/JPN/No.9/2010/G3P[12] 2010 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 25 RVA/Horse-tc/JPN/No.13/2010/G3P[12] 2010 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 26 RVA/Horse-tc/JPN/No.32/2010/G3P[12] 2010 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 27 RVA/Horse-tc/JPN/No.52/2009/G3P[12] 2009 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 28 RVA/Horse-tc/JPN/No.55/2009/G3P[12] 2009 G3 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 29 RVA/Horse-tc/JPN/JE77/1997/G14P[12] 1997 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 30 RVA/Horse-tc/JPN/JE81/1997/G14P[12] 1997 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 31 RVA/Horse-tc/JPN/JE84/1996/G14P[12] 1996 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 32 RVA/Horse-tc/JPN/JE87/1996-1997/G14P[12] 1996 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 33 RVA/Horse-tc/JPN/JE115/1997/G14P[12] 1997 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 34 RVA/Horse-tc/JPN/No.24/2010/G14P[12] 2010 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 35 RVA/Horse-tc/JPN/No.28/2010/G14P[12] 2010 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015) 36 RVA/Horse-tc/JPN/No.29/2010/G14P[12] 2010 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c) 37 RVA/Horse-tc/JPN/No.50/2010/G14P[12] 2010 G14 P[12] I2 R2 C2 M3 A10 N2 T3 E2 H7 Nemoto et al. (2015c)

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Evolving Views on Enteric Viral Infections of Equines: An Appraisal of Key Pathogens. S188 The NSP4 gene of G14 strains and also G3 strains clustered restricted its transmission to other continents. But no such along with bovine RVAs and bovine-like equine strain OH-4 geographical barrier is present between the African and and HH-22, whereas rest all other genes were identical to HO- European equine RVA strains as two strains (04V2024 and 5 vaccine strain (Nemoto et al., 2015c). The common EqRV-SA1) shared nearly identical genotype G14P[12] backbone of the equine RVAs has been shown in Table 2 identified in South Africa and Ireland, respectively. comprising all the whole genome isolates. Presence of bovine RVA like NSP4 gene in conserved equine Almost all the common whole genome equine RVA strains backbone from Japan points towards the probability of equine characterized previously showed conserved genotypic rotavirus as an acceptor of gene segments from different host constellation among them and were divergent in nature to the species and vice-versa (Nemoto et al., 2015b). Detection of other RVA isolates (Matthijnssens et al., 2012b). Whereas few equine origin G3 genotype of VP7 in humans from Asia, equine RVA strains characterized showed unique genetic Australia and Europe confirms the possible host species jump makeup and elaborated the possibility of interspecies jumping event of equine rotavirus to human hosts (Malasao et al., 2015; of RVAs from one species to the other (Figure 1). The strains Cowley et al., 2016). Lately, similar reports emerged from showing the possibility of interspecies transmission were Spain and Hungary concerning the emergence of unusual equine G3P[18] RVA strain L338 (Matthijnssens et al., G3P[8] type rotaviruses in humans wherein the VP7 gene 2012b), H-1 strain believed to be transmitted from pigs to represents an equine like G3 (Arana et al., 2016; Dóró et al., equines (Ghosh et al., 2012), whereas strain E3198 seemed to 2016). These evolutionary phenomenon i.e. reassortment and be resultant of canine/feline RVA strain to equines (Mino et species jumping leads to rotavirus genetic diversity which al., 2013). Further, strengthening the need to analyze more eventually pose a threat to human and other animal host common and uncommon equine RVA strains around the world species. Therefore, a more rigorous surveillance programs are the analysis done by two research groups were remarkable prerequisite to establish geographical relationship among the (Matthijnssens et al., 2012b; Ghosh et al., 2013). These reports equine rotavirus strains. established the fact that more or less out of the 11 segments of RVAs the interspecies transmission event are constantly 5 Picobirnavirus changing the nature of equine RVA being detected in the horse population worldwide. While a report from UK reported that Picobirnaviruses (PBVs) are small, un-enveloped viruses the strain L338 has no closeness with any other rotavirus and containing bi-segmented dsRNA genome. They contains ~2.5 thus confirms that few strains remains solely adaptable to kbp segments 1 and ~ 1.7 kbp for the segment 2 (Malik et al., equine with no sign of interspecies transmission (Ciarlet et al., 2014). The segment I encodes for the single structural capsid 2001). protein gene and segment 2 encodes the viral RNA polymerase (RdRp). It is placed in a new viral family namely Although out of the eleven segments of RVA, eight showed Picobirnaviridae and is assigned a new taxonomic order which the conserved nature among the equine RVAs, whereas only contains a single genus Picobirnavirus. The genus include only VP6, VP7 and NSP4 genes showed existence of 2 diverse two species i.e. Human Picobirnavirus and Rabbit genotypes: I2/I6, G3/G14 and E2/E12, respectively. These Picobirnavirus (Malik et al., 2014). The human PBV serves as reports also hypothesized the presence of distinguished the type species whereas the rabbit PBV serves as the lineages present among the common equine genotype designated species according to the ICTV. Based on the RdRp constellations. At least three lineages of equine genomic gene segment 2, PBVs are grouped into 2 genogroups constellation are circulating among equine rotaviruses since (genogroup-I and II). Recent studies also proposed possible 1990s including G3-P[12]-I6-E2 lineage I, G14-P[12]-I2-E2 new genogroup namely III, IV and V based on the diversity of lineage I, and G14-P[12]-I2-E2 lineage II (Nemoto et al., RdRp sequences (Smits et al., 2014; Li et al., 2015). The 2015c). Though these lineages are interim therefore more proposed new groups include strains from human (group III complete information will be needed to decipher the and V), dromedary (group V) and equine (group IV and V). relationship of these lineages. Unusually the three complete Equine PBV strains have been identified in horse blood plasma genome reports of equine RVA from Argentina showed the (Li et al., 2015). presence of E12 NSP4 genotype which were recently recovered from guanacos and the unusual bovine RVA strain With the advancement of molecular diagnostic techniques such (G15P[11]) from Argentina (Matthijnssens et al., 2009). as RT-PCR (Ganesh et al., 2012; Malik et al., 2013; Takiuchi et al., 2016) and qRT-PCR (Malik, unpublished data) for Further, this E12 genotype was also detected in all analyzed picobirnaviruses, they have been detected in faecal and isolates from 1998 to 2008 in Argentinian foals (Garaicoechea respiratory samples from over 20 animal species including et al., 2011). Certain unpublished data also shown the rodents, aves and large animals like rats, hamsters, guinea pigs, incidence of E12 NSP4 genotype in Argentinean goat and giant ant eater, dogs, pigs, bovine calves, buffalo calves, foals cattle population. But it was surprising to find that NSP4 E12 camels, snakes worldwide and exhibit vast genetic diversity genotype is only confined to equine RVA strains to much of (Ganesh et al., 2011; Malik et al., 2011; Smits et al., 2011; extent which may be the success indicator of quarantine Gillman et al., 2013; Malik et al., 2013; Malik et al., 2014; Ng procedures involved in the transportation of horses which et al., 2014; Ribeiro et al., 2014; Woo et al., 2014, Verma et

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S189 Sircar et al al., 2015; Takiuchi et al., 2016). Despite the broad host range, Acknowledgments pathogenicity of PBVs alone or as co-infecting agents remains unclear. All the authors acknowledge their thanks for support to their respective institutions and universities. In equines, PBVs have been reported for the first time in the year 1991 in foals in Ireland and Britain (Browning et al., Conflict of interest 1991b), followed by the RT-PCR detection in a female foal from Kolkata, India (Ganesh et al., 2011). Recently, equine Authors would hereby like to declare that there is no conflict of PBVs co-infections were detected in plasma of a horse interests that could possibly arise. showing depression, loss of appetite (Li et al., 2015). Sequence analysis of viral RdRp and capsid genes showed four highly References diverse picobirnaviruses including a novel fused picobirnavirus genome. These few reports of equine PBVs deserves attention Anzai T, Fukunaga Y, Matsumura T, Imagawa H, Oikawa MA regarding their epidemiological study and towards the (2001) Serological Examination for Viral Infection among development of diagnostic tools. Young Racehorses Transported by Vehicle over a Long Distance. Journal of Equine Science 12: 135-137. DOI: 6 Miscellaneous Enteric Viral Infections 10.1294/jes.12.135

