Preliminary Laboratory Outbreak Investigation of Acute Sudden Death Syndrome in Mule herds of Udayapur 2063 Dr.Kedar Karki Dr. Purnima Manandhar Dr.Pragya Koirala Objective of Study  Identification of the possible cause of the Syndrome.  To identify the causative agent involved. To correlate similar outbreak To Suggest the possible treatment and prevention Methods.   Material and Methods  Field investigation.  Laboratory investigation of the relevant sample.  Review of clinical and laboratory finding. History/Epidemiology The DLSO Udayapur reported the periodic acute sudden death of total 31 adult mules in a herd of 900, during May – October,2006 History: Symptoms Reported before death Increased respiratory rate, Trembling, Tremor, Excessive , Sweating, Sudden death, Review of World wide Epidemiology of Syndrome  In 1995, 38 horses died to moldy corn poisoning in Kentucky and Virginia (U.S.A). From 1995-2004, 75 horses affected by neurological disorder in Norway due to feeding of moldy silage Purdue Indiana,(U.S.A.)1/6 horse died due to moldy corn poisoning from1997-2004   World wide Epidemiology of syndrome Southern Brazil (spring of 1996)  3 males and 1 female; died (3 to 4 years old thoroughbred horses)  native grass supplemented with 2 kg of corn/horse/day. World wide Epidemiology of syndrome Brazil (1994)  41/58 died  caused by fumonisin concentration lower than 10 ppm. Oaxaca, Mexico (1998)  donkeys died showing a neurological syndrome, World wide Epidemiology of syndrome  Iran (2003), Equine Leukoencephalomalacia  Spain(1988) leukoencephalomalacia (eaten corn with a pinkish fungal growth) The fumonisins were discovered in 1988 following the 1970 outbreak of Equine Leukoencephalomalacia in South Africa.  World wide Epidemiology of syndrome  North Carolina (1989) white-tailed deer Leukoencephalomalacia moldy field corn (Fusarium moniliforme) Preliminary outbreak Investigation On field investigation/ physical examination  mule/horses - normal health,  fed 20-25% cracked and moldy corn. Preliminary Field Investigation Post-mortem examination  Severe congestion of liver, lung, spleen, heart,  Serosangqinus fluid in thoracic cavity.  Hemorrhage in stomach mucosa. Preliminary cause of sudden death was suspected for moldy grain poisoning. Samples for Laboratory from dead/healthy mule  Bacteriological examination Nasal swab, blood (healthy mules) Histopathology examination Liver, lung, spleen, heart tissue Parasitological examination Blood, Fecal samples (healthy mules)   Samples for Laboratory from dead/healthy mule  Toxicological analysis Liver, lung, spleen, heart, intestine (dead) Serological examination Serum (healthy mules)   Mycological culture Feed grains: Maize, Gram Result and Discussion  Bacteriological culture of tissue specimen, swab blood - revealed no growth of any bacteria. Blood parasite - Negative. Chemical toxin in tissue – Negative Intestinal parasite - Mixed Stongyles spp.    Laboratory Investigation: Results  Penicillium, Aspergillus, Candida spp on mycological media (CVL).  60-110 CFU/gm Penicillium colonies recorded in feed samples. (Department of Food Tech and Quality Control Kathmandu) Unacceptably High Fungal Levels Identified in Horse Feeds Common fungus (Aspergillus, Fusarium, and Penicillium) – Equine feed (proposed limit of 104 cfu per gram). The Mycobiota and Toxicity of Equine Feeds mould counts - <1× 102 to 1× 105 cfu/g.  Frequent isolated genus - Aspergillus (40.54%), Penicillium (18.38%) and Fusarium (16.22%).  