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SCVMJ, XIII (1) 2008 69 CLINICOEPIDEMIOLOGICAL STUDIES AND EVALUATION OF PCR ON DIAGNOSIS OF LUMPY SKIN DISEASE OUTBREAK, EGYPT 2005 Mahmoud, M. M., Ibrahiem, K. S., Ghaniem, F. M., El-HeeG, M. Infectious Diseases - Animal Med. Dept., Faculty. of Vet. Med, Suez Canal University ABSTRACT In this study the last outbreak of lumpy skin disease (LSD) was investigated from June 2005 to April 2006 over two Egyptian governorates (Ismailia and Damietta). A total number of 954 (103 native and 851 mixed) cattle breed were examined clinically for LSD. Different clinical forms of the disease were reported and ranged from acute, mild and inapparent forms. Total morbidity and mortality rates were 50.5% and 1.1% among examined cattle respectively. Different field samples were collected (Buffy coat 20, skin biopsy 15 and lymph nodes biopsy 5) for virus isolation and polymerase chain reaction technique (PCR) and the isolated virus was identified using virus neutralization test (VNT) and indirect fluorescent antibody technique (IFAT), while 200 sera samples were collected from both diseased and contact healthy cattle for serological investigations. The present study showed that the old age groups, male and native breeds were more resistant to infection. The results of serological examinations revealed that the incidence of infection was 58% and 49% by Serum Neutralization Test (SNT), while by Enzyme Linked Immune Sorbant Assay (ELISA) were 62%, and 55% in Ismailia and Damietta governorates respectively. ELISA test was more sensitive than SNT in early detection of infection as it detect the infection by 7 day while SNT by 10 days after fever. The results of PCR test was more sensitive and rapid for detection of the virus as compared with isolation and identification technique when applied on blood and tissue samples therefore we can use it as rapid means for LSD diagnosis. tible than females, caused by NeethINTRODUCTION ling virus which belongs to family Lumpy skin disease (LSD) is a Poxviridae (Alaa, 2000, Wallace, cattle disease of all ages specially in 2001 and Tuppurainen et al., 2005). summer season, males more suscep- The disease usually associated with 70 severe economic losses like emaciation, hide damage, male infertility, mastitis, loss of milk production, with mortalities (40%) and morbidities (100%) (Barnard et al., 1994). The severity of the clinical manifestations range from acute to inapparent infection, and characterized by eruption of skin nodules, pyrexia, anorexia, dysagalactia and pneumonia. Most cattle were seen with edema of legs and brisket (Khalafalla et al., 1993; Younis and Aboul Soud, 2005). Complications include respiratory manifestation, mastitis, dehydration, abortion and later on recumbence (Abdallah and Gawad, 1992). In May 1988, LSD was clinically recognized in the Suez-Egypt, where it was thought to be arrived at the local quarantine station via cattle that imported from Africa. The disease locally spread in summer, 1988 and apparently over wintered with little or no manifestations of clinical disease. It reappeared in 1989 along a period of five to six months, spread to 22 out of 26 governorates of Egypt, (Ali et al., 1990 and House et al., 1990). In 2005, the disease returned and reappeared in Damietta-Egypt, and affected large number of cattle populations then spread to other provinces. A presumptive diagnosis of the disease can be based on clinical signs, Mahmoud et al., however, mild and inapparent cases may be difficult to be detected. Therefore Rapid laboratory methods are needed to confirm diagnosis, which can be done either by isolation and identification of the virus, or by detection of antibody using serological tests (Tuppurainen et al., 2005). AS virus isolation is very difficult and time consuming we can use a rapid and sensitive test to confirm the diagnosis as PCR (Ireland and Binepal, 1998). Therefore, the aim of the present study was to monitor some epidemiological parameters, clinical signs, serological diagnosis with comparison between PCR and virus isolation as a diagnostic means for LSD in the last outbreak in Egypt. MATERIAL & METHODS 1. Investigated animals: A total number of 954 cattle (native & mixed), aged 6 months to 5 years, were examined from June, 2005 to April, 2006 at Ismailia and Damietta governorates. 