Galvijų spongiforminės encefalopatijos ir virusinių ligų paplitimo, diagnostikos ir prevencijos retrospektyvi analizė Lietuvoje ; Retrospective analysis of bovine spongiform encephalopathy and prevalence, diagnostics and prevention of viral diseases in cattle in Lithuania
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The doctoral thesis was prepared at the Lithuanian National Veterinary La-LITHUANIAN VETERINARY ACADEMY boratory, Institute of Immunology of Vilnius University and Veterinary Institute of the Lithuanian Veterinary Academy in the period 2001–2005. The thesis is to be defended externally. Scientific supervisor – Prof. Dr. Habil. Vytas Tamoši ūnas (Institute of Immunology of Vilnius University, biomedical sciences, veterinary medicine – 12B). Veterinary Medicine Dissertation Defence Board: Chairman – Acting Prof. Dr. Habil. Saulius Petkevi čius (Lithuanian Veterinary Acade- my, biomedical sciences, veterinary medicine – 12B). Members: Acting Prof. Dr. Habil. Vytautas Špakauskas (Veterinary Institute of the Jonas Milius Lithuanian Veterinary Academy, biomedical sciences, veterinary medicine – 12B); Prof. Dr. Bronius Bakutis (Lithuanian Veterinary Academy, biomedical sciences, veterinary medicine – 12B); Acting Prof. Dr. Vida Juozaitien ė (Lithuanian Veterinary Academy, biome-RETROSPECTIVE ANALYSIS OF BOVINE dical sciences, zootechnics – 13B); SPONGIFORM ENCEPHALOPATHY AND PREVALENCE, DIAGNOSTICS AND PREVENTION Dr. Vilimas Sereika (Veterinary Institute of the Lithuanian Veterinary Aca-demy, biomedical sciences, veterinary medicine – 12B). OF VIRAL DISEASES IN CATTLE IN LITHUANIA Opponents: Acting Assoc. Prof. Dr.
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01 janvier 2006
LITHUANIAN VETERINARY ACADEMY
Jonas Milius RETROSPECTIVE ANALYSIS OF BOVINE SPONGIFORM ENCEPHALOPATHY AND PREVALENCE, DIAGNOSTICS AND PREVENTION OF VIRAL DISEASES IN CATTLE IN LITHUANIA Summary of doctoral thesis Biomedical sciences, veterinary medicine (12B) Kaunas, 2006
The doctoral thesis was prepared at the Lithuanian National Veterinary La-boratory, Institute of Immunology of Vilnius University and Veterinary Institute of the Lithuanian Veterinary Academy in the period 20012005. The thesis is to be defended externally. Scientific supervisor Prof. Dr. Habil. Vytas Tamoiūnas (Institute of Immunology of Vilnius University, biomedical sciences, veterinary medicine 12B). Veterinary Medicine Dissertation Defence Board: Chairman Acting Prof. Dr. Habil. Saulius Petkevičius (Lithuanian Veterinary Acade-my, biomedical sciences, veterinary medicine 12B). Members: Acting Prof. Dr. Habil. Vytautas pakauskas (Veterinary Institute of the Lithuanian Veterinary Academy, biomedical sciences, veterinary medicine 12B); Prof. Dr. Bronius Bakutis (Lithuanian Veterinary Academy, biomedical sciences, veterinary medicine 12B); Acting Prof. Dr. Vida Juozaitienė(Lithuanian Veterinary Academy, biome-dical sciences, zootechnics 13B); Dr. Vilimas Sereika (Veterinary Institute of the Lithuanian Veterinary Aca-demy, biomedical sciences, veterinary medicine 12B). Opponents: Acting Assoc. Prof. Dr. Marija Stankevičienė Veterinary Aca- (Lithuanian demy, biomedical sciences, veterinary medicine 12B); Prof. Dr. Habil. Aniolas Sruoga (Institute of Ecology of Vilnius University, biomedical sciences, biology 01B). The dissertation is due to be defended at the session of Veterinary Medicine Dissertation Defence Board on December 28 2006 at 1 p.m. in room No 1 of the Lithuanian Veterinary Academy. Address: Tilės 18, LT-47181 Kaunas, Lithuania. The dissertation report was circulated on November 28 2006 to the confir-med listed addresses. The dissertation is available at the libraries of Lithuanian Veterinary Aca-demy and Veterinary Institute of Lithuanian Veterinary Academy.
LIETUVOS VETERINARIJOS AKADEMIJA Jonas Milius GALVIJŲSPONGIFORMINĖS ENCEFALOPATIJOS IR VIRUSINIŲLIGŲPAPLITIMO, DIAGNOSTIKOS IR PREVENCIJOS RETROSPEKTYVI ANALIZĖLIETUVOJE Daktaro disertacijos santrauka Biomedicinos mokslai, veterinarinėmedicina (12B) Kaunas, 2006
Disertacija rengta 20012005 metais Nacionalinėje veterinarijos laboratori-joje, Vilniaus universiteto Imunologijos institute ir Lietuvos veterinarijos akademijoje ir LVA Veterinarijos institute. Disertacija ginama eksternu. Mokslinis konsultantas prof. habil. dr. Vytas Tamoiūnas (Vilniaus universiteto Imunologijos insti-tutas, biomedicinos mokslai, veterinarinėmedicina 12B). Veterinarinės medicinos mokslo krypties disertacijos gynimo taryba Pirmininkas e. prof. p. habil. dr. Saulius Petkevičius (Lietuvos veterinari-jos akademija, biomedicinos mokslai, veterinarinėmedicina 12B). Nariai: E. prof. p. habil. dr. Vytautas pakauskas (Lietuvos veterinarijos akademijos Veterinarijos institutas, biomedicinos mokslai, veterinarinė medicina 12B); Prof. dr. Bronius Bakutis (Lietuvos veterinarijos akademija, biomedicinos mokslai, veterinarinėmedicina 12B); E. prof. p. dr. Vida Juozaitienė(Lietuvos veterinarijos akademija, biomedi-cinos mokslai, zootechnika 13B); Dr. Vilimas Sereika (Lietuvos veterinarijos akademijos Veterinarijos institu-tas, biomedicinos mokslai, veterinarinėmedicina 12B). Oponentai: E. doc. p. dr. Marija Stankevičienė veterinarijos akademija, bio- (Lietuvos medicinos mokslai, veterinarinėmedicina 12B); Prof. habil. dr. Aniolas Sruoga (Vilniaus universiteto Ekologijos institutas, biomedicinos mokslai, biologija 01B). Disertacija bus ginama Veterinarinės medicinos mokslo krypties tarybos posėdyje 2006 m. gruodio 28 d. 13 val. Lietuvos veterinarijos akademijos I auditorijoje. Adresas: Tilės g. 18, LT-47181 Kaunas, Lietuva. Disertacijos santrauka isiuntinėta 2006 m. lapkričio mėn. 27 d. pagal pat-virtintąadresųsąraą. Disertacijągalima periūrėti Lietuvos veterinarijos akademijos ir LVA Ve-terinarijos instituto bibliotekose.