Several other viruses have also been shown to induce diarrhea Arana A, Montes M, Jere KC, Alkorta M, Iturriza-Gómara M, like symptoms in neonatal or young foals but their relevance to Cilla G (2016) Emergence and spread of G3P [8] rotaviruses enteritis less studied. Parvovirus in animals is usually known possessing an equine-like VP7 and a DS-1-like genetic for its diarrheal symptoms but in equines it is reported in backbone in the Basque Country (North of Spain), 2015. aborted equine fetus (Wong et al., 1985). Of late, parvovirus Infection, Genetics and Evolution 44: 137-144. was recorded in cerebrospinal fluid (CSF) of a horse exhibiting neurological signs (Li et al., 2015). The study also enlist some Ardans A, Pritchett RF, Zee YC (1973) Isolation and other enteric viruses with no relevance to enteritis in equine characterization of an equine adenovirus. Infection and namely anelloviruses, cyclovirus, kirkovirus etc. The scanty Immunity 7: 673–677. data on these viruses especially in equines is still lacking. Arguedas MG (2007) Coronavirus Infections: Equine Conclusion Coronavirus. In: Sellon DC, Long MT (Eds.) Equine Infectious Diseases, Elsevier, Philadelphia, USA. Many diseases have their multifactorial causes with different symptoms and etiologies. A peculiar type of immune response Bailey KE, Gilkerson JR, Browning GF (2013) Equine is prompted by a particular pathogen whereas, other co- rotaviruses—Current understanding and continuing challenges. infecting pathogens make it complex. In veterinary medicine, Veterinary Microbiology 167: 135-144. the documentation of GIT infections with co-infecting pathogens has not been well studied. Especially in equines, the Barr B (2016) Nutritional management of the foal with knowledge of co-infections is very scarce. Different enteric diarrhoea. Equine Veterinary Education DOI: viruses have different symptoms and their spread can be 10.1016/j.vetmic.2013.07.010 controlled by better diagnosis and intensive care of the foals. Prognosis for a young foal with diarrhea varies according to Barrandeguy M, Parreno V, Lagos M, Lezica F Pont, Rivas C, the causative virus and hence the severity of clinical signs also Valle C, Fernandez F (1998) Prevention of rotavirus diarrhoea changes. in foals by parenteral vaccination of the mares: field trial. Developments in Biological Standardization 92: 253–257. Apart from vaccine development other options such as herbal preparations can be also tried as potential preventive Bass EP, Sharpee RL (1975) Coronavirus and gastroenteritis in alternative. Since enteric infections are a big challenge foals. The Lancet 2: 822. worldwide we need to explore better preventive options available as anything might prove efficient and miraculous in Biermann U, Schmitt K, Krauss H (1991) Electron preventing these infection in days to come. For inactivation of microscopic virus diagnosis in dogs, cats, calves, swine and the environmental contamination through virus shedding, foals in the year 1989. Berliner Und Munchener Tierarztliche proper disinfectants must be opted as well as selection of better Wochenschrift 104: 117-119. hand sanitizers must be taken care. Therefore, better management practices, nutritional management, diagnosis and Boileau MJ, Kapil S (2010) Bovine coronavirus associated treatment strategy according to the causative virus is important syndromes. Veterinary Clinics of North America. Food Animal for the equine health. Practice 26: 123-146. DOI: 10.1016/j.cvfa.2009.10.003

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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

LYME BORRELIOSIS IN THE HORSE:

A MINI-REVIEW

* J.H. van der Kolk

Division of Clinical Veterinary Medicine, Swiss Institute for Equine Medicine (ISME), Vetsuisse Faculty, ALP Haras, University of Bern, Länggassstrabe 124, 3012 Bern, Switzerland

Received – November 06, 2016; Revision – November 20, 2016; Accepted – December 05, 2016 Available Online – December 17, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S196.S202

KEYWORDS ABSTRACT Lyme borreliosis

Horse Lyme borreliosis is a multisystemic tick borne disease (also called Lyme disease in humans) which is caused by the Borrelia burgdorferi sensu lato species complex. It is a thin, elongated Gram-negative bacterium and exhibiting motility with flagellar projections. Affected animals mainly show cranial or peripheral neuropathies and uveitis as the more commonly seen extraneural manifestation. Serological evidences confirm its higher occurrence in elderly horses than young ones. Although, incidence of equine Lyme borreliosis is low, its diagnosis is a real challenge. As no indisputable test exists for detecting antibodies to B. burgdorferi, histopathology remains the gold standard and might reveal vascular sclerosis and pleocellular inflammatory infiltrates in neural tissue. Despite antibiotic treatment clinical signs might progress or recur. This review converses with the disease etiology, pathobiology in brief and its mangment from publich health point of view.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (J.H. van der Kolk) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

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S197 Kolk

1 Introduction In Austria, B. afzelii is the predominant genospecies in clinically normal horses (Muller et al., 2002). Lately, B. Lyme borreliosis caused by the Borrelia burgdorferi sensu lato lusitaniae infection has been noticed in horses in Italy species complex, is a multisystemic tickborne disease (also (Veronesi et al., 2012). called Lyme disease in humans). About a century back, Afzelius was the first to notice expanding skin lesion in a 3 Epidemiology human patient, and named it as erythema migrans, which has now become clear to be as the initial skin manifestation of In Europe, Ixodes ricinus is the main vector of B. burgdorferi Lyme disease. Subsequently, in 1976 Burgdorfer and his s.l. in comparison to black-legged ticks (Ixodes scapularis) associates first time reported the etiologic agent of this disease which is more common in the USA. The small rodents also act and also performed an epidemiological evaluation on a cluster as reservoirs (Gern et al., 1998, Humair et al., 1998). of children suffering with arthritis in the city Old Lyme, Additionally, birds have been found to play a major role in the Connecticut, USA. This became the first report on Lyme ecology of Lyme borreliosis, where ticks have been transported disease (Mast & Burrows 1976; Steere et al., 1977; Burgdorfer over large distances and across geographical barriers by avian et al., 1982; Butler et al., 2005; Steere 2006; Staneck et al., hosts (Humair, 2002). To note, particular vector adaptations 2012). determine the geographic distribution range of genospecies (Becker et al., 2016). Hitherto studies have revealed geographical restiction of this bacteria, as in Europian countries, five genospecies of B. It has been noticed that B. burgdorferi s.s. may persist in burgdorferi s.l. have been found to affect humans, namely B. clinically healthy horses (Chang et al., 2000). The afzelii, B. burgdorferi s.s., B. garinii, B. spielmanii and B. seroprevalence of Lyme borreliosis in horses varies with Bavariensis. Among these, the first three are the predominant geographical areas. In some areas of the north-eastern USA it species. While in North America, B. burgdorferi s.s. is the only is about 33-50% (Funk et al., 2016; Magnarelli et al., 2000), species that is pathogenic for humans. In Asian region, B. 9.8-42.8% in Brazil (Basile et al., 2016), 31-48% in France garinii is more commonly reported (Staneck et al., 2012). (Maurizi et al., 2010), 29% in Denmark (Hansen et al., 2010), 26% in Poland (Stefanciková et al., 2008), 24% in Italy (Ebani In the equine species, diagnosis of Lyme borreliosis remains a et al., 2012), 6% in Turkey (Bhide et al., 2008), and 5.5% in challenge like in other species. As a consequence, the question Korea (Lee et al., 2016). Older horses are more prone for if Lyme borreliosis in horses is overdiagnosed remains valid positive test response than younger ones (Ebani et al., 2012; even to date (Bartol, 2013). This review converses with the Funk et al., 2016). Of note, no significant differences in the recent information available on Lyme borreliosisin the horse. mean seroprevalence were observed in the respective years in Italy (Ebani et al., 2012). It has been indicated that the majority 2 Etiology of horses that were positive on initial testing did not have a different test result 5-17 months later (Funk et al., 2016). Borrelia sps. are gram-negative, flagellated, thin and elongated Recently, Lee et al. (2016) showed statistically significant motile bacteria. This bacterium possess 21 plasmids (nine differences according to breed and region where variances circular and 12 linear), which is the largest number of plasmids might be attributed to the ecology of vector ticks and climate to be found in any known bacterium. Moreover, it also shows conditions. genetic complexity with at least 132 functioning genes, intracellular localisation, immune evasion, and auto-regulation, The presence of viable B. burgdorferi spirochetes observed in which makes this spirochaete a formidable infectious pathogen clinically healthy horse’s urine in an endemic region (Manion (Qiu et al., 2004; Stricker et al., 2005).These bacteria belong to et al., 1998) has raised concern whether non-tick transmission the phylum Spirochaetes and are grouped in the B. burgdorferi of this bacterium may occur by direct urine/mucosal contact s.l. genospecies complex that contains at least 20 proposed (Butler et al., 2005). genospecies such as B. afzelii, B. garinii, B. burgdorferi sensu stricto, B. andersoni, B. japonica, B. lusitaniae, B. sinica, B. 4 Pathophysiology tanuki, B. turdii, B. valaisiana, and B. bissettii (Stanek et al., 2004; Wang et al., 1999; Becker et al., 2016). Phylogenetically The attachment of an outer-coat protein (OspA) displayed on all these genospecies are classifed into two major clades: one the lumenal side of the gut of ticks (like black-legged ticks that represents Europian and Asian species and another Ixodes scapularis or Ixodes ricinus) to a receptor (TROSPA) consists of species found in North America and Europe. favours B. burgdorferi to persist in the gut and avoid Furthermore, the two groups in the “American” cluster (B. elimination (Pal et al., 2004). The infection may be acquired burgdorferi s.s. and B. Bissettiae) occur in Europe as well as in through larvae or nymphs feeding on small to medium sized North America. This raises concern over the common ancestor wild animals harbouring the B. burgdorferi as reservoir. Adult of this cluster whether it originated in North America or ticks have been found to only engorge on larger animals (deer, Europe (Becker et al., 2016). sheep, cows and horses). Of the note, B. burgdorferi s.l. DNA has been more commonly detected in female ticks, followed by