2- Samples for virus isolation: 2. A. Blood samples: 20 whole blood samples from clinically affected cases were drained on anti-coagulant (EDTA) in vacationer tubes to separate buffy coat for virus isolation and PCR test. 2. B. Other samples: 20 skin and lymph nodes biopsies from diseased cattle were aseptically collected in sterile clean MacCarteny bottles cont- SCVMJ, XIII (1) 2008 aining maintenance medium with antibiotics, transported on ice to the laboratory and stored at -70ºC until used for virus isolation and PCR test. 3. Serum samples for serological examination: A total 310 serum samples were collected, 200 sera samples from the two Egyptian governorates were collected from both clinically and apparently healthy contact animals postappearance of signs according to Hedger and Hamblin (1983). Another 110 sera sample were daily collected from 10 cattle suffered from acute clinical signs (6th till day16th after fever). All samples were transported on ice to the laboratory and stored at -20ºC until used for serological examination. 4. Virus isolation and identification: the virus was isolated in fertile eggs according to Michael et al., (1991) and in cell lines according to OIE Manual (1992). 5. Polymerase chain reaction (PCR) assay: The extraction of DNA was carried out according to Sambrook et al. (1989). The PCR reactions were carried out in a final volume of 25 µl in PCR tubes, the reaction mixture consists of 1 µl (200 ng) of the extracted DNA template, 5 µl of 10X PCR buffer (75M Tris-HCL, pH 9.0, 2mM MgCL2, 50mM KCL, 20mM (NH4)2SO4), 2 µl dNTPs (10mM) 71 (Biotools), 1µl Taq DNA polymerase (Bio tools) and 0.5 µl (50 pM) from the forward primer (5'TTTCCTGATTTTTACTAT3') and reverse primer (5'AAATTATATACGTAAATAAC3') (Integrated DNA Technologies, Inc. Coralaville) of the gene for viral attachment protein according to (Ireland and Binepal, 1998). The volume of the reaction mixture was completed to 25 µl of DDW. The thermal cycler (Omni-Gene, USA) was adjusted as follows; initial denaturation 94oC for 10 minutes, followed by 40 cycles of 1minute denaturation at 94oC, 30 sec. annealing at 50oC and 1 minute extension at 72oC, followed by final 72°C extension for 1min, then hold at 4°C. Five micro liters of PCR products, negative control, positive control (kindly supplied from microbiology department, Vet Med., Cairo University) and 100bp DNA marker (Promega, Madison, WI, USA) were electrophoresed at a constant current of 40 V for 1hours in 1% agarose gel stained with 0.5ug of Ethidium bromide/ml to determine the size of the product. The gels were visualized under U V illumination (Eagle Eye II, Startagene, Germany) and thereafter photographed using digital camera. The sizes of the amplified product were determined by comparison to DNA marker. 72 RESULTS & DISCUSSION Now LSD is considering as enzootic disease in Egypt whereas several mild outbreaks recorded after 1988 (Radostitis et al., 2007). In the present study, the total number and percentage of examined cases in Ismailia and Damietta governorates during 2005 outbreak were shown in (Table 1). The clinical observations based on fever and numbers of skin nodules classify the disease into acute, mild and inapparent forms. Acute form showed signs of fever (41.5ºC), nasal and ocular discharges. Two days later, the characteristic skin lumps developed and covered the whole body. The cattle suffered from mild form showed a few skin lesions or transient fever while, inapparent form showed complete absence of clinical signs and diagnosis based on serological tests. The observed clinical findings were in similar to results obtained by Alaa, (2000); Hamoda et al., (2002); Younis and Aboul soud, (2005) and Khadr et