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Clinical and Molecular Study of Bovine Ulcerative Mammillitis in Cattle in AL- Qadisiyah Province

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Clinical and Molecular Study of Bovine Ulcerative Mammillitis in Cattle in AL- Qadisiyah Province IV Republic of Iraq Ministry of Higher Education and Scientific Research University of AL-Qadisiyah College of Veterinary Medicine Clinical and Molecular Study of Bovine Ulcerative Mammillitis in Cattle in AL- Qadisiyah Province A Litter submitted to the Council of the College of Veterinary Medicine / University of AL-Qadisiyah in Partial Fulfillment of the Requirements for the Degree of Bachelors in Science of General Veterinary Medicine and surgery. By Assmaa Juad Khadem Supervised by Instructor. Dr. Yahia Ismail Khudhair University of AL-Qadisiyah / Veterinary Medicine College 2016 A.D. 1437 A.H. V سورة طه VI Abstract Bovine ulcerative mammillitis (BUM) is endemic disease of cattle in many countries, including Iraq. It is causes by alpha-herpersvirus that induces an infection of skin of cattle, which develop in several possible pathological forms: server acute ulcerative form, superficial dermatitis with alopecia and crusts, and pseudolumpy skin disease. It is important to be studied because of its transmissible nature and has economic impact on the dairy industries. The current study focused on two parts, the first part was to determine epidemiological and clinical elements, and the second part concerned on the molecular diagnosis and phylogenetic study in AL-Qadisiyah province of Iraq, but the narrow period of study and delayed of sequence results prevent included some results. In the first part of study, clinical examination and observations to determine the BUM in 200 adult lactating cattle (100 crossbred and 100 local breed) and 50 calves from Al-Qadisiyah province, clinically, the results showed that 18 (7.2%) animals out of 250 examined cattle were having teat, udder and muzzle lesions. Thirteen (6.5%) of dairy cows were infected, all infected cattle were at lactation period. Five (10%) calves aged from 1 week to 3 months were have muzzle and mouth lesions the lesions teat and udder in dairy cattle and muzzles of calves. Lesions superficial dermatitis caused by bovine herpesvirus 2 (BoHV-2) in dairy breed calves. Clinically, all of the affected cattle were 2-6 years of age, had ulcerations and crusts on the clinical signs that have been observed included sever from acute clinical lesion, characterized by enlargement and increased in size of one or more teats, usually the infection effected four teats and extended the lesion to skin of udder. in general the severity of lesions ranged from several ulcers covered with tightly adherent of yellow to orange color scabs to sever acute lesions involved whole the teats with irregular shape of ulcers, scabs and cracks of teats. VII Polymerase chain reaction results detected BoHV-2 gene in all clinical samples (7.75%). All 18 of the seropositive samples were PCR positive, molecular detection was targeting gb enes which gave band corresponded to 607bp segment. In this survey, depending on the molecular results, some epidemiological elements as age and breeds to BoHV-2infected were also investigated. To analyze the risk related with cattle breed associated with the BoHV-2 infection, two breeds (crossbred and local breed) of Iraqi cattle were infected. Both breeds were infected at percentage 8 (8%) and 5(5%) for local breed and crossbred respectively. Statically the results revealed that there were no significant differences them for their susceptibility to BoHV-2infection. The results of this study showed that the number of infected animals decrease with age The infection rate among the cattle of 2-4 years of age group was 9/110 (2.8%), whereas of cattle over than 5 years old 4/90(4.4%). Also results revealed that there were significant differences between ages of cattle for their susceptibility to BoNV-2. In conclusion, these isolated virus in these Iraqi isolates are members of the BoHV- 2 family and indicated by clincal, molecular, These Iraqi BoHV-2 isolates represents an invaluable tool as a source for future studies and provide a reproducible cell culture system for studying the herpesviruses. VIII List of Contents NO. Subjects Page Abstract I List of Contents V List of Figures X List of Tables 8 List of Abbreviations XVII 1 Chapter One:- Introduction 1-1 Introduction 1 1-2 Aims of study 3 2 Chapter Two Literatures Review 2 Literatures Review 4 2-1 History of Bovine ulcerative mammillitis (BUM) 4 2-2 Bovine ulcerative mammillitis 5 2-3 Etiology 5 2-4 Epidemiology 6 2-4-1 Transmission 6 2-4-1-1 Horizontal Transmission 7 2-4-2 Occurrence of BoHV-2 8 2-4-4 Risk Factors 11 2-4-5 Economic Impacts 11 2-5 Clinical Signs 14 2-6 Diagnosis 16 2-6-1 Serological assays 17 2-6-1-1 Enzyme linked immunosorbent assay (ELISA) 17 2-6-1-2 Agar gel immunodiffusion test (AGID) 18 2-6-3 Molecular Assays (Polymerase chain reaction: PCR): 19 2-6-4 Isolation 20 2-7 Treatment and Control: 21 2-7-1 Treatment: 21 2-7-2 Control 21 2-7-2-1 Eradication strategies: 22 2-7-2-2 Vaccination 23 2-7-2-3 Management practice 24 3 Chapter Three Materials and methods 3 Materials and methods 25 3-1 Materials 25 3-1-1 The equipment's that were used in the study. 