INTRODUCTION Relevance of the present research Bovine viral diseases are widespread throughout the world and inflict huge economical losses. Contagious bovine diseases also periodically occur in Europe. Assessment of the health state of animals and precise laboratory diagnostics are indispensable for prevention and control of these diseases (Biokas ir kt., 2002). After accession to the EU, where animal trade migra-tion is free, it became necessary to control the health state of imported and exported animals and in this way to avoid their infection with viral diseases and to pre-empt the spread of mentioned diseases in Lithuania and in other European countries. After Lithuanias becoming member of various international organiza-tions, development of import and export of stock-breeding products and changing of farming conditions, the attitude towards bovine viral diseases (especially the ones controlled by the World Organization for Animal He-alth OIE and regulated by the veterinary legislation of the EU) also changed. Early diagnosis of viral diseases help to avoid huge economic los-ses incurred by reduced production of milk and meat, loss of animals, the get and core of pedigree herds. Great losses also are incurred by export limi-tations of cattle and their products and expenditures on compensations for the dead animals or on eradication of viral diseases (Oszvary, 2004). Diagnostics and control of bovine viral diseases and liquidation meas-ures are regulated and implemented in Lithuania by the State Food and Vet-erinary Service (SFVS). Owners of animals and state and private veterinary surgeons (who follow the veterinary laws of the Lithuania and SFVS orders) also participate in the process of disease control and eradication. The Lithu-anian veterinary laboratories are in charge of timely laboratory investiga-tions of these diseases and the SFVS is responsible for implementation of sanitary measures in farms, control of herd import and export and develop-ment and implementation of the programs for eradication of diseases and vaccination (Kliučinskas et al., 2004). In many countries of the world a great attention is paid to investigations of infectious diseases in cattle. The research of viral diseases in cattle em-braces the findings about the causes of disease, its agents and their propaga-tion ways and sources and evaluation of animal immunity. Laboratory diag-nostic examinations create preconditions for assessment of actual spread of viral diseases in the country, expenditures on veterinary measures and re-lated economic losses. Prevention measures against viral diseases in cattle not always are effective. Some viral diseases can spread among cattle and wildlife populations,
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e.g. rabies occur in domestic and wild animals as well as viral diarrhoea, whose virus may infect cattle and wildlife roe, mouflons and fallow-deer (Frolich et al., 2002; Kalman and Egyed, 2005). Often infectious diseases in cattle show manifest through untypical symptoms or show no symptoms at all. This depends on the resistance capacity of animal organism, keeping conditions, tolerance of agents towards environmental conditions, their viru-lence, persistence and other conditions. Therefore, it is expedient to know what virus strains are in circulation, how they transform and what the pre-ventive implications of their transformation are. Permanent epizootic studies of bovine viral diseases in the Lithuanian stock-breeding farms and investi-gation of the biological properties of their agents are necessary. Methods enabling examination of virus structure should be used. This is especially relevant in investigations of the spread of bovine viral diseases in Lithuania and in other countries. Development and improvement of the programmes for observing herds and disease control should be based on comparison of research data and assessment of specific features of virus circulation in herds. A database of serological investigations of cattle and application of the most up-to-date research methods are of paramount importance in evalu-ating the efficiency of vaccination. Epidemiological investigations of some infectious diseases in cattle, e.g. bovine viral diarrhoea (Mockeliūnienėet al., 2004) and preliminary investi-gations of the occurrence of viruses of bovine infectious rhinotracheitis and some viruses causing respiratory diseases in the cattle get have been per-formed in Lithuania (alomskas et al., 1998). Though investigations of oc-currence of enzootic leucosis in cattle were started more than thirty years ago (Glamba, 1999), there appeared no recent publications of comprehen-sive analysis of cattle leukaemia viruses. So far, laboratory diagnostics of another potentially very dangerous for human health disease, spongyformal encephalopathy, have not been undertaken in Lithuania either. It can be also pointed out that there have been no generalizing diagnostic, occurrence and prevention efficiency investigations in Lithuania related with cattle disease viruses included in the list of diseases controlled by OIE. Aim of research The aim of the present research is to carry out investigations of occur-rence and diagnostics of economically relevant cattle diseases caused by viruses and prions, to evaluate preventive measures and to analyse financial expenditures. Tasks 1. To investigate the occurrence and epidemiological features of bovine viral diarrhoea, infectious bovine rhinotracheitis, rabies, enzootic bovine