al., (2006). The field investigation revealed the total morbidity and mortality rates among examined cattle were 50.5 % and 1.1% respectively (Table 2). The finding came in the same line of results recorded by Castro and Heuschele (1996). The highest rate recorded for the morbidity among cattle populations in Ismailia and Damietta governorates indicated that Mahmoud et al., the outbreak was severe. This observation could be related to decrease of immunity or vaccination with inadequate dose, or improper route of vaccination (Younis and Aboul soud, 2005). Table (2) also showed high morbidity in Ismailia than Damietta that could be explained by different breed and climatic conditions between the two governorates or related to the affected farms. The observed high mortalities rate among calves less than six months (Tables 3) may be attributed to insufficient active and passive immunity (Ali et al., 1990). It was confirmed from the obtained result that the age, sex, season and breeds play an important role in epidemiology of the disease (Tables 3, 4 and 5). Old age, male and local breeds were more resistant to infection than others and these results could be referred to a previous exposure of these animal groups via routine vaccination or emergency vaccinations during the outbreak, or natural resistant among native breeds (Younis and Aboul soud, 2005). A tentative diagnosis of the disease is usually based on clinical signs but, animals with few skin lesions or transient fever may be not detected or confused with other diseases (Barnard et al., 1994). The results of serological examinations (Tables 6), revealed that the incidence of infection was 58% SCVMJ, XIII (1) 2008 and 49% in Ismailia and Damietta governorates respectively by SNT, while by ELISA were 62%, and 55% respectively, and the ELISA was more sensitive than SNT and this agree with Hamoda et al., (2002) and Ibrahim et al., (2006). In study of the efficiency of SNT and ELISA in detection of early infection (Table 7), ELISA was more sensitive as it detect the infection by 7- 13 days post fever while SNT started to detect increased antibody by 10 – 15 days and this indicate that the sensitivity of ELISA in detection of early infection.This observation supported the findings of (Tuppurainen et al., 2005) who stated that SNT started to detect increased antibody titers 13 – 18 days post infection after onset of fever but, ELISA demonstrated a rise in antibody titers as early as 8 days post infection after onset of fever. Indirect diagnosis of LSD by virus isolation on embryonated chicken egg (Table 8) the virus was isolated from buffy coat 50% and 40% and by 60% and 50% from tissue samples in Ismailia and Damietta governorates respectively, the failure of virus isolation from buufy coat may be due to the absence of viraemia during collection of samples or low number of virus in the blood, the observations supported byin accordance with Tuppurainen et al., (2005) who stated that length of viraemia varies from 1-12 73 days and not correlate with severity of clinical signs. the highest rate of virus isolation from tissue than buffy coat may be due to long persistence of virus in tissue Tuppurainen et al., (2005) who reported that virus can be isolated up to 39 days post infection. The virus detection by PCR on both blood and tissue samples (Tab. 8 and Fig. 1) revealed positive cases 60% and 60% in Ismailia and Damietta governorates respectively, while in tissue samples positive cases were 80% and 70% respectively The results is in the same line with Ibrahim et al., (2006), the highest percent positive cases in tissue samples than blood samples may be attributed to the long persistence of the virus in tissue than blood (Tuppurainen et al., 2005). In comparison between virus isolation and PCR as shown in (Table 8) there was high positive cases in PCR than isolation and this can be explained due to the sensitivity of PCR as the ability of the PCR to detect small numbers and / or inactive virus. It is recommended that all animals should be vaccinated intradermal with recommended dose of vaccine and the new cattle comers to the herd should be examined using the PCR assay as adequate control measures of the disease and we can advice to use ELISA and PCR for the rapid diagnosis. 