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Lyme borreliosis in the horse: A mini-review S198 nymphs and larvae, and least in adult male ticks (Wodecka, The variation of the clinical presentation in B. burgdorferi 2003). After the attachment of ticks to a host, the spirochetes infected horses might be unapparent due to co-infection with present in the midgut of ticks move through the midgut wall pathogens like A. phagocytophylum. Such variation might also and haemocoel, reach the salivary glands and get inoculated occur due to infection with different genospecies of B. with the saliva of ticks into the host 2-3 days after attachment burgdorferi as has been seen in case of human beings (Butler (Piesman et al., 1987). On few occasions, inoculation may et al., 2005). occur earlier if spirochetes are already present in the salivary glands of the infected tick (Alekseev et al., 1995). Though, for 6 Differential Diagnosis proper B. burgdorferi transmission to occur an infected tick must attach for at least 24 hours on the animal (Thanassi & The differential diagnosis is large not only due to the great Schoen 2000), its transmission to the host has been seen to variety in clinical signs but also associated with different B. occur as early as 18 hours after attachment (Alekseev et al., burgdorferi genospecies and possible co-infection. 1995). Concurrent infection with other tick-borne pathogens like Anaplasma phagocytophilum (Persing 1997) and Theileria 7 Diagnosis equi (Basile et al., 2015) can occur. The diagnosis of borreliosis in horses as well as in other The predominant migration of B. burgdorferi within species remains a challenge as persistent B. burgdorferi connective tissues may provide protection to this bacterium infections without any clinical symptoms have been from humoral antibodies (Divers et al., 2001). documented in horses too (Chang et al., 2000). Antibodies can be detected at 5-6 weeks in Ponies exposed to ticks infected 5 Clinical Presentation with B. burgdorferi, with the highest antibody levels induced at 3 months after exposure (Chang et al., 2000). Preference Of the 16 equine cases with histologically confirmed Lyme might be given to culture of B. burgdorferi from equine skin borreliosis recently reviewed, 12 were geldings, while the biopsies (Chang et al., 2000) combined with a two-step remainder was mares. Breeds included 6 Thoroughbreds, 2 serology protocol (ELISA or IFAT supplemented by protein Paints, 2 Ponies, 2 Quarter Horses, and one each of Haflinger, immunoblotting like Western blot or reverse line blot) (Trevejo Arabian, and Morgan. The breed was not known in one of the et al., 1999; Magnarelli et al., 2000; Butler et al., 2005). cases studied. The horses were not vaccinated against Borrelia (Johnstone et al., 2016). The development and validation of a new fluorescent bead- based multiplex assay for the detection of antibodies to outer Incubation period of this bacterium in the equine species has surface protein A (OspA), OspC and OspF antigens of B. not been established yet. Clinical signs in horses attributed to burgdorferi in horse serum has been reported. This assay has B. burgdorferi include low grade fever and lethargy (Burgess improved analytical and diagnostic sensitivities compared to & Mattison 1987b; Magnarelli et al., 1988; Johnstone et al., Western blot analysis. Multiplex analysis is a valuable 2016), weight loss (Johnstone et al., 2016), changes in quantitative tool that simultaneously detects antibodies behavior (Johnstone et al., 2016), dysphagia (Johnstone et al., indicative for natural infection with and/or vaccination against 2016), lameness (Browning et al., 1993), arthritis (Burgess et the Lyme pathogen (Wagner et al., 2011). al., 1986; Hahn et al., 1996; Passamonti et al., 2015; Johnstone et al., 2016), neck stiffness (Johnstone et al., 2016), episodic Commercial C6 testing identified most infected horses but also respiratory distress (Johnstone et al., 2016), muscle tenderness resulted in false positive and false negative interpretations (Divers et al., 2003) and fasciculations (Johnstone et al., 2016), (Johnson et al., 2008; Wagner et al., 2013; Schvartz et al., anterior uveitis (Burgess et al., 1986; Hahn et al., 1996; 2015b). A recent study indicated that the available serologic Johnstone et al., 2016), cranial nerve deficits (Johnstone et al., tests (a point-of-care C6 enzyme-linked immunosorbent assay 2016), ataxia (Johnstone et al., 2016), meningo-encephalitis (ELISA), an whole-cell IFAT, an ELISA confirmed with (Burgess & Mattison 1987b; James et al., 2010; Imai et al., Western blot, and the Lyme multiplex assay for antibodies 2011), abortion (Sorensen et al., 1990), cardiac arrhythmias against B. burgdorferi) all lacked agreement when used to (Johnstone et al., 2016) and foal mortality (Burgess et al., assess the exposure to B. burgdorferi of horses from a low- 1987a). Ataxia was characterized by general proprioceptive prevalence population. Samples found positive by whole-cell deficits and was frequently reported in conjunction with limb IFAT and Lyme multiplex assay, detecting antibodies against paresis. Signs of generalized lower motor neuron weakness, Osp C during early antibody responses, could yield negative facial nerve deficits with paresis or muscle fasciculations have results by ELISA. The differences between the diagnostic tests been observed less frequently. Dysphagia, tongue paresis and owes to varying sensitivities for Osp C antibodies detection, fasciculations are also clinically evident. The variation supported by the low anti-Osp C titers in the Lyme multiplex reported in presenting complaints reflects the multisystemic assay. Caution against the use of serologic “screening” in the nature of Lyme borreliosis. Uveitis has been reported to be the absence of clinical suspicion has been advocated accordingly most frequent extraneural manifestation of Borrelia infection (Schvartz et al., 2015a). (Johnstone et al., 2016).