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leukosis, and spongiform encephalopathy in cattle. 2. To evaluate the investigation methods applied in laboratory diagnos-tics and control measures of bovine viral diseases. 3. To carry out financial analysis of expenditures on diagnostics and con-trol of diseases caused by viruses and prions. Scientific novelty Assessment of occurrence and diagnostic methods of viral diseases in cattle viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), ra-bies, enzootic bovine leukosis (EBL), and spongiform encephalopathy (BSE) was carried out for the first time in Lithuania. It was established that viruses of rabies, infectious rhinotraheitis and viral diarrhoea are most widespread in the country. It was determined that occurrence of rabies in cattle is parallel with the infection of wildlife with rabies virus. Analysis of eradication programme of enzootic bovine leucosis was done. It revealed that only combined application of diagnostic and preventive measures allo-wed reducing the cattle infection up to 0.2%. Though bovine spongiform encephalopathy has not been recorded in Lithuania, it is feasible to imple-ment its diagnostic and prevention programme. An overall financial analysis of expenditures on BSE and viral diseases diagnostics and control was for the first time done in Lithuania. It showed that BSE and EBL occupied the leading positions in the structure of expen-ditures on viral diseases. In 2001, expenditures on BSE investigations ac-counted for 76.68% and in 2004 for 86.74% of the total. Expenditures on EBL investigations relatively reduced from 86.98% in 2000 to 8.47% in 2004. During the time under consideration, expenditures on investigations of other viral diseases changed but little. It was determined that consistent and wide-scale preventive vaccination created preconditions for diagnostic investigations of rabies; preventive vaccination of cattle against BR is applied to 30 thou animals every year. A few hundred are examined on suspicion of infection. The annual expenditu-res related with IBR and BVD amount up to 45 thou yet in the context of economic unit these expenditures are comparatively low from 8.7 to 13.5 Lt. Practical value An overall analysis of sample examination results was performed. For assessment of epidemiological situation of bovine viral diseases, 12 modern research methods, introduced in the National Veterinary Laboratory (NVL) and veterinary laboratories of counties, were used. Analysis of occurrence of viral diseases in separate cattle groups (cows,
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heifers and bulls) revealed that most severe infections occur in cow herds predetermined by high concentration of animals, their older (compared to heifers) age, insufficient control of movement from farm to farm, and no-nobservance of control measures. It was also determined that measures now in use for prevention of the spread of IBR and BVD are insufficient and it is necessary to develop national long-term programmes for eradication of these diseases. For prevention of rabies it is necessary to implement planned vac-cination over the country. Analysis of veterinary expenditures showed that the state plays an ever increasing role in the field of cattle health prophylaxis and care even in the private sector. The state covers an increasing part of expenditures of stock-breeders related to viral diseases (in 2004, more than 90% of expenditures were covered by the state budget). Approbation of the doctoral dissertation The dissertation results were published in 5 scientific articles and repor-ted in scientific presentations at the National Veterinary Laboratory (2004), State Food and Veterinary Service (SFVS) (2005), Ministry of Agriculture and Parliament of the Republic of Georgia (2005), and scientific conference at the Lithuanian Veterinary Academy (2005). Extent and structure The doctoral dissertation is composed of Introduction, Review of Litera-ture, Materials and Methods, Presentation and Discussion of obtained re-sults, Conclusions, List of Publications and List of References (212 entries). The work contains 29 Tables and 28 Figures. MATERIAL AND METHODS The investigations were carried out in conformity with the legal acts: Law of animal care, keeping and use No 8-500 of the Republic of Lithuania adopted in 06 11 1997 (Valstybė1997 11 28, Nr. 108) and subsi-s inios, diary documentation: orders of the National Veterinary Laboratory of the Republic of Lithuania On the veterinary requirements for breeding, repro-duction, care and transportation of laboratory animals (1998 12 31, Nr. 4-361) and On the use of laboratory animals for scientific experiments (1999 01 18, Nr. 4-16). Examined groups of animals. The blood for analysis of BVD virus ant-ibodies and antigen was taken from 19 566 cows of variable age, heifers and bulls kept in the Lithuanian industrial and breeding farms. Analysis was performed at the National Veterinary Laboratory in the period 19972004.
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In order to evaluate the comparative efficiency of PCR and antigen ELISA, parallel investigations using both methods were carried out. For this purpo-se, 107 samples of blood, organs and virus isolates were examined at the National Veterinary Laboratory and Veterinary Institute of Lithuanian Ve-terinary Academy. For investigation of rabies samples of brain matter of domestic and wild animals were collected in different Lithuanian regions irrespective of animal age, species or sex. The total of 11 267 samples were collected. Diagnostic analysis was performed at the National Veterinary Laboratory and 9 county veterinary laboratories (Alytus, Kaunas, Marijampolė, Teliai, Klaipėda, Tauragė, Utena, Panevėys, and iauliai). For BSE analysis, brain matter of animals was collected in various Lit-huanian regions. The samples were taken taking into account the age risk groups. The total of 9 667 samples were collected. Diagnostic analysis was performed at the National Veterinary Laboratory in 19952004. For investigation of EBL antibodies, the blood was taken from 7 389 641 cows and bulls of variable age. Diagnostic analysis was performed at the National Veterinary Laboratory (NVL) and 9 county veterinary laboratories (Alytus, Kaunas, Marijampolė, Teliai, Klaipėda, Tauragė, Utena, Panevė-ys, and iauliai) in 19952004. Collection and preparation of samples for laboratory examination. The blood(for determining BVD, IBR and EBL antibodies, antigen and nucleic acids of viruses)was taken from the jugular vein using sterile vacu-um 10 ml test-tubes. The blood without anticoagulant was used for BVD, IBR and EBL antibody ELISA tests and the blood with anticoagulants was used for BVD antigen ELISA (Ag ELISA) tests. The sera before the tests were kept at a temperature of -20ºC or tested on the day of delivery. A leu-kocyte extract was prepared from the blood samples with anticoagulant for BVDV Ag ELISA and PCR tests. Forinvestigation of rabiesby the fluorescent antibody test (FAT), sam-ples from different parts (medulla, cerebellum, cerebral cortex of hemisphe-res, and Ammons horn of animal brain were taken by opening the scull and bringing out the brain. A suspension of animal brain was prepared for infec-tion of laboratory mice. For investigation of the efficiency of oral vaccina-tion against rabies, the carcasses of hunted foxes were collected and delive-red to the NVL. The jawbone was taken from the animal head without da-magingangulus mandibulaeand 150 microns thick slice of bone was taken with osteotom. The tetracycline test was performed at the NVL. ForBSE investigationby the Enfer and BioRad tests, a thin slice (weig-hing 1 g) of the tissue of the CNS with the grey brain material was cut and manually or automatically homogenized. The samples were kept at a tempe-