74 Mahmoud et al., Fig (1): Results of PCR product for detection of Lumby Skin Disease virus M: 100 bp marker, 1,2: Control positive and negative, 3-7: test samples. SCVMJ, XIII (1) 2008 75 Table (1): Number and percentage of examined animals. Locality Ismailia Sex Age /month No of cattle Native Mixed male female >6 6-30 <30 Sum. Wint. Aug. 2005 Sep. 2005 Oct. 2005 Dec. 2005 Jua. 2006 80 - 80 32 48 - 30 50 80 - 120 20 100 35 85 3 67 50 120 - 75 15 60 22 53 4 32 39 - 75 50 15 35 10 40 1 15 34 - 50 25 10 15 7 18 - 20 5 - 25 350 60 290 106 244 8 164 178 200 150 85 5 80 20 65 4 50 31 85 - 200 - 200 69 131 10 80 110 200 - 110 18 92 37 73 9 60 31 110 - 100 - 100 33 67 6 52 52 - 100 65 20 45 20 45 - 35 30 - 65 44 - 44 19 25 - 22 22 - 44 604 43 561 198 406 29 299 276 395 209 954 103 851 304 650 37 463 354 595 359 Total Damietta Species Field trip Jul. 2005 Aug. 2005 Sep. 2005 Oct. 2005 Mar 2006 Apr. 2006 Total Total per two provinces Table (2): Morbidity and Mortality rates among examined cattle. Locality No of examined cattle Ismailia Damietta Total 350 604 954 Morbidity rate No % 205 58.6 277 49.9 482 50.5 Mortality rate No % 3 0.9 7 1.2 10 1.1 76 Mahmoud et al., Table (3): LSD forms in relation to age groups. locality Age/month Ismailia Damietta >6 Ismailia Damietta 6-30 Ismailia Damietta <30 Acute Mild Inapparent deaths No % No % No % No % 6 75 2 25 - - 2 25 18 62.1 8 27.6 10.3 5 17.2 40 24.4 60 36.6 64 39 1 0.6 43 14.4 55 18.4 201 67.2 1 0.3 34 19.1 63 35.4 81 45.5 - - 42 15.2 111 40.2 123 44.6 1 0.4 3 Table (4): LSD' forms in relation to sex groups. LSD' forms sex Female Male Locality/NO Acute Mild Inapparent No % No No % Ismailia (244) 54 22.1 90 36.9 % 100 41 Damietta(406) 66 16.3 108 26.6 232 57.1 Ismailia (106) 26 24.5 35 33 45 42.5 Damietta(198) 37 18.7 66 33.3 95 48 Table (5): LSD' forms in relation to breed groups. LSD' forms Breed Locality/NO Acute No Mix Native Mild % No % Inapparent No % 113 39 Ismailia (290) 72 24.8 105 36.2 Damietta (561) 98 17.5 161 28.7 302 53.8 Ismailia (60) 8 13.3 20 33.3 32 53.4 Damietta (43) 5 11.6 13 30.2 25 58.1 SCVMJ, XIII (1) 2008 77 Table (6): Result of antibodies against LSD in collected sera samples using ELISA and SNT. Positive sera Total sera Locality ELISA No. SNT % No. % Ismailia 100 62 62 58 58 Damietta 100 55 55 49 49 Table (7): comparison between of means days. Days after onset of fever Test No of ELISA mean 7 8 9 10 11 12 13 14 15 1 3 2 1 1 1 1 - - 9.3 - - - 1 1 3 1 3 1 12.7 positive animals SNT (10) Table (8): Comparison between the results of virus isolation and PCR. isolation PCR Locality samples Ismailia Blood (10) 5 50 5 50 6 60 4 40 Tissue (10) 6 60 4 40 8 80 2 20 Blood (10) 4 40 6 60 6 60 4 40 Tissue (10) 5 50 5 50 7 70 3 30 Damietta + ve % - ve % + ve % - ve % 78 REFERENCES Abdalla, M.A. and Gawad S.M. (1992): Characterization of serum lysosomal enzymatic activities II. Dutch Tierarztl Wochenschr. Aug; 99(8): 347-9. Alaa, (2000): Some studies on LSD in cows and buffaloes. PhD, thesis (infectious diseases), Zagazig University. Ali, A. A.; Esmat, M.; Attia, H.; Selim, A. and Abdel-Hamid, Y. M. (1990): Clinical and pathological studies on lumpy skin disease in Egypt. Vet Rec. 1990 Dec 1; 127(22): 549-550. Barnard, B. J. H.; Munz, E.; Dumbell, K. and Prozesky, L. (1994): LSD in Coetzer JAW, Thomson GR, Tustin RC, Kriek NPJ. Infectious diseases of livestock with special references to southern African cape town Oxford University Press p: 604-612. Castro, A. E. and Heuschele, W. P. (1996): Veterinary diagnostic virology (LSD). A practitioner guide. Mosby Year Book, Inc. pp. 106-108. Hamoda, F. K.; Aboul Soud, E. A.; Magda, M. S.; Shahein, M. A.; Michael, A. and Daoud, A. M. (2002): Field and laboratory studies on lumpy skin disease. j. Egypt. Vet. Med. Assoc. 