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Overall, 94% of the suspected horse samples were seropositive 5/10 horses with histologically confirmed Lyme borreliosis. Of by luciferase immunoprecipitation systems (LIPS) test, and the 5 cases that had a negative PCR, 4 were positive with heat map analysis revealed that seropositive samples often Warthin–Starry stain and one showed an indication of were immunoreactive with at least two of the three antigens intrathecal antibody production (Johnstone et al., 2016). The (against the synthetic VOVO antigen, comprising repeated distribution and histologic features including vascular sclerosis immunodominant C6 epitopes as well as OspC (indicating chronicity) and pleocellular inflammatory infiltrates immunodominant epitopes). These results suggest that LIPS present in horses with histologically confirmed Lyme tests employing multiple recombinant antigens offer a borreliosis are rather characteristic (Johnstone et al., 2016). promising approach for the evaluation of antibody responses in Lyme borreliosis (Burbelo et al., 2011). 9 Management/Treatment (including prognosis)

Due to limitations of false negative results of serum tests In comparison to oral administration of doxycycline or during B. burgdorferi associated uveitis and their failure to parenteral sodium ceftiofur, tetracycline @ 6.6 mg/kg BW IV identify an active infection, a combination of cytologic bid for 3 weeks has been found to be superior for treatment of assessment, antibody, and/or PCR testing of ocular fluids has B. burgdorferi infected ponies (Divers et al 2003). Besides, been suggested to be worthwhile when the clinical suspicion is avoiding tick-infested areas as well as careful grooming of the high for Lyme uveitis. horse for early removal of ticks are the best preventive measures. For prevention of tick-infestation, various kind of Of note, horses treated with antibiotics revealed a decline in insecticidal sprays can be used but most of these have not been ELISA titres as compared to control horses (P ≤ 0.05) while approved for horses and also their efficacy is unproven yet the untreated horses showed increased ELISA titres (OR = 0.5; (Butler et al., 2005). However, the use of canine tick sprays on 95% C.I. = 0.3-0.9). Such decline in ELISA titres was low in horses till so far has not revealed any adverse effects (Divers et comparison to the previously reported results in al., 2001). experimentally infected and treated ponies. Horses exposed in the field with B. burgdorferi having high ELISA values when Of interest, a 12-year-old thoroughbred horse with B. treated with either oxytetracycline or doxycycline may show burgdorferi infection responded well to doxycycline treatment only a small decline in ELISA values (Divers et al., 2012). (10 mg/kg BW PO q 12 h for 60 days) and returned to normal exercise. However, the horse was found to again develop a stiff It should be realized that negative serology and normal CSF neck and rapidly progressive neurologic deficits along with analysis do not exclude the diagnosis of Lyme borreliosis and severe ataxia and vestibular deficits after 60 days of treatment. it has been stated that histopathology might represent the most The condition of the horse got deteriorated rapidly despite definitive test for borreliosis in horses. However, when administering IV oxytetracycline, and it was euthanatized presented with a horse displaying ataxia, cranial nerve deficits, (James et al., 2010). and weight loss, with historic or current evidence of uveitis, collapse, or dysphagia, one should consider Lyme borreliosis Eight horses out of sixteen horses with histologically regardless of CSF analysis or serological results (Johnstone et confirmed Lyme borreliosis received antibiotic treatment, al., 2016). including doxycycline, minocycline, oxytetracycline, or ceftiofur. In these cases, clinical signs either continued to 8 Pathology progress or, despite an initial improvement, plateaued or showed recurrence and subsequent progression (Johnstone et Duration of disease before death has been observed to range al., 2016). from 2 to 730 days with a median of 120 days (IQR 33–180 days) (Johnstone et al., 2016). Spirochetes can be visualized in Of importance, persistently high serum titres observed after affected tissues of the horses with Lyme borreliosis by Steiner treatment of Lyme disease in horses, without the presence of silver impregnation and immunohistochemistry, predominantly clinical signs, may not be a reason to follow more prolonged within the dense collagenous tissue of the dura mater and treatment (Divers et al., 2012). leptomeninges (Imai et al., 2011). Leptomeningitis, lymphohistiocytic leptomeningeal vasculitis, cranial neuritis, 10 Public Health Significance and peripheral radiculoneuritis with Wallerian degeneration are the lesions observed during histopathology, which are Lyme borreliosis is regarded as an important tick borne consistent with a diagnosis of neuroborreliosis (James et al., zoonosis although the equine species is not considered as a 2010; Johnstone et al., 2016). main reservoir for human infection. However, the presence of viable B. burgdorferi spirochetes has been observed in urine of In comparison, lesions in B. burgdorferi s.s. infected ponies clinically healthy horses (Manion et al., 1998). Lyme have been reported to be limited to the skin, observed as borreliosis is the most common human tick-transmitted disease perivascular and perineural lymphohistiocytic aggregates in the in the northern hemisphere. A complete presentation of the superficial and deep dermis (Chang et al., 2000). B. disease is an extremely unusual observation in which a skin burgdorferi PCR of nervous tissue obtained positive results in lesion results from a tick bite and is followed by heart and

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Lyme borreliosis in the horse: A mini-review S200 nervous system involvement, and later on by arthritis. Late Burgess EC, Gillette D, Pickett JP (1986) Arthritis and involvement of eye, nervous system, joints, and skin can also panuveitis as manifestations of Borrelia burgdorferi infection occur. The only sign that enables a reliable clinical diagnosis in a Wisconsin pony. Journal of the American Veterinary of Lyme borreliosis in humans is erythema migrans (Stanek et Medical Association 189: 1340-1342. al., 2004). Burgess EC, Genchon-Fitzpatrick A, Mattison M (1987a) Foal Conflict of interest mortality associated with natural infection of pregnant mares with Borrelia burgdorferi (abstract). 5th International Authors would hereby like to declare that there is no conflict of Conference of Equine Infectious Diseases pp: 217. interests that could possibly arise. Burgess EC, Mattison M (1987b) Encephalitis associated with References Borrelia burgdorferi infection in a horse. Journal of the American Veterinary Medical Association 191: 1457-1458. Alekseev AN, Arumova EA, Vasilieva IS (1995) Borrelia burgdorferi sensu lato in female cement plug of Ixodes Butler CM, Houwers DJ, Jongejan F, Kolk JH van der (2005) persulcatus ticks (Acari, Ixodidae). Experimental and Applied Borrelia burgdorferi infections with special reference to horses. Acarology 19: 519-522. A review. Veterinary Quarterly 27 : 146-156.

Bartol J (2013) Is Lyme disease overdiagnosed in horses? Chang YF, Novosol V, McDonough SP, Chang CF, Jacobson Equine Veterinary Journal 45: 529-30. RH, Divers TJ, Quimby FW, Shin S, Lein DH (2003) Experimental infection of ponies with Borrelia burgdorferi by Basile RC, Rivera GG, Del Rio LA, de Bonis TC, do Amaral exposure to Ixodid ticks. Veterinary Pathology 37: 68-76. GP, Giangrecco E, Ferraz G, Yoshinari NH, Canola PA, Queiroz Neto A (2015) Anaphylactoid reaction caused by Divers TJ, Chang YF, Jacobson RH, McDonough SP (2001) sodium ceftriaxone in two horses experimentally infected by Lyme disease in horses. Compendium on Continuing Borrelia burgdorferi. BMC Veterinary Research 11: 197. Education for the Practicing Veterinarian 23: 375-380.

Basile RC, Yoshinari NH, Mantovani E, Bonoldi VN, Macoris Divers TJ, Chang YF, McDonough PL (2003) Equine Lyme DD, Queiroz-Neto A (2016) Brazilian borreliosis with special disease: a review of experimental disease production, treatment emphasis on humans and horses. Brazilian Journal of efficacy, and vaccine protection. 49th Annual Convention of Microbiology. pii: S1517-8382(16)30902-9. doi: the American Association of Equine Practitioners, November 10.1016/j.bjm.2016.09.005. 2003, New Orleans, Louisiana, USA.