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rature of +2- +8ºC when purification was performed in 24 hours. Frozen samples can be stored for a few months. Samples are suitable for testing when the number of freezing-thawing cycles does not exceed 3. For BSE histological examination, samples were taken from a fixed (at least for 14 days) cerebral stem in the required areas (naholepncseme,pedunculus cere-bellaris caudalis, andobex). The histological sections were stained with haematoxylin and eosin. Diagnostic methods.BVD ELISA antibody method. diag- Commercial nostic ELISA kits standardized at the Institute Pourquier (France) and de-signed for testing of antibodies to virus protein p80 in blood serum, milk or plasma were used following the manufacturers instruction. The method is based on the principle of competition between the antibodies in cattle and monoclonal anti-p80 antibodies combined with peroxidise. Protein p80 is bound in microplate wells by specific monoclonal antibodies WB103. Serum samples were diluted and incubated on microplates. When specific antibodies to BVDV are present in a sample, protein p80 and conjugate cannot bind with a corresponding epitope but conjugate can bind with p80. After rinsing, substrate TMB is poured into wells. In the course of reaction, the substrate changes colour from blue to yellow. The colour intensity is inversely proportional to anti-p80 antibody concentration in the sample. Results are evaluated according to the optical density (OD) of the sample measured at light wave length 450 nm. BVD ELISA antigen method. For identification of BVDV antigens, commercial standardized ELISA diagnostic kit able to identify p80 BVD virus proteins in the blood serum and leukocyte fraction was used. The pro-cedures were implemented following the manufacturers instruction for use. The results were evaluated according to the OD and read at the light wave length of 450 nm. BVD PCR method. The clinical samples for PCR examination were se-lected from the risk group of serologically negative animals following H. Houes recommendations (Houe, 1999). The pestiviral RNA for one tube nested RT-PCR investigation was isolated from EDTA uninucleated stabili-zed blood cells or MDBK cell cultures using the isolation kit Total RNA Prep Plus, whose performance is based on a modified Chomczynskis me-thod (Chomczynski, Sacchi, 1987). The reaction was performed and the obtained products identified in an agarose gel following A. Stankevičius modified RT-PCR method (Stankevičius et al., 2002). Rabies fluorescent antibody test (FAT). For examinations by this me-thod, commercial diagnostic kit Bioveta (Czech Republic) was used. Sa-mples of medulla, cerebellum, cerebral cortex of hemispheres, and Am-mons horn were taken. Nine clean degreased object glasses were taken. Six
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of them were stained with a positive conjugate and the remaining three with a negative conjugate. Three smears for each part of brain (two positive and one negative) were made. The objective glasses with the smears were air dried and fixed by flame. After that they were inactivated for 510 min un-der the UV rays. 0.1 ml of working conjugate solution was dripped on the fixed smear and covered with a glass. The samples were incubated in a wet chamber of thermostat (37º C) for 30 min. Afterwards the smears were three times rinsed with phosphate buffer solution (PBS) for 10 minutes each time and then washed with distilled water, dried and examined by a luminescent microscope (magnifying capacity 600x) using chemically clean immersion oil. The controls used during the test were: positive conjugate and negative preparation; negative conjugate and positive preparation. If the FAT shows specific radiance of antigen and antibodies, cerebral brain is frozen at a tem-perature of -20ºC and the examined preparations are kept at a temperature of +4° C in a fridge for 10 days. If the FAT does not show any specific radian- ce of antigen and antibodies, the brain is stored for one month in the cold storage and the examined smears are stored at a temperature of +4° C in a fridge for 30 days before the biological examination. Biological method for rabies diagnostics. a negative result was When obtained by the FAT, a method of in of laboratory mice was used for con-firmation of the negative result. For this purpose, the prepared brain suspen-sions were injected into the area above the cerebral cortex of six mice (weighing 1214 g). The state of mice was observed for 28 days. Microsco-pic analysis of the brain of mice that died in the first 48 hours was not pe-rformed. Smears were made from the brains of mice that died later. The smears were stained and microscopically examined as described above. In positive cases, the surviving mice were subjected to euthanasia in negative cases, the experiment was continued. Twenty eight days after infection, the surviving mice were killed by dislocation of vertebra and burned in autocla-ve together with the hutch. GSE Enfer method.By chemiluminescent immunassay using Enfer TSE kit (version 2.0) and following the manufacturers instruction for use it is possible to identify resistant prion protein PrPres in the CNS tissues typical for spongiform encephalopathys. Positive and negative control of brain tis-sues were used during the investigation. Results were evaluated by determi-ning the positive (peptide indicators), positive and negative control lumines-cence values and calculating the average empty control value. BSE Bio-Rad method. of spongiform encephalopathys by Investigation res express Bio-Rad method (a sandwich immunoassay for PrP in the tissues of CNS of cattle, sheep and goats) includes two stages: A. Purification of BSE marker PrPresfrom the nervous tissue using aTeSeE®Purification Kit.