Feed Mycoflora of the toxic feeds associated with moldy corn poisoning in equine outbreaks in Brazil  The Mycoflora of 39 feed samples associated with 29 EquineLeukoencephalomalacia (ELEM) outbreaks was studied from in Brazil. most frequent mold which occurred Fusarium spp. in 97.4% of samples followed by Penicillium spp. in 61.5% and Aspergillus spp. in 35.9%, (1988 to 1990,) Molds, Mycotoxins and Their Effect on Horses Pasture grasses, hay, grain, straw and stubble can all support the growth of various fungi. Aspergillus, Claviceps, Stachybotrys, Fusarium and Penicillium. Ontario Canada (September 2003 ) Horse Feed Recall Affects East Coast U.S.A.  The products might contain an ingredient with an unacceptably high level of aflatoxin. Aflatoxins are a form of mycotoxins, and are secondary metabolites produced by Aspergillus spp. fungi. by: Erin Ryder, TheHorse.com News Editor May 05 2008, Article # 11810 Histopathological changes Liver • Fatty degeneration of hepatocytes • Mononuclear cells infiltration in the form of few nodules. Lungs • Perivascular cuffing • Infiltration of mononuclear cells in the form of nodules. Histopathological changes Kidney • Deep medullary region reveals infiltration of mononuclear cells. Spleen • The number of white cells seems to be increased in the area of white pulp. Suggestive of chronic/viral disease. (C.V.L.Patholigy Unit) Animal Disease Diagnostic Laboratory Purdue University  Histological description of hepatocytes necrosis and vacuolization with bile duct proliferation is more characteristic of poisoning by the mycotoxins, Aflatoxins, also produced primarily by Aspergillus sp. molds. In many species other than horses, fumonisin does cause liver damage, however in horses, the lesions are primarily in the brain and can often be seen at post-mortem and histologically.. Animal Disease Diagnostic Laboratory Purdue University It is unfortunate that there still is not a postmortem test for fumonisins in tissues and that we still must rely on analysis of feed for confirmation of the presence of fumonisins. Steve Hooser  Stephen B. Hooser, DVM, PhD, DABVT Head, Toxicology Sect. & Asst. Director, Animal Disease Diagnostic Laboratory Purdue University Cardiovascular change with Moldy corn poisoning  Interfere the metabolism of fat -sphingolipids.  Increased serum, sphingolipids, decreased cardiac function preceding neurological signs. Uncontrolled dilatation of blood vessels of brain,swelling,increased itracranial pressure, brain damage  Laboratory Investigation: Results Serum sample Revealed positive for Japanese's Encephalitis (Elisa) Treatment and Control Measure advised  Advised for proper drying of grain before feeding.  Commercial toxin binders, (Varishta, Toxicurb) @ 1kg/tone of grain for 15days along with Liver tonic, (Hepatocare,bioliv), Immunomodulaters, (immunocare), Promin,Vitamin B complex). Final Diagnosis  Moldy Corn Poisoning in Mule  Situation Today- Mortality under control  1200 mules plying on Udayapur Khotang Rout Conclusion  Findings of this investigation indicates that moldy feed grains and ingredients are infested with toxic fungus is emerging as a new health hazard for livestock and poultry. Seasonal prevalence of mycobiota of feed forage need to be assessed. Simultaneous use of toxinbinder,adaptogen like livertonic,mineralmixture and Immunomodulaters drugs promises to help in minimizing health risk in livestock and poultry production should be looked into.  Acknowledgement Dr.Rebatiman Shrestha,  Dr.Sadananda Deo,  Dr.Ganesh Raj Panta,  Dr.Salina Manandhar,  Dr.Vinaya Kumar Karna,  Dr.Keshab P.Shah,  Dr.Shanker Yadav  Mr.Sonfi Shah,  Mr. .B.B.Kunwar,  Mr.Tek B.Air.  Mr.Gyan B.Bogti.  Mr.Samir Gurung Polchem Hygiene lab Pune  Mr.Nishant Bhattarai Varsha Multi tech.Banglore For their contribution in Field and laboratory investigation and control of this disease syndrome 