Becker NS Margos G, Blum H, Krebs S, Graf A, Lane RS, Divers TJ, Grice AL, Mohammed HO, Glaser AL, Wagner B Castillo-Ramírez S, Sing A, Fingerle V (2016) Recurrent (2012) Changes in Borrelia burgdorferi ELISA antibody over evolution of host and vector association in bacteria of the time in both antibiotic treated and untreated horses. Acta Borrelia burgdorferi sensu lato species complex. BMC Veterinaria Hungarica 60 : 421-429. Genomics 17:734. Ebani VV, Bertelloni F, Pinzauti P, Cerri D (2012) Bhide M, Yilmaz Z, Golcu E, Torun S, Mikula I (2008) Seroprevalence of Leptospira spp. and Borrelia burgdorferi Seroprevalence of anti-Borrelia burgdorferi antibodies in dogs sensu lato in Italian horses. Annals of Agricultural and and horses in Turkey. Annals of Agricultural and Environmental Medicine 19 : 237-240. Environmental Medicine 15: 85-90. Funk RA, Pleasant RS, Witonsky SG, Reeder DS, Werre SR, Browning A, Carter SD, Barnes A, May C, Bennett D (1993) Hodgson DR (2016) Seroprevalence of Borrelia burgdorferi in Lameness associated with Borrelia burgdorferi infection in the Horses Presented for Coggins Testing in Southwest Virginia horse. Veterinary Record 132: 610-611. and Change in Positive Test Results Approximately 1 Year Later. Journal of Veterinary Internal Medicine 30 : 1300-1304. Burbelo PD, Bren KE, Ching KH, Coleman A, Yang X, Kariu T, Iadarola MJ, Pal U (2011) Antibody profiling of Borrelia Gern L, Estrada-Pena A, Frandsen F, Gray JS, Jaenson TGT, burgdorferi infection in horses. Clinical and Vaccine Jongejan F, Kahl O, Korenberg E, Mehl R, Nuttall PA (1998) Immunology 18: 1562-1567. European reservoir hosts of Borrelia burgdorferi sensu lato. Zentralblatt für Bakteriologie 287: 196-204. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP (1982) Lyme disease-a tick-borne spirochetosis? Hahn CN, Mayhew IG, Whitwell KE, Smith KC, Carey D, Science 216 : 1317-1319. Carter SD, Read RA (1996) A possible case of Lyme borreliosis in a horse in the UK. Equine Veterinary Journal 28:84-88.

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Hansen MG, Christoffersen M, Thuesen LR, Petersen MR, horses. Journal of Veterinary Diagnostic Investigation 10: 196- Bojesen AM (2010) Seroprevalence of Borrelia burgdorferi 199. sensu lato and Anaplasma phagocytophilum in Danish horses. Acta Veterinaria Scandinavica 52: 49. Mast WE, Burrows WM (1976) Erythema chronicum migrans and "lyme arthritis". JAMA 236 : 2392. Humair PF, Rais O, Gern L (1999) Transmission of Borrelia afzelii from Apodemus mice and Clethrionomys voles to Maurizi L, Marié JL, Aoun O, Courtin C, Gorsane S, Chal D, Ixodes ricinus ticks: differential transmission pattern and Davoust B (2010) Seroprevalence survey of equine Lyme overwintering maintenance. Parasitology 118: 33-42. borreliosis in France and in sub-Saharan Africa. Vector Borne and Zoonotic Diseases 10: 535-537. Humair PF (2002) Birds and Borrelia. International Journal of Medical Microbiology 291: 70-74. Muller I, Khanakah G, Kundi M, Stanek G (2002) Horses and Borrelia: immunoblot patterns with five Borrelia burgdorferi Imai DM, Barr BC, Daft B, Bertone JJ, Feng S, Hodzic E, sensu lato strains and sera from horses of various stud farms in Johnston JM, Olsen KJ, Barthold SW (2011) Lyme Austria and from the Spanish Riding School in Vienna. neuroborreliosis in 2 horses. Veterinary Pathology 48 : 1151- International Journal of Medical Microbiology 291:80-87. 1157. Pal U, Li X, Wang T, Montgomery RR, Ramamoorthi N, James FM, Engiles JB, Beech J (2010) Meningitis, cranial Desilva AM, Bao F, Yang X Pypaert M, Pradhan D, Kantor neuritis, and radiculoneuritis associated with Borrelia FS, Telford S, Anderson JF, Fikrig E (2004) TROSPA, an burgdorferi infection in a horse. Journal of American Ixodes scapularis receptor for Borrelia burgdorferi. Cell 119: Veterinary Medical Association 237: 1180-1185. 457-468.

Johnson AL, Divers TJ, Chang YF (2008) Validation of an in- Passamonti F, Veronesi F, Cappelli K, Capomaccio S, clinic enzyme-linked immunosorbent assay kit for diagnosis of Reginato A, Miglio A, Vardi DM, Stefanetti V, Coletti M, Borrelia burgdorferi infection in horses. Journal of Veterinary Bazzica C, Pepe M (2015) Polysynovitis in a horse due to Diagnostic Investigation 20: 321-324. Borrelia burgdorferi sensu lato infection--Case study. Annals of Agricultural and Environmental Medicine 22 : 247-250. Johnstone LK, Engiles JB, Aceto H, Buechner-Maxwell V, Divers T, Gardner R, Levine R, Scherrer N, Tewari D, Persing DH (1997) The cold zone: a curious convergence of Tomlinson J, Johnson AL (2016) Retrospective Evaluation of tick-transmitted diseases. Clinical Infectious Diseases 25 :S35- Horses Diagnosed with Neuroborreliosis on Postmortem S42. Examination: 16 Cases (2004-2015). Journal of Veterinary Internal Medicine. 30 : 1305-1312. Piesman J, Mather TN, Sinsky RJ, Spielman A (1987) Duration of tick attachment and Borrelia burgdorferi Lee SH, Yun SH, Choi E, Park YS, Lee SE, Cho GJ, Kwon transmission. Journal of Clinical Microbiology 25: 557-558. OD, Kwak D (2016) Serological detection of Borrelia burgdorferi among horses in Korea. Korean Journal Qiu WG, Schutzer SE, Bruno JF, Attie O, Xu Y, Dunn JJ, Parasitology 54 : 97-101. Fraser CM, Casjens SR, Luft BJ (2004) Genetic exchange and plasmid transfers in Borrelia burgdorferi sensu stricto revealed Magnarelli LA, Anderson JF, Shaw E, Post JE, Palka FC by three-way genome comparisons and multilocus sequence (1998) Borreliosis in equids in northeastern United States. typing. Proceedings of the National Academy of Sciences of American Journal of Veterinary Research 49: 359-362. the United States of America 101: 14150-14155.

Magnarelli LA, Flavell RA, Padula SJ, Anderson JF, Fikrig E Schvartz G, Epp T, Burgess HJ, Chilton NB, Lohmann KL (1997) Serologic diagnosis of canine and equine borreliosis: (2015a) Comparison between available serologic tests for use of recombinant antigens in enzyme-linked immunosorbent detecting antibodies against Anaplasma phagocytophilum and assays. Journal of Clinical Microbiology 35: 169-173. Borrelia burgdorferi in horses in Canada. Journal of Veterinary Diagnostic Investigation 27 : 540-546. Magnarelli LA, Ijdo JW, Andel AE van, Wu C, Padula SJ, Fikrig E (2000) Serologic confirmation of Ehrlichia equi and Schvartz G, Epp T, Burgess HJ, Chilton NB, Pearl DL, Borrelia burgdorferi infections in horses from the northeastern Lohmann KL (2015b) Seroprevalence of equine granulocytic United States. Journal of the American Veterinary Medical anaplasmosis and lyme borreliosis in Canada as determined by Association 217: 1045-1050. a point-of-care enzyme-linked immunosorbent assay (ELISA). Canadian Veterinary Journal 56 : 575-80. Manion TB, Khan MI, Dinger J, Bushmich SL (1998) Viable Borrelia burgdorferi in the urine of two clinically normal Stefanciková A, Adaszek Ł, Pet'ko B, Winiarczyk S, Dudinák V (2008) Serological evidence of Borrelia burgdorferi sensu

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Lyme borreliosis in the horse: A mini-review S202 lato in horses and cattle from Poland and diagnostic problems Trevejo RT, Krause PJ, Sikand VK, Schriefer ME, Ryan R, of Lyme borreliosis. Annals of Agricultural and Environmental Lepore T, Porter W, Dennis DT (1999) Evaluation of two-test Medicine 15: 37-43. serodiagnostic method for early Lyme disease in clinical practice. Journal of Infectious Diseases 179: 931-938. Sorensen K, DP Neely, Grappell PM, Read W (1990) Lyme disease antibodies in Thoroughbred broodmares, correlation to Veronesi F, Laus F, Passamonti F, Tesei B, Piergili Fioretti D, early pregnancy failure. Journal of Equine Veterinary Science Genchi C (2012) Occurrence of Borrelia lusitaniae infection in 10 :166-168. horses. Veterinary Microbiology 160 : 535-538.