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B. Qualitative PrPresdeterminationin vitroby ELISA method using aTeSe-E®Detection Kit. With the aid of ELISA kit created by the CEA (France) and manufactured by Bio-Rad we attempted to identify the typical marker for infectious SE prion protein PrPres. The investigation was carried out fol-lowing the manufacturers reaction performance protocol. IBR ELISA antibody method. Blood samples were examined using com-mercial ELISA manufactured by the Institute Pourquier, France. They are designed for identification of antibodies to bovine herpesvirus type 1 (BHV-1) gB antigen in blood serum and milk. Investigation was carried out follo-wing the manufacturers instruction. The optical density was measured by light wave length 450 nm (OD450). The results were evaluated when the minimal value of OD450of positive control serum was 0.350 and the ratio of corrected positive control OD450and negative control OD450 higher or was equalled 2.5% of S/P (S sample, P positive control) ratio of each sample was calculated: S/P% =optical density of the sample OD450/positive control OD450 x 100. The sample, where the mentioned ratio S/P% 85%, was < considered taken from an animal that had no contact with BHV-1. The sam-ple, where the ratio S/P% between 85% and 115%, was considered was suspicious. The sample, where S/P% 115%, was considered taken from > an animal that was infected with BHV-1. EBL agar gel immunodiffusion antibody method.Standard methods were used for performing reactions. Agar gel was made of the gel provided in the kit. In a Petri dish, agar layer was made to be 3 mm thick (12.5 ml of agar were poured into Petri dish 100 mm in diameter). The wells in agar were made with a special stencil flower in the following way: one well in the centre and the remaining six around the central one. The well diameter was 7 mm. The wells were spaced 3 mm. Antigen was poured into the central well. The external wells were filled with control positive precipitating serum and examined sera. The wells were filled with sera and antigen to the rim of agar. Meniscuses of the liquid were convex. The dishes were covered and incubated at room temperature for 48 hours. The reaction was evaluated after 24 hours and 48 hours. Due to specific interaction of antigen and anti-body, an insoluble combine precipitate forming a line of antigen and anti-bodies developed. The final evaluation of reaction and its recording were done after 72 hours. EBL ELISA method.For diagnostic investigation of EBL, a commercial standard ELISA kit was used (Institute Porquier, France). The kit is desig-ned for determining specific antibodies to EBL virus antigen gp51 in blood and milk. Microplates covered with EBL virus antigen were used. The es-sence of the reaction is that antibodies against EBL viruses form an immuni-ty complex with EBL virus and remain bound in the microplate where they
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bind with enzyme conjugate changing the colour of reaction. The cut off point of positive reaction is determined using control serum included in the kit (positive control). The optical density is read by spectrometer at light wave length 450 nm (OD450). The results were evaluated when the minimal value of OD450 ofcontrol serum was not less than 0.350 and the positive ratio of corrected positive control OD450and negative control OD450 was higher or equalled 3. BSE histological examination method. sections were chec- Histological ked by microscopic analysis of the contrast of luminescence phases (micros-cope Zeiss, 40500x) It was observed whether vacuolization (typical of BSE) of neurophyleme (thinnest nervous fibres), when vacuoles localize in the nuclei of grey brain material, would occur. The samples were also chec-ked for typical transformations inobex, nucleus tractus solitariiandtractus spinalis nervi trigemini. Investigation methods of oral rabies vaccination efficiency.Six oral vaccinations of wild animals were implemented during the study period. Baits with rabies vaccine were manually scattered from a plane. Vaccines SAG-1 (France), Lysvulpen (Czech Republic) and Rabifox (Germany) were used. Vaccinations were carried out seasonally: in spring and in autumn. Baits were composed of one dose of rabies vaccine in a capsule within a food mass each. Vaccine efficiency was estimated according to fluorescence of tetracycline marker under ultraviolet light in teeth and bone tissues. Bone sections were put under the objective of luminescent microscope and chec-ked for yellow fluorescent tetracycline rings and lines. Analysis of preventive measures against cattle diseases.The preventi-ve measures against BVD, IBR and EBL were evaluated by analysis of or-der No 281, 16 10 2000, of Director of SFVS On confirmation of the cont-rol and eradication measures for bovine infectious rhinotracheitis and viral diarrhoea (in., 2001, No 4120), order No B1-349, 20 04 2004, of Direc-tor of SFVS On confirmation of veterinary requirements for cattle and pig trade and order No B1-325 of 2004 04 15 of Director of SFVS On requi-rements for cattle semen and other relevant documents. Territorial informa-tion of SFVS sub-units about BVD and IBR preventive measures and infor-mation about diagnostic investigations of NVL also were analysed. The rabies control measures were studied based on statistical data and annual reports of SFVS. Preventive measures against BSE were evaluated in the context of regulations adopted by the European Parliament and European Council 2001/999 EC, and results obtained by NVL and reports of SFVS sub-units on the issues of BSE prevention. Methods of calculating expenditures and their economic evaluation. Methods of calculating expenditures. Expenditures were calculated follo-