62(5): 183-199. Hedger, R. S. and Hamblin, C. (1983): Neutralizing antibodies to lumpy skin disease virus in African wildlife.Comp Mahmoud et al., Immunol Microbiol Infect Dis., 6(3): 209-13. House, J.A.; Wilson, T.M.; El Nakashly, S.; Karim, I.A.; Ismail, I.; El Danaf, N.; Moussa, A.M. and Ayoub, N.N. (1990): The isolation of lumpy skin disease virus and bovine herpesvirus-4 from cattle in Egypt. J Vet Dig. Invest.; 2(2): 111-115. Ibrahiem, A. K; Ahmed, S. A.; Darderi, M. A. and Amin, A. S. (2006): Comparative studies on conventional and molecular assays used for diagnosis of Lumpy skin disease. Egypt. J. Comp. Path. & clinic. Path, 19(3): 227-239. Ireland, D. C. and Binepal, Y. S. (1998): Improved detection of capripox virus in biopsy samples by PCR. J. Virol. Meth., 74: 1-7. Khalafalla, A. I.; Gaffar Elamin, M. A. and Abbas, Z. (1993): Lumpy skin disease: observations on the recent outbreaks of the disease in the Sudan. Rev Elev Med Vet Pays Trop.; 46(4): 548-50. Khadr, A. M.; E-Fayoumy, M. M.; Abou-Elsoud, E.; El-Ballal, S. and Zaghawa, A. (2006): An outbreak of lumpy skin disease in Egyptian farm during 2005-2006. Clinical, epidemiological, laboratory and electron microscopic investigations. Menofia Vet. J, 4(1): 1-13. SCVMJ, XIII (1) 2008 79 Michael, A; Salama, s. A.; Soliman, S. M.; Mousa, A. A.; Bachoum, M. E.; Barsoum, G. W.; Osman, A. O.and Nassar, M. I. (1991): The determination of the immune state of cattle to LSD in Egypt. J. Egypt Vet. Med. Assoc., 51(2): 427-434. Office International Des Epizooties (OIE) (1992): Manual of recommended diagnostic techniques and requirements for biological products for lists A and B diseases. Vol.1:1-5. OIE 12, reu du Prony, 75017 Pairs- france. Radostits, O. M.; Gay, C. C.; Blood, D. C. and Hinchxliff, K. W. (2007): Veterinary medicine A textbook of the disease of cattle, sheep, pigs, goats and horse., 10th Ed. W. B. Saunders Company Ltd. Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989): Molecular cloning: A laboratory manual 2nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Tuppurainen, E. S.; Venter, E. H. and Coetzer, J. A. (2005): The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques. Onderstepoort J. Vet. Res.; 72(2): 153-64. Wallace, H. P. D. (2001): LSD in southern Africa, a review of the disease and aspects of control. J. Afr. Vet. Assoc., 72: 68-71. Younis, E and Aboul Soud, E. (2005): Some studies on outbreak of LSD, Egypt 2005. Mansoura Vet. Med. J., VII(2): 37-59. الملخص العربي دراسات إكلينيكية وبائية وتقييم اختبار تفاعل البلمرة المتسلسل في تشخيص وباء التهاب الجلد العقدي مـ5002 مصر عام مي في محافظيات5002 أجريت الدراسة على الوباء األخير لمرض التهاب الجلد العقدي في مرير عيا ( 429 عييدد وقييد ت ي فح ي5002 إل ي رييهر أبري ي5002 ( اإلسييماعيلية ودميييا ) مييش رييهر يوني ي في%303 ونسيبة الوفييات%2002 خليي ) ميش األبقيار وقيد اانيت نسيبة اإلريابة123 محل و301 وقييد ت ي تسييجي عييدم رييور للمييرض منهييا الرييورم الحييادم والرييورم ال ي يية.األبقييار الت ي ت ي فحرييها عينية32 مسيحة افيية ميش ايرات اليد البي ياء و50 ( والرورم الخ ية وقد ت تجميع عينيات مختل ية وقيد تي عينات ميش الديدد الليم اويية) لعيل ال ييروس وإجيراء اختبيار ت اعي البلميرم المتسلسي2جلد و علي ي عييش ريييد اختبييار تعيياد ال يييروس واختبييار المري ال لورسييينتى ييير عييل ال يييروس والتعيير 80 Mahmoud et al., المباريير و قييد ت ي تجميييع 500عينيية مييش مر ي الييد للحيوانييات المرييابة والسييليمة المخال يية لل ح ي السيييرولوج .وقييد أرييارت النتييا للمرض وقد أظهرت نتيا ال حي إل ي أش األعمييار الابيييرم والييااور والسييليت المحلييية أاثيير مقاوميية السييرولوج أش نسيبة اإلريابة ي 21و %94باسيتخدا اختبيار التعاد المرل 25و % 22باستخدا اختبار اإلليلا ف محيافظت اإلسيماعيلية ودمييا عليى التيوال و االك إل أش اختبار اإلليلا أاثر حساسية مش اختبار التعاد المرل للار أش اختبار اإلليلا قد أظهر نتا إيجابية للمرض ف اليو السابع بينميا اختبيار التعياد المريل في الييو العارر بعد ظهور الحمى وقد اانت نتا بييال رد التقليدييية لعييل وترييني سريعة لترخي المرض. المباير عيش الميرض حيي اختبار ت اع البلميرم المتسلسي أاثير سيرعة وحساسيية مقارنية ال يييروس وبالتييال نسييت يع إجييراء اختبييار البلمييرم المتسلس ي اوسيييلة