Stanek G, Gray J, Strle F, Wormser G (2004) Lyme Wagner B, Freer H, Rollins A, Erb HN, Lu Z, Gröhn Y (2011) borreliosis. Lancet Infectious Diseases 4: 197-199. Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation Stanek G, Wormser GP, Gray J, Strle F (2012) Lyme using Bayesian and conventional statistical methods. borreliosis. Lancet 379: 461–473. Veterinary Immunology and Immunopathology 144 : 374-381.

Steere AC (2006) Lyme borreliosis in 2005, 30 years after Wagner B, Goodman LB, Rollins A, Freer HS (2013) initial observations in Lyme Connecticut. Wiener Klinische Antibodies to OspC, OspF and C6 antigens as indicators for Wochenschrift 118: 625-633. infection with Borrelia burgdorferi in horses. Equine Veterinary Journal 45 : 533-537. Steere AC, Malawista SE, Snydman DR, Shope RE, Andiman WA, Ross MR, Steele FM (1997) Lyme arthritis: an epidemic Wang G, Dam AP van, Schwartz I, Dankert J (1999) of oligoarticular arthritis in children and adults in three Molecular typing of Borrelia burgdorferi sensu lato: connecticut communities. Arthritis and Rheumatology 20 : 7- taxonomic, epidemiological, and clinical implications. Clinical 17. Microbiology Reviews 12: 633-653.

Stricker RB, Lautin A, Burrascano JJ (2005) Lyme disease: Wodecka B (2003) Detection of Borrelia burgdorferi sensu lato point/counterpoint. Expert Review of Anti-Infective Therapy DNA in Ixodes ricinus ticks in North-Western Poland. Annals 3: 155-165. of Agricultural and Environmental Medicine 10: 171-178.

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Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

AN OVERVIEW OF OZONE THERAPY IN EQUINE- AN EMERGING HEALTHCARE SOLUTION

1,* 1 2 3 Jyotsana Bhatt , Abas Rashid Bhat , Kuldeep Dhama and Amarpal

1Research Scholar, Division of Surgery, Indian Veterinary Research Institute, Izatnagar-243122 (UP), India 2Principal Scientist, Division of Pathology, Indian Veterinary Research Institute, Izatnagar-243122 (UP), India 3Head, Division of Surgery, Indian Veterinary Research Institute, Izatnagar-243122 (UP), India

Received – November 11, 2016; Revision – November 26, 2016; Accepted – December 10, 2016 Available Online – December 22, 2016

DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S203.S210

KEYWORDS ABSTRACT Ozone therapy The significance of ozone therapy has been increasingly realised in recent times particularly in equine Equine medicine. The beneficial effects of ozone therapy are basically engendered by the mild oxidative stress it creates upon interacting with the extra-cellular and intracellular components. However therapeutic Oxidative stress benefits of treatment could be obtained only when it is used within the therapeutic window. Higher doses may be counterproductive and lower doses ineffective. It is now well proved that it up regulates Antioxidant the antioxidant system of the patient and may provide relief from many chronic degenerative diseases upon prolonged use. Ozone therapy has shown encouraging results in the treatment of wide spectrum of Hydrogen peroxide diseases and disorders in equines including bacterial and viral infections. Obvious benefits of ozone therapy have been reported in Equine infectious anemia, chlamydial abortions, lymphomas and equine ehrlichiosis. This article provides an insight into the mechanism of action involved in ozone therapy and reviews various conditions which could be treated with the use of ozone therapy in equines.

All the article published by Journal of Experimental * Corresponding author Biology and Agricultural Sciences is licensed under a E-mail: [email protected] (Dr Jyotsana Bhatt) Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org. Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/ ). All ______rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

S204 Bhatt et al

1 Introduction provision for temperature and humidity control as temperature and humidity also play important role in ozone production. A Ozone molecule comprises of three atoms of oxygen arranged variety of ozone generators are available nowadays. However, in a dihedral shape. This allotropic form of oxygen is less three types of ozone generators are commonly used for stable than oxygen due to the presence of mesomeric states production of medical ozone. (Elvis & Ekta, 2011) and reacts with many other compounds like carbon and nitrous oxide. Ozone is 1.6 fold denser and 10- I. Corona discharge type- This type of ozone generators fold more soluble in water than oxygen. It is the third most use corona discharge tube (high voltage electric field). potent oxidant after fluorine and per-sulfate. Ozone is an They are cost effective and use ambient air as a source of unstable gas that cannot be stored and should be used at once oxygen. This type of ozone generators produce a because it has a half life of 40 min at 20°C and 140 min at 0°C concentration of 3-6% of ozone, however they also (Cakir,2014). produce nitrous oxide as a by-product. A research showed the use of meshed–plate electrode in place of In nature ozone is produced by the effect of electrical conventional plate has advantage of decreasing corona discharge on atmospheric oxygen during lightening or by onset voltage and reduced decomposition of ozone ultraviolet radiation. It is a powerful oxidant and has many thereby providing maximum ozone generation applications owing to its oxidant property. It is a highly concentration and ozone generation efficiency (Park reactive molecule which is able to inactivate microorganisms, etal., 2006) boost the immune system and is also able to induce analgesic effect (Duricic et al., 2015). Ozone has many health benefits if II. Ultraviolet lamp type- This type of ozone generators used within the therapeutic window but may cause tissue utilise a light source that generates a narrow-band damage if used above it. Ozone oxygenates every cell of the ultraviolet light with a wavelength of approximately 185 body and increases the stability of healthy cells. Medical ozone nm. They produce ozone with concentration of 0.5% or is a mixture of oxygen and ozone having less than 5% of ozone less but this type of ozone generators are cost effective at maximum concentration and rest is pure oxygen (Bocci, than the corona discharge type generators as they 2006). consume less electricity. Added advantage of such type of generator is that it doesn’t produces nitrous oxide Unfortunately many clinicians and veterinary professionals are (http://www.silvermedicine.org/ozone-therapy- unaware of the therapeutic benefits and mechanism of action of generators.html). ozone upon its interaction with biological fluids. As in most of the cases it is the dose which would decide the therapeutic or III. Cold plasma type- In this type of generator oxygen gas is harmful effects of ozone. But it is now possible to perfectly exposed to a plasma created by dielectric barrier tame the cytotoxicity of ozone by potent antioxidant system of discharge. These ozone generators breaks oxygen the body. During last two decades scientists have made great molecule in atoms of oxygen, which are very reactive efforts to understand the scientific mechanisms underlying the and combines the available oxygen molecules to form a beneficial effects of ozone therapy at both basic research and molecule of ozone. A maximal concentration of 5% is clinical level. Ozone therapy has now been established as produced in such generators treatment of choice for many equine diseases like Equine (http://www.silvermedicine.org/ozone-therapy- infectious anaemia. Ozone therapy is also able to boost the generators.html). antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase etc. in the body (Mandhare et al., 2012). Ozone molecule is very unstable so it must be prepared immediately before use. Within less than an hour after 2 Ozone productions preparation only half of the mixture remained ozone while the rest half transformed into oxygen. Because of this As in nature, ozone is produced by the action of lightening on characteristic it is impossible to store medical grade ozone for oxygen, ozone generation can be achieved by passing oxygen long period of time (Nogales et al., 2008). across an electric arc having a potential difference of about 10,000 Volt in an ozone generator according to the following 3 Mechanism of action of ozone reaction- Ozone on coming in contact with the blood acts on different

3O2 + 68,400 cal------2O3 targets and initiates a cascade of reactions thereby producing several beneficial effects. Unlike oxygen, ozone is very active An ozone generator must be made up of high quality ozone gas and reacts as it comes in contact with blood or any other resistant material like teflon, glass, 316 stainless steel, silicone biological fluid. In order of preference, ozone reacts with etc to resist oxidation (Mandhare et al., 2012). It should also be polyunsaturated fatty acids (PUFA), protiens, antioxidants equipped with a photometer to measure the accurate such as ascorbic acid and glutathione. concentration of ozone produced. There must also be a

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An overview of ozone therapy in equine- an emerging healthcare solution S205

Figure 1 Schematic representation of ozone therapy procedure in horses.