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wing the Veterinary Law of the Republic of Lithuania ((in., 1992, No 2-15), order No 93 of 14 07 1993 of State Food and Veterinary Service of the RL On specification of orientational tariffs, order No 90, 14 02 2002, of the SFVS of the RL On confirmation of the tariffs of laboratory investiga-tions, and order No B1-894, 14 10 2004, of the SFVS of the RL On the methods of price-fixing for laboratory investigations carried out by the VVL. Investigations are related with expenditures on purchasing test equipment, sampling and sample transportation, implementation of tests, and storage and utilization of samples. Evaluation included only the direct expenditures on documented laboratory examination services which only partly include the above mentioned expenditures. Methods of financialeconomic assessment of expenditures.Analysis of feasibility of expenditures was based on correlation and discounted money flow criteria financial existing net value and economic existing net value as integral indices of correct analysis, comparison of expenditures, calcula-tion of interdependence of laboratory examinations, and financialeconomic effect. For data analysis Pearsons correlation coefficient was used, which in the probability theory and statistics is a measuring unit of statistical relation between variables. RESULTS Epidemiology of viral diseases in cattle. Bovine viral diarrhoea. The first record that cattle in Lithuania are infected with BVDV was made in 19941995. In 19952004, 23.547.6% of examined cattle were seropositi-ve with respect to antibodies of BVD viruses. The highest number of sero-positive cattle was recorded in the Klaipėda 54.97%, Marijampolė48.20%, iauliai 45.80% and Teliai 39.46% counties. In the Alytus, Tauragė and Utena counties, where cattle-breeding and dairying is not developed, the proportion of seropositive cattle was statistically significantly lower. Com-parison of results from different counties allows assuming that BVDV infec-tion is dominant in South-West. West, Middle and North Lithuania, where cattle concentrations are highest and the number of large herds is greatest. Rabies.Investigations revealed that rabies in wild (foxes, raccoon dogs, martens, polecats, etc.) and domestic animals (cattle, cats, dogs, etc.) is wi-despread over the country. The epidemiology can be evaluated as unfavou-rable. The highest number of rabies was recorded in the Lazdijai, Klaipėda, Panevėys and Tauragė districts and the lowest number in Maeikiai, Ak-menė, Kaiiadorys, Jonava, Vilkavikis and Trakai districts. According to the spread of rabies in wild and domestic animals in 19942003, the Lithua-nian regions were conditionally divided into three groups: Group I classified
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districts with registered 190 cases of rabies, Group II 91180 cases, and Group III more than 180 cases. Group I included 21 districts: Akmenė, Birai, Jonava, Kaiiadorys, Kretinga, Kupikis, Maeikiai, Molėtai, Plungė, Prienai, Raseiniai, Rokikis, Skuodas, akiai, alčininkai, ilalė, venčio-nys, Teliai, Trakai, Vilkavikis, and Zarasai; Group II (91180 cases) inc-luded 19 districts: Alytus, Anykčiai, Ignalina, Jonikis, Jurbarkas, Kaunas, Kelmė, Kėdainiai, Marijampolė, Pakruojis, Pasvalys, Radvilikis, iauliai, ilutė, irvintos, Ukmergė, Utena, Varėna, and Vilnius; and Group III (more than 180 cases) included four districts: Klaipėda, Lazdijai, Panevėys and Tauragė(Fig. 1).
Fig. 1.rabies in wild and domestic animals in countryOccurrence of districts in 19942003(Group I - light grey 190 cases; Group II- grey 91180 cases; Group III - dark grey more than 180 cases) 1 pav.Naminiųir laukiniųgyvūnųpasiutligės paplitimas alies rajo-nuose 19942003 metais(tamsiai pilka daugiau negu 180 atvejų, pilka 91180 atvejų, viesiai pilka 190 atvejų) Bovine spongiform encephalopathy. to the geographical risk According of BSE, the countries of the world are classified into four categories (I-IV). The geographical risk is evaluated taking into account the BSE risk factors in a country, practical implementation and management of national control programmes, implementation of active and passive observance of BSE pro-
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grammes and infection of cattle with BSE. Category I include the countries of least risk, category II includes the countries of least but not dismissed risk and category III includes the countries where BSE risk is presumptive yet unconfirmed or BSE was identified in a small number of animals. Lithuania belongs to the third category. Category IV includes the countries where BSE was identified in many animals. In view of this, overall diagnostic investiga-tions were carried out in all counties. The highest number of samples was examined from Kaunas, Klaipėda, Panevėys, iauliai and Tauragė coun-ties. The total of 90 000 samples were examined and not a single animal infected with PrPres identified. Thus it may be assumed that Lithuania was was fortunate to avoid the outbreak of BSE and that the epidemiological situation is good. Infectious bovine rhinotracheitis.Exhaustive investigations of IBR were started in Lithuania in 1992. Investigations revealed that irrespective the still valid control measures, from 3 % to 22 % of all examined animals were infected with bovine herpes virus of type I (BHV-1) which is the agent of infectious rhnotraheitis. Analysis of obtained data shows that the greatest numbers of infected animals occur in the counties where concentration of animals is greatest and the number of cattle-breeding and dairying farms is highest. The greatest number of seropositive animals in 20032004 was recorded in Kaunas (28.21%) and iauliai (16.53%) counties. In other coun-ties the numbers of seropositive animals were a few times as low: Panevėys 6.42%, Teliai 3.53%, Marijampolė3.05%, Vilnius 0.79%, Utena 0.77%. In some counties (Alytus, Klaipėda and Tauragė) seropositive animals were not identified. Thus based on investigation data it is possible to assume that BHV-1 are most widespread in Middle and North Lithuania, i.e. in the dist-ricts with higher concentrations of animals and greater number of industrial and dairying farms keeping large high productivity herds. Enzootic bovine leukosis.Though laboratory investigations of EBL were carried out and national control programmes implemented already in 1967, relevant immunological diagnostic methods had not been not developed until 1988. For this reason, the epidemiological situation of EBL was unc-lear. After introduction of agar gel immunodiffusion (AGID) method in laboratory practices, the EBL eradication programme became more effecti-ve. Application of new diagnostic methods led to considerable reduction of EBL occurrence in Lithuania. In 2004, only solitary cases of EBL infection were registered in herds of different Lithuanian districts. The greatest nu-mber of infected animals was identified in Maeikiai, Naujoji Akmenė, Radvilikis, Vilnius, and alčininkai districts. No cases of EBL were recor-ded in the Alytus, Varėna and irvintos districts. According to these data it is possible to assume that in 2004 EBL infection was most widespread in the