25 3-1-2 The chemical used in epidemiological study. 25 3-1-3 The solutions that used in used in study. 26 IX 3-1-4 I. The Kits used in PCR 26 3-1-5 Primers: 27 3-1-6 Animals: 27 3-2 Methods: 28 3-2-1 Animals Examination 28 3-2-2 Tissue Samples Collection 29 3-2-3 Detection of BoHV-2 by Molecular Techniques, (PCR) 29 3-2-3-1 Preparation of Tris-Borate –EDTA (TBE) buffer: 29 3-2-3-2 Tris EDTA (TE) buffer 29 3-2-3-3 Total PBMCs Genomic DNA Extraction 29 3-2-3-4 Assessing DNA yield and purity: 31 3-2-3-5 Polymerase Chain Reaction (PCR) protocols 32 3-2-3-6 Gel electrophoresis preparation and sample loading: 33 4 Chapter Four Results 4 Results 34 4-1 Results of clinical study 34 4-2 Molecular Results 36 4-3 Breed, Age and Sex relationship 37 5 Chapter Five Discussion 5 Discussion 39 6 Chapter Six Conclusions and Recommendations 6-1 Conclusions 44 6-2 Recommendations 45 7 References 7 References 46 X List of Figures NO. Figure Page BoHV-2 infected cow 0.5-1 cm diameter teat and udder lesions, adherent 4-1 scabs 35 4-2 teat ulceration in BoHV-2 infected cows 35 sever BoHV-2 infected cow appeared enlargement of teat size (swelling 4-3 teats) 35 4-4 BOHV-2infected crossbred cow sever infection and cracks of teats 35 4-5 calve appeared muzzle lesion of BoHV-2infected. 35 BoHV-2infected calve appeared small vesicles and pustules with escapes 4-6 lesions of muzzle 36 Loaded of 15ul of amplification resulted in the predicted 609- 4-7 bp amplification product of the glycoprotein B (gB) gene of 37 BoHV-2 through1% agarose stained with ethidium bromide List of Tables NO. Table Page 3-1 The equipment that were used in the study 25 3-2 The chemicals used in study 25 3-3 the solutions that used epidemiological study 26 3-6 The kits used in the in PCR. 26 The primers sits with sequence and flanked portion of genome 3-7 27 which were give positive results used in conventional PCR. The animals of study depending on location and their breed, age 3-8 28 and sex. 3-10 The thermocyclar conditions in Conventional PCR system 33 XI 1-1- Introduction: Bovine ulcerative mammillitis (BUM) is a chronic transmissible viral disease of cattle (Lairmore, 2014). It is characterize by a skin and skin associated strictures disorder (EFSA, 2015). The disease is associated with infection by Bovine ulcerative mammalitisvirus (BoHV-2), which also recognized as a long-latent infection virus in cattle (Kettmann, et al., 1994 and Miura, et al. 2015). Bovine ulcerative virus is characterized by a very long latent period, most infected cattle appeared asymptomatic carriers (Tsutsui, et al., 2016). Most of BoHV-2 infected cattle develop persistent infection. The disease is transmitted by horizontal way, horizontal occurs by direct contact, and inappropriate management practices (Kobayashi, et al., 2010, Ooshiro et al. 2013, Lairmore, 2014 and Kobayashi, et al., 2015). Bovine ulcerative mammillitis virus is an economically important viral infection of cattle (Erskine, 2012). The estimated costs of BoHV-2 infection in United States were 525 million dollars annually, due to lost productivity and premature death, while the average reduction in annual value of production was $ 59 per cow for BOHV-2-positive herds (Ott et al. 2003). In mid- Atlantic dairy herds, (Rhodes, et al., 2003) referred to that 412 dollars the cost of case of LP, while mean annual cost of subclinical infection at a 50% prevalence of infection was 6,406 dollars per 100 milking cows. Other causes were reported that associated with the decreased milk production, the National Animal Health Monitoring System (NAHMS) study determined that herds with BoHV-2 produced 3% less milk, than non-BoHV-2 herds (Ott et al. 2003). Decreased reproductive efficiencies, But a major indirect cost associated with infection is the restriction of the sale of animals (Pelzer, et al., 1997). The cost of control and XII eradication varied with herd size, the mean annual cost of a test-and-manage control program was 1,765 dollars with a 50% prevalence of BoHV-2 infection (Rhodes et al. 2003). In Canada, a total annual treatment costs of EBL infected 50 cows was $ 806 dollars (Chi, et al., 2002).
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