On reaction with bio molecules it produces one molecule of with NO regulates the vasodilation by activating cGMP reactive oxygen species (ROS) mainly hydrogen peroxide and (Bocci,2006). two molecules of lipid oxidation products(LOP). The main reaction with bio molecules is depicted below: The mechanism of beneficial effects of ozone therapy may be summarised in the following ways-

R-CH=CH-R + O3+H2O ----- R-CH=O + R-CH=O + H2O2(Bocci et al.,1993) I. It increases availability and delivery of oxygen, glucose and ATP within ischemic tissues. The by-products of the reaction tend to act in two different II. It enhances implantation of bone marrow stem cells at ways. ROS act immediately by reacting with erythrocytes the site of lesion, which can provide angiogenesis, which are available in the blood stream and disappear. This neovascularization and tissue regeneration. may be termed as early phase reaction, which is short lived. III. It activates a neurohumoral reaction responsible for LOP, on the other hand, are distributed in the tissues and act on improving quality of life. receptor molecules located at different locations in body, IV. It induces up-regulation of the expression of antioxidant undergoing marked dilution within the circulatory system and enzymes and heme-oxygenase I and extends thus their action may be termed as late phase reaction, which preconditioning benefits (Bocci,2006). lasts longer. Ozone however could be toxic to the respiratory system, if ROS, particularly hydrogen peroxide, activates pentose inhaled, because respiratory tract lining has minimal amount of phosphate pathway which is determined by the significant antioxidant coverage. In contrary to that blood has adequate increase in the ATP formation (Bocci, 2005). LOPs produced amount of antioxidants to completely tame up the ozone in the reaction are mainly malonaldehyde and 4- toxicity, if used within the limits of therapeutic range. It is hydroxynonenal, which are very stable and toxic as well in advisable that to enhance the safety of ozone therapy, prior to vitro as compared to ROS. Fortunately, they undergo marked initiation of the therapy, antioxidant level of the patient body dilution in the circulation and gets metabolised upon blood should be measured and should be strengthened by distribution and redistribution in the body thus they reach at administration of antioxidants like vitamin C , α-tocopherol etc their target sites only in the submicromolar concentration (Sagai & Bocci,2011). thereby minimising their toxic effects. 4 Methods of administration of ozone Small concentration of LOPs extend their beneficial effect by up-regulation of the antioxidant enzymes like superoxide 4.1 Auto-hemotherapy dismutase, glutathione peroxide etc (Iles & Liu,2005). They also induce oxidative stress protein like Hemoxygenase-1(HO- Autohemotherapy was first described by Wherli & Steinbart in 1) or Heat shock protein. HO-1 degrade the heme molecule 1954. It may be major or minor autohemotherapy. into CO and bilirubin (Snyder & Barañano, 2001). Bilirubin acts as a powerful antioxidant molecule whereas CO along

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S206 Bhatt et al

In major autohemotherapy method around 250ml of blood is treated. In this method superficial lesions can be treated as collected from the animal in heparin or 3.13% sodium citrate ozone is absorbed through the skin. The method has been anticoagulant and the blood is ozonated outside the body of the applied for the treatment of cutaneous infections like chronic animal for 5-10 minutes and then this ozonated blood is slowly wounds and ulcers (Bocci, 2013). infused back into the animal’s body in over 15 minutes by intravenous routes. Ozonated autohemotherapy (O3-AHT) was 4.4 Ozonated oil used in the treatment of atherosclerotic ischemia of lower limb in human patients (Tylicki et al., 2004).While in minor In ozonated oil method the ozone is used with oil as a carrier of autohemotherapy method, around 5ml of venous blood is ozone. Ozone is bubbled in oil like olive, sesame or sunflower collected from the patient without anticoagulant and ozonated oil until it forms a gel like consistency, the gel can be used to outside the body for 1 minute. The ozonated blood is then treat several conditions like skin infections, insect stings, injected intramuscularly (Borrelli &Bocci, 2009). This minor ulcers, vulvovaginitis and periodontitis (Shoukheba & Ali, autohaemotherapy approach had been used in treatment of 2014 ). chronic laminitis in a 10 years old mare (Coelho et al., 2015) and for mechanical lumbar pain in riding horses (Vigliani et 4.5 Ozony blanket al., 2005). In ozony blanket method an ozonated silicone blanket is placed 4.2 Insufflation around the horse body to ozonate the whole body of the animal. It could be used to treat several local and systemic Insufflation of ozone is done in the body spaces like rectal, conditions. This system proved to be very effective in treating vaginal and ear canal. Rectal insufflation of ozone is most various equine diseases (http://ozonyozone.weebly.com/ozony- commonly practiced. Humidified ozonated gas can be blanket.html (accessed on 24/11/2016) introduced through the rectal opening to treat conditions like diarrhoea and inflammatory bowel disease caused by infections 5 Medical application of ozone therapy in equine such as Rotavirus and Ehrlichia in horses. Rectal sufflation of medical ozone was also used in the treatment of patients with Owing to its versatile biological action, ozone therapy is able type 2 diabetes and diabetic feet (Martínez-Sánchez et al., to treat a wide variety of diseases and conditions. Equine 2005). medicine is a potential field for therapeutic exploitation of this modality as it has a capacity to reverse many serious illnesses. 4.3 Ozone bagging Initial studies suggested that ozone therapy could be very effective in treating many equine diseases by controlling In this method an oxygen-ozone mixture is pumped into an infection, mitigating inflammation and improving anti-oxidant ozone resistant bag which is then placed around the area to be status.

Table 1 Some of the important studies showing beneficial outcome of ozone therapy in clinical cases of equine.

S.no. Scientist / Disease condition Ozone Site of administration After effects Investigator concentration 1 Coelho et al., Chronic laminitis 19mg/L IM, at suprascapular Recovered to obel grade IV 2015 region, Near deep digital to grade II of lameness flexor tendon 2 Vigliani et al., Mechanical lumbar pain 30µg/ml IM, at interspinous and Effective analgesic effect 2005 paravertebral level

3 Shinozuka et Mastitis 0.8mg/l Into the affected quarter Signs of clinical mastitis al., 2008 reversed 4 Akey & Inactivation of 0.025 mg/L Physical exposure for 45 reduction of 99.99997% of Walton, 1985 Venezuelan equine minutes the viral particles encephalomyelitis virus 5 Sechi et al., Cutaneous wound 1·18– Topically as ozonised oil Better healing with increased 2001 healing 9·5 mg/ ml production of various growth factors 6 Ozbay et al., Facial nerve paralysis 1.1 mg/kg Intraperitoneally Lower stimulation thresholds 2016 body weight in ozone treated group 7 Ouf et Antifungal effect 4 µg/ml As ozone gas spray Better recovery with least al.,2016 0.5 and 0.25 As ozonised oil side effects µg/ml