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northern and south-eastern Lithuanian districts. Diagnostics of bovine viral diseases. Diagnostics of bovine viral diar-rhoea. 19972004, immunological investigations of antibodies against In BVD viruses were carried out in all Lithuanian counties. In 19972004, the total of 13 909 animals was examined. 6 201 had antibodies against BVDV, i.e. 44.58% of the total. Analysis of diagnostic examination dynamics sho-wed that, the smallest number of animals was examined in 2000 185 inc-luding 21 seropositive animals or 11.35%. In 1997, 1999 and 2001, the ex-tent of examinations was stable. The number of seropositive animals ranged from 30.61% to 66.95%. The greatest number of seropositive animals was identified in 19971999 when the portion of BVDV infected animals even reached 74.13%. In 20022004 the number of examinations slightly increa-sed and in 2004 more than 8 000 samples were examined. Since 2002, the percent of seropositive cattle have been reducing and in 2004 it was 29.88%. For more detailed analysis of the spread of BVD virus, the examined ani-mals were divided into three groups according to the age and gender: cows, heifers and bulls. Analysis of the obtained results showed that the highest numbers of seropositive animals were found in cows. In 19972004, the total of 5 801 sample of cow blood serum was examined. The number of seropositive cows was 3 658, i.e. 63.06% of the total number of examined cows. Lowest numbers of seropositive individuals were determined in the group of bulls. In 19972004 the total of 2 477 bulls were examined. Sero-positive individuals amounted to 850 (34.31%). In the group of heifers, the number of seropositive individuals was 1728 (32.33%). Thus, it is possible to assume that the probability of contact and infection with BVDV increases with age of the animals, but also to depend on the breeding technologies, management practice, cattle movement within and between farms, and com-paratively high concentration of cattle in the studied districts. As it is known that persistently infected (PI) and sick viremic animals are the main source of BVD viruses, in 2001 cattle blood was started to be examined by antigen ELISA. Tests were carried out in many Lithuanian farms. The obtained results showed that the greatest number of viremic individuals was in the group of bulls 0.74% on the average. The number of viremic individuals was twice as low in the groups of cows and heifers (0.39% and 0.36%, res-pectively). In order to evaluate PCR and Ag ELISA comparative efficiency parallel investigations by both methods were carried out. For this purpose clinical samples, standard and isolated in Lithuania BVD virus isolates were used. Comparative studies of diagnostic methods showed that RT-PCR me-thod was considerably more sensitive compared to the Ag ELISA on patho-logical material and virus isolates while the results of examination of leuko-cytes in cattle blood samples were identical.
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Investigations of rabies by FAT and laboratory mice inoculation me-thods.In 19952004, investigations of rabies were carried out over Lithua-nia. Diagnostic methods applied were two: fluorescent antibody test (FAT) and laboratory mice inoculation. The laboratory mice infection method was used for proving the negative result obtained by the FAT. In 19952004, the total of 11 257 FAT examinations of animal brain samples including 1 474 of cattle brain samples were carried out. The highest prevalence of infection was found in foxes and in raccoon dogs 58.5% and 70.7%, respectively. Among the examined 12 species of domestic animals most susceptible to infection were cattle 52.1% of the total examined animals. The rates of infection among other species (dogs, cats and horses) were lower and ranged from 17.29% to 46.51%. Presumably, cattle most often get infected during the grazing period because at that time the conditions for contact with wild animals are most favourable. According to our data, rabies in cats and dogs is most widespread in stray animals. At the same time (19972004), 6 155 laboratory investigations by mice inoculation method were carried out using material in which rabies viruses were not identified by the FAT. The inves-tigations yielded no positive case. Thus, we may assume that the FAT is sufficiently effective, reliable and applicable in rabies diagnostics in Lithua-nia. Investigations of bovine spongiform encephalopathy.An investigation of BSE in Lithuania was carried out in 20012004. These include 83 673 heal-thy animals aged >30 months; 420 animals aged >24 months; 155 sick ani-mals aged >24 months, which were slaughtered and investigated. In addi-tion, 6 419 dead animals aged >24 months were checked. Enfer TSE, Bio-Rad immunoassays and histopathological method for diagnosis of BSE were implemented. All 90 thousand investigated samples were negative to BSE. Investigation of IBR by ELISA.In 19952004 30 334 individuals of cat-tle were tested for IBR and 5 057 (16.67%) were found seropositive. The highest numbers of samples were examined in 19971998, 2002 and on-wards (more than 3 000 samples per year). Further analysis results showed that the highest prevalence of BHV-1 was registered in cows followed by heifers, and in bulls were registered only sporadic cases. Majority of infec-ted bulls were identified either during quarantine before getting into farms or during control examinations prior to sale for breeding. In 19972004, 85 bulls and 948 pedigree heifers were discarded as infected with BHV-1 (Fig. 2).
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7,85
33,12
14,78
35 30 25 20 % 15 10 5 1,53 0 Bulls / Buliai Heifers / Telyčios Cows / Karvė / Vidutinikais Mean Fig. 2.The prevalence of infectious bovine rhinotracheitis in Lithua-nia 2 pav.Galvijųinfekcinio rinotracheito paplitimas skirtingose galvijų grupėse Our laboratory investigations showed that BHV-1 is widespread in Lit-huanian farms and may be the cause of respiratory diseases and reproductive disorders in cows and, rarer, in heifers. However, only sporadic cases of IBR in bulls were registered. Due to the practice of laboratory examination during quarantine, there is a minimal risk that BHV-1 can be spread with semen. Laboratory investigations of enzootic bovine leukosis. diag- Laboratory nostic examination of EBL was carried out in the National Veterinary Labo-ratory and in subunits in the counties. The total of 7 387 641 animals were examined in 19952004. It appeared, that the prevalence of leukosis was identified in 2.58 % of animals, which were discarded and slaughtered. Hence, the number of seropositive animals has been reducing since 1996. The highest number of positive animals (7%) was registered in 1996. Sub-sequently, in 1997 the number of positive animals reduced to 2-fold diffe-rence. However, in 2004, only 0.23% of animals (mostly cows) were positi-ve.
