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Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis (IBR/IPV) are diseases that affect cattle population of all breeds, categories and age. Both diseases, especially infectious bovine rhinotracheitis (IBR), poses severe health threat and causes major economic losses and is considered one of the “most costly” disease in cattle industry. The causal agent of the disease is a virus and any detection of IBR/IPV specific antibodies in non-vaccinated cattle, either in blood or milk, indicates that animal is infected and represents the source of infection. Countries with developed and intensive cattle breeding have been developed and implemented their national eradication programs to control IBR/IPV in accordance with international regulations. In this article, we outlined the needs and program for the eradication of IBR/IPV in the Republic of Serbia. The eradication program for IBR/IPV is an extensive process that requires systematic strategy involving different phases and activities. The eradication process from the moment of implementation until obtaining IBR/IPV-free status can last over several years and requires joint work and considerable financial resources that will be compansated with the elimination of IBR/IPV from the herd. This article gives an overview of all stages and activities regarding eradication of the disease and certification and maintaining of IBR/IPV-free herd status.

West Nile virus (WNV) is a neurovirulent mosquito-borne Flavivirus with zoonotic potential. Virus is maintained in nature in an enzootic transmission cycle between avian hosts and mosquito vectors, but occasionally infects other vertebrates. The infection in horses and humans can be asymptomatic or it can have different clinical manifestations ranging from light febrile diseases to fatal meningoencephalitis. Recently, the number, frequency and severity of outbreaks with neurological consequences for birds, humans and horses have increased dramatically throughout central and south Europe, including Serbia, posing a serious veterinary and public health problem. The emergency of WNV infections in Serbia is described through the current epidemiology situation based on recent data on the incidence of WNV infection among virus natural hosts and vectors; sentinel (horses) and other animal species, and in human population. The results of the WNV serology studies conducted on horse blood samples collected in different occasions during the last six years, and the results of the serology studies conducted among other animal species like pigs, wild boars, roe deer and dogs in Serbia are presented and discussed. Also, the results of the first studies on WNV presence in mosquito vectors and in wild birds as virus natural hosts in Serbia are presented and analyzed. In addition, the data on the WNV serology studies conducted in human population in Serbia in the last few years, and the existing data of WNV outbreaks in 2012 and 2013 are included. Regarding the existing knowledge on WNV epidemiology situation, the crucial role of veterinary service in early detection of WNV presence and ongoing national program of WNV surveillance in sentinel animals, mosquitoes and wild birds are discussed.

The titer of specific antibodies against equine herpesvirus-1 in blood serum was tested in two groups of mares and their foals. The first group consisted of 12 mares, Standardbred and Serbian Trotter breed, who were vaccinated against equine herpesvirus-1 and 4 in the 5th, 7th and 9th month of pregnancy. On the contrary, 12 mares from the second group, of Lipizzaner breed, were not vaccinated. The mares’ blood samples for antibodies titer investigation were taken 30, 15 and 7 days before the expected partus, then immediately after the partus, while their foals’ blood samples were taken immediately after foaling, then just before colostrum intake, and finally 1, 2, 3 and 7 days later. The titer of antibodies against equine herpesvirus-1 was tested by the method of virus - neutralization, on microtiter plates with constant dose of the virus and serial double dilutions of the serum. In unvaccinated mares, titer of antibodies against equine herpesvirus-1 was either low or not present, but on the contrary, in the vaccinated ones the antibodies titer ranged from 1:32 to 1:256. In the foals originating from both vaccinated and unvaccinated there were not found specific antibodies in the serum before colostrum intake. After the colostrum intake, the values of specific antibodies against equine herpesvirus-1 significantly increased in the foals originating from the vaccinated mares, and ranged from 1:8 to 1:32.

The objective of our work was to investigate some biological characteristics of purified glycoprotein subunits of Newcastle disease virus strain PHY-LMV.42 isolated from pigeons for the purpose of vaccine production. PHY-LMV.42 strain of Newcastle disease virus was multiplied by successive passages in embryonated eggs and identified by the methods of Reverse transcriptase PCR and Real-Time PCR along with F gene sequencing. Proving the presence of HN and F antigene in the virus subunits samples was carried out by hemagglutination inhibition method with referent immune sera. Biochemical characterization of glycoprotein subunits was performed by SDS-PAGE method as well as liquid chromatography with mass spectrometry (LC ESI-TOF-MS/MS). Testing for the virus subunits immunogenicity was carried out in biological experiment on 75 laying hens Tetra-SSL and 25 chickens Isa Brown by inducing an artificial infection with Hertz 33 strain of the virus. Low concentrations of the virus antigens of 0.36 mg/ml along with glycoprotein fractions of 77 i 58 kDa manifested a strong hemagglutination activity of 4096 HJ/0,1ml. The subunit vaccines of 256 and 128 HJ/0.5 ml induced a protective immune response in all the vaccinated animals. Based on the obtained results it can be concluded that low concentrations of purified virus subunits of PHY-LMV.42 strain can be used for preparing of effective vaccines. [Projekat Ministarstva nauke Republike Srbije, br. TR 31008: Development and application of molecular methods based on polymerase chain reaction (PCR) in quick and direct identification of Newcastle disease virus strains and investigation of immunogenicity of subunit vaccine prepared of their antigens]