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An overview of ozone therapy in equine- an emerging healthcare solution S207 Several reports and clinical trials have appeared in the recent 5.3 Clinical mastitis in mares literature to prove the efficacy of ozone therapy as an adjunct therapy as well as a sole therapeutic agent. The following text Mastitis in mare is not quite common as in dairy cattle but can describes various medical applications of ozone therapy in affect the health and productivity of mare adversely. Gram treating equine diseases. negative organisms accounted for mastitis in 42% of cases including Klebsiella and E.coli in 11.8% and 5.9% of cases, 5.1 Chronic laminitis respectively (McCue & Wilson, 1989). These gram negative microbes tend to develop endotoxic shock upon treatment with Chronic laminitis is a crippling disease of equine. It does not antibiotics. A study was conducted to compare the amount of easily respond to standard medical therapy. Ozone therapy endotoxin produced upon antibiotic therapy and ozone could be a solution for such cases. A case of chronic laminitis therapy.Results showed that in comparison to antibiotic in a 10 year mare diagnosed with Obel grade IV chronic therapy, ozone therapy (0.8mg/L) resulted in less amount of laminitis on right forelimb was successfully treated with ozone endotoxin production (Shinozuka et al., 2008). It is observed therapy (Coelho et al., 2015). On presentation the horse that ozone therapy helps in reversing the local and systemic exhibited signs of laminitis like shifting lameness, high signs of acute clinical mastitis in animals. The therapeutic temperature, pain on palpation etc. On radiological effects of ozone therapy could be attributable to increase in examination distal phalanx of right limb was displaced by 30 leukocytic function and increased respiratory burst. Ozone degrees. Therapeutic regimen comprised of hoof trimming therapy can be preferred over antibiotic therapy in treating followed by intramuscular, peritendinous and intrarectal clinical mastitis as no milk withdrawal time is required with administration of medical ozone. ozone therapy (Ogata & Nagahata, 2000). However, it is not clear whether ozone functions by killing the pathogen or by The injection of ozone was made at two suprascapular points improving the host defence mechanism. located cranial and caudal to the scapula on both sides, two points were selected in scapular region, one point in the middle 5.4 Enhancement of antioxidant capacity of the radial region and one near the deep digital flexor tendon. The selected points were clipped and aseptically prepared and Oxidative stress can adversely affect the physical ability of a 10 ml mixture of ozone-oxygen having a concentration of 19 performing horses. Ozonated autohemotherapy (OAHT) has mg ozone per ml was injected at each point. No NSAIDs or shown to increase the antioxidant capacity of the blood. In a any other medication was used in the treatment regimen. study 10 thoroughbred horses were examined for their Intrarectal insufflation of ozone was also performed. Ozone biological antioxidant potential (BAP) after treating with ozone therapy was given twice a week for 10 weeks. Six months after at the concentration of 20µg/kg body weight .Same horses therapy mare was able to walk properly along with normal were used as control and for treatment. Control blood and relationship between the dorsal hoof wall and the distal serum samples were collected one month before the OAHT phalanx on radiographic examination. Animal recovered up to and for treatment group 1, 2, 3, 7 and 14 days later. Diacron- the Obel grade II lameness with the help of this therapy reactive oxygen metabolites (d-ROMs) and biological (Coelho et al., 2015). antioxidant potential (BAP) was measured from serum samples to calculate oxidative stress index (OSI).BAP was increased 5.2 Mechanical lumbar pain and spinal muscle disorders in significantly on day 3 and 7 in OAHT group as compared to riding horse control group (Tsuzuki et al., 2015).

Riding and thoroughbred horses which are commonly used for 5.5 Inactivation of Venezuelan equine encephalomyelitis virus racing purpose frequently suffer from back pain. Its probable causes include bone or soft tissue injuries. In a study 30 horses Humidified ozone gas is used as a sterilizing agent for medical suffering from back pain were treated with local infiltration of instruments (Faddis, 1993). Ozone in liquid phase application 15 ml of oxygen-ozone mixture at the ozone concentration of at a concentration of 0.025 mg per liter was able to inactivate 30µg/ml into the affected muscle at interspinous and arbovirus within 45 min of exposure. This study showed a paravertebral level. This study proved efficacy of ozone reduction of 99.99997% of the viral particles as compared to therapy in pain management. Hence, ozone therapy could be the control levels. Hence ozone proved to be an effective considered as an alternative to NSAIDS treatment in cases of candidate as a sterilizing agent in some applications for lumbar pain (Vigliani et al., 2005). It has been observed that biological safety cabinets and other equipment used in vector during its administration, ozone produces an itchy sensation studies with arboviruses (Akey & Walton, 1985). which is replaced by the analgesic effect as animal does not feel pain on palpation at the later stage. Supplementation with 5.6 Cutaneous wound healing antioxidant in the form of vitamin C to maintain oxidant- antioxidant balance could benefit the patient during ozone Ozonated olive oil proved to be effective in healing of therapy (Ballardini, 2005). cutaneous wounds.

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In a study using guinea pigs as animal model the ozonated applied as gas and was 0.5 and 0.25 µg/ml in the case of olive oil showed better wound healing in terms of lesser ozonized oil. Application of ozone in the form of ozonized oil residual wound area, increased number of collagen fibres and appears to be more efficacious than gaseous ozone. fibroblast along with upregulation of various growth factors like PDGF, TGF-β and VEGF in comparison to the group 5.9 Other applications treated with simple olive oil and control group (Kim et al., 2009). Ozone used for wound treatment causes reduction in Several studies have been conducted to develop a non invasive septic process and accelerates wound healing and also technique of ozone therapy in which an ozone blanket is used decreases the cost of antibiotic therapy (Białoszewski & to ozonate the whole body of the horse Kowalewski, 2003).A research showed increased angiogenesis, (http://ozonyozone.weebly.com/ozony-blanket.html accessed an enhanced vascular endothelial growth factors and cyclin D1 on 24/11/2016). This method has shown promising results in a expression as a result of using ozonated sesame oil in wide range of ailments in horses. The method was developed cutaneous wound healing study model in SKH1 mice by South African scientist Gail Pedra (Valacchi et al., 2011). In a study, effect of Oleozon (ozonised (http://ozonyozone.weebly.com/ozony-blanket.html accessed sunflower oil) was tested on Mycobacteria, staphylococci, on 24/11/2016). These researchers treated a foal suffering from streptococci, enterococci, Pseudomonas and Escherichia coli, African Horse Sickness Virus in 2006 by ozonation of the which showed encouraging antimicrobial effects of oleozon on whole body. This method is applied to treat illness by covering tested organisms (MIC range 1·18–9·5 mg/ ml) (Sechi et al., the animal inside an ozony blanket. Several conditions in 2001) . which ozony blanket yields significant outcomes are sarcoma, African horse sickness, lyme disease, pro-active sport therapy 5.7 Neurological malfunctions (facial nerve paralysis) etc. The ozony blankets have been used widely in South Africa and approximately1000 horses have been successfully treated In a case record of 450 horses with signs of neurological for disease control, prevention or enhancement of sports disease, facial nerve paralysis was the most common type of performance. This technique has various benefits as it is a cranial nerve injury (Tyler et al., 1993). Ozone therapy completely portable device, even recumbent animal is able to provides promising results in facial nerve regeneration. In a receive treatment and multiple horses can be handled at one study comprised of fourteen Wistar albino rats, all animals time. underwent surgery in which the left facial nerve was exposed and crushed. Treatment with saline or ozone began on the day Conclusion of the nerve crush. The ozone group received an ozone dose of 1.1 mg/kg/d intraperitoneally (IP) for 30 days. Left facial nerve The benefits of ozone therapy have been documented in stimulation thresholds were measured before crush, numerous studies. It has been proved to be effective as an immediately after crush, and after 30 days. Post-crushing, the alternative treatment or as an adjunct treatment for many ozone-treated group had lower stimulation thresholds than the equine diseases like lameness, mastitis, bacterial and viral saline group. In this study regeneration of the facial nerve was diseases, neurological and musculoskeletal disorders etc. evaluated by assessing electrophysiological thresholds and by Ozone acts to induce mild oxidative stress that can induce histopathological examination. This proves that ozonetherapy production of several anti-oxidant enzymes to benefit the exerted beneficial effect on the regeneration of crushed facial patients and gradually being accepted as therapeutic modality nerves (Ozbay et al., 2016). in orthodox medicine. Medical grade ozone generators and photometers are essential for its optimum production and 5.8 Antifungal potential of ozone potential useful effects. There could be several methods of its administration, which should be standardised for maximum Fungal infection possesses a serious threat to animal health therapeutic benefits. eventually leading to decreased productivity and work potential. Trichophyton equinum was recognized as the most Conflict of interest common cause of equine ringworm in the United States, Canada, South America, and Europe (Georg et al., 1957). M. Authors would hereby like to declare that there is no conflict of canis, T. Mentagrophytes varmentagrophytes, T. verrucosum, interests that could possibly arise. M. Praecox and M. Gypseum are also reported to be associated with equine ringworm (De Vroey et al., 1983). According to References Ouf et al. (2016) Ozone therapy proved to be a better alternative in curing fungal infection and also prevents side Akey DH, Walton TE (1985) Liquid-phase study of ozone effects of synthetic antifungal drugs due to their long term inactivation of Venezuelan equine encephalomyelitis virus. application. Fungal wall comprised of approximately 80% Applied and Environmental Microbiology 50: 882-886.doi carbohydrates and 20% of proteins and glycoproteins with 0099-2240/85/100882-05$02.00/0 multiple disulfide bond making it possible site for oxidative inactivation by ozone. MIC for growth and spore germination Ballardini E (2005) Oxygen-Ozone Therapy for spinal muscle for M. Gypseum and M. canis was 4 µg/ml in the case of ozone disorders in the horse. RivistaItaliana di Ossigeno-

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