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% of positive animals / Teigiamų, %
8 7 6 5 4 3 2 1 0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Fig. 3.The prevalence of bovine enzootic leucosis 3 pav.Galvijųenzootinės leukozės paplitimo dinamika Prevention of viral diseases in cattle. Prevention of bovine viral diar-rhoea. Within the implementation programme of preventive measures against BVD virus, all pedigree bulls were examined and only 1 sample was found positive to BVDV antigen. This bull was immediately discarded as were the samples of its semen. Simultaneously, all stored (in liquid nitrogen) sperm samples of pedigree farms were examined by PCR for contamination with BVDV. All samples were negative. However, the situation regarding BVDV in dairy cattle is rather different. In 2004, there were 181.3 thousand dairying farms in Lithuania with average of 2.6 cows per farm. In 138.6 thousand farms (76.5%) were kept only one or two cows. However, only in 280 farms (0.15%) were kept pedigree cattle. Until the end of 2005, only 13 farms (4.6%) participated in the BVD eradication and control programme, and only 3 farms (1.1%) out of 280 had an official status of BVDV free. Prophylaxis of rabies.In Lithuania an obligatory vaccination of cats and dogs was carried out in 19942004. All other domestic animals were vacci-
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nated after a contact with sick or potentially sick animals and in the foci of rabies. According to the 1995-2000 Lithuanian National Rabies prevention program was organized in Central and North-western regions of Lithuania. In 1995 first oral vaccination against rabies in wildlife was conducted in the area of 940 km2in Panevėys, Pakruojis and Jonikis districts. In these areas 19,000 SAG-1 vaccine baits containing tetracycline marker were distributed. Continuously, in 1996 a spring vaccination campaign was arranged in the area of 4000 km2in 13 districts of northern Lithuania, when 200,000 doses of rabies vaccine were used (20-25 baits per km2). The baits were placed by hand in forests and bushes near densest. The distribution by aircraft was used in Birai district. In 1997 two vaccination campaigns in spring (May) and autumn (October - November) in an area of 5349 km2in 22 districts of Lithuania were performed and 200,000 SAG-1 vaccine baits (20-25 per km2) were distributed. Afterwards, in 1998 the vaccination campaign was arranged in an area of 6375 km2in 26 districts of northern and western parts of Lithuania. During that period 170,000 baits of a new type rabies vaccine Lysvulpen (Bioveta) were used. Continuously, in 1999 170,000 SAG-1 vaccine baits in the area of 7000 km2in 22 districts were used. Two-vaccination campaigns (April-May and October-November) were carried out. In 2000, the Rabifox (Desau) oral rabies vaccine was used in an area of 8000 km2in 20 districts of Lithuania, when 200,000 baits (15-20 per km2) were distributed in two-vaccination campaigns. Because of lack of funds in 2001-2004 oral vaccination of wildlife in Lithuania was discontinued. Pre-sumably, oral vaccination was not very efficient because only in 30% of vaccinated foxes tetracycline markers were identified. Prevention of bovine spongiform encephalopathy.BSE prevention is as-sociated with control measures, identification of disease, preventive slaugh-tering and destruction of animals. The obtained results revealed that all BSE prevention measures are implemented in Lithuania in strict conformity with the regulations for control of BSE (EC No 999/2001, May 22, 2001). Lithu-ania has developed and is implementing a BSE monitoring programme. The cattle are marked by individual seals and the herds are registered based on the health status. Dead animals aged > 24 months, culled animals and ani-mals from higher risk groups are examined. The BSE investigations are carried out at NVL, which ensures the reliability of examination results and control of BSE. For consumers protection beef and its products are marked for possibility to trace their origin. It is forbidden to feed cattle with animal proteins. Laboratory investigation data confirmed,that BSE prevention pro-gramme was successfully implemented in Lithuania. Prevention measures against infectious bovine rhinotracheitis.In Lithu-ania the control of IBR started in 1993. All bulls in artificial insemination
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(AI) centres were examined for specific antibodies. It appeared, that 49.8% of bulls were infected with BHV-1 and 16.9 % of these bulls semen samples were infected with BHV-1. Until 1997, all seropositive bulls and all infected semen lots had been destructed. Subsequently, all bulls of AI centres are negative. Thus, only sporadic cases of seropositive bulls in quarantine are registered. However, the situation regarding IBR in dairy cattle is rather different. Until the end of 2005, only 14 farms (5%) participated in the IBR eradication and control programme, and only 3 farms (1.1%) out of 280 had an official status of IBR free. Prophylactic measures of enzootic bovine leukosis. Lithuania applies strict prevention measures against EBL. Permanent control of the clinical state and serological examinations of cattle blood serum are carried out. Farms are banned to accept animals that have not been examined for leuko-sis and virus carriers are culled. All bulls in AI centres are examined serolo-gically. Bulls-virus carriers are culled and the semen lots of infected bulls are destroyed. In infected farms it is banned to regroup animals without a permission of veterinarian. Special attention is paid to strict sterilization of instruments and equipment. All infected animals have to be isolated and culled in 15 days. Eradication measures are applied taking into account the prevalence of EBL in a herd, breeding technologies, number of farms etc. Transportation of animals is allowed only following strict veterinary requi-rements for extraordinary transportation. The mentioned strict preventive measures are targeted to minimize the number of EBL positive animals. Financial analysis of expenditures related with viral diseases in cat-tle.showed that expenditures on diagnostics and prevention of Analysis diseases are very uneven. Generalized data on diagnostic and preventive measures against various diseases are given in Table 1. The total diagnostic and vaccination expenditures based on current pri-ces from 2000 to 2004 increased by 8-fold: from 0.72 million to 5.81 million Lt (Table 1). The increase of expenditures based on actual prices was slight-ly by 7.8-fold lower. There are no significant differences between the inc-rease based on current and actual prices. Although the highest rise of expenditures was observed in the last year of the study period (2004). The analysis of expenditures (Fig. 4) shows that the structure of expenditures has considerably changed since year 2000. While in 2000 the relatively greatest portion of expenditures included EBL diagnostic costs. From 2001, the ma-jority of expenses were related to BSE investigations.
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