Bovine herpesvirus 1 (BHV-1) is one of the most siginificant causes of infections of the respiratory tract of cattle and immunoprophylaxis has a key role in curbing this infection. The intensity of the immune response against BHV-1 following immunization using inactivated commercial vaccines varies depending on the type of vaccine, but it is generally believed that they provide good protection from the development of the clinical form of the infection, and that they are safe. The paper present the development of the humoral immune response in fattening calves that were immunized against bovine herpesvirus 1 (BHV-1) at different time intervals between the primary and the secondary immunization. Calves were administered a commercial vaccine, and then they were divided into two groups which were revaccinated on days 14 or 21. Over a course of the 120 days of the duration of the experiment, blood and nasal mucus were sampled 11 times. The blood serum samples were examined for antibodies to BHV-1 using the virus neutralization (VN) test, and the nasal mucus samples were analyzed using the VN test and the ELISA method. Following revaccination, it was established that there was an increase in the antibody titer in blood of all experimental animals, and it was maintained at a high level up until the very end of the experiment (day 120). In the blood serums, maximum mean values for the antibody titer were determined on day 30 in the group that was revaccinated on day 14, and on day 45 in the group of calves revaccinated on day 21. In nasal mucus, antibodies were established at the earliest, using the virus neutralization test, on day 14 following vaccination, and using the ELISA method only after revaccination. The highest antibody titer in nasal mucus was established on day 45 in the group revaccinated on day 21, and on day 120 in the group revaccinated on day 14. Based on the established antibody titer values, calves can be revaccinated using the inactivated BHV-1 vaccine already on day 14. [Projekat Ministarstva nauke Republike Srbije, br. 31084]

The paper presents the results of investigations of the presence of the bovine herpesvirus-1 (BHV-1) in samples of blood serum from 106 cows and 107 of their calves (one cow had twins). Blood was sampled from the cows immediately after parturition, and from the calves before feeding on colostrum. The examined cows and their calves originated from 5 herds in which previous investigations had shown infection with the bovine herpesvirus-1. The determination of antibodies against BHV-1 was performed using the method of virus neutralization in culture of MDBK cells with 100 TCID/50 viruses (BHV-1, TN-41 Am. Bio Research, USA). Antibodies against BHV-1 were determined in all blood serum samples of cows and in 16 samples of precolostral blood serums of calves. The antibody titer values in cows ranged from 1:4 to 1:512, and in calves the determined values were from 1:2 to 1:16. The results indicate that cows that are seropositive to BHV-1 can deliver calves seropositive to BHV-1 in about 15% cases. This must be kept in mind in selecting cows for the production of breeding material, in particular bulls for reproduction centers, as well as in making a programme for the immunoprophylaxis of calves against BHV-1. .

Bovine Respiratory Syndrome (BRS) is a multifactorial disease caused by the interaction of infective agents, the environment and the individual immunological response of animals in the herd. Despite five decades of research on BRS, no clear understanding of how environmental factors influence pathogenic outcomes of the disease has been defined. As such, the development of immunoprophylaxis and vaccine programmes to prevent outbreaks of BRS in cattle has not been successful. The current paper discusses vaccination programmes for all categories of cattle and presents a review of existing vaccines being used for immunoprophylaxis of respiratory syndrome in cattle and discusses the advantages and disadvantages of the currently used vaccines and vaccination programmes. Lastly, a discussion detailing the design of future perfect vaccines is presented.

Verotoxin-producing Esherichia coli (VTEC) is one of six pathogenicity groups of Eschericha coli. The reservoir for VTEC is the intestinal tract of domestic animals, primarily ruminants. Investigations in our geographic region have also demonstrated that domestic animals are a significant VTEC reservoir. In spite of this, sporadic diseases in humans caused by these agents are rare, and no epidemics have been registered so far. The question is, therefore, what percentage of VTEC isolated in our region have the characteristics of enterohaemorrhagic E. coli (EHEC) which cause intestinal diseases in humans. The aim of this study was to test the isolated bovine strains for some phenotypic and genotypic traits and to determine the percent of strains that belong to EHEC. A total of 105 VTEC strains isolated from cattle were tested for the presence of verotoxin (vtx) genes by PCR (polymerase chain reaction) method. All of them possessed one or more vtx genes. In accordance with our investigations, only four (3.8%) strains, of a total of 105 VTEC strains belong to groups of EHEC. It may be concluded that the majority of strains isolated from cattle in this part of the world do not have phenotypic traits typical for EHEC. Therefore human VTEC associated diseases in Serbia are rare, despite the fact that domestic animals frequently harbor VTEC.

Parvoviral infection of swine is a disease which is manifested in reproductive disorders in sows and gilts in the form of anestria, premature births, miscarriages, mummified fetuses, the birth of poorly vital piglets and/or a reduced number of piglets in the litter. The infection is present in farms with intensive breeding conditions in the form of an endemic infection, all over the world, and also in our country. Timely diagnostics and adequate prophylaxis prevent the occurrence and spread of this disease. Experimental investigations covered 21 sows, divided into two experimental and a third, the control, group. Animals of the first experimental group were vaccinated once before exposure to the boar using an inactivated vaccine, Porcilis Parvo, and animals of the second experimental group were vaccinated twice at an interval of 3 weeks, also using an inactivated vaccine, Parvovax. Sows of the control group were not vaccinated. Blood samples were taken from all animals four times during the course of the experiment, and specific antibodies against the swine parvovirus were determined using the method of hemagglutination inhibition (HI test). The results of the investigations indicate that there was an increase in the titre of specific antibodies following the vaccination of persistently infected sows with the swine parvivirus, and that the present antibodies did not prevent the creating of an immune response. It was established following a comparison of the geometric mean values of antibody titres of vaccinated sows that there was a significant increase in the antibody level following the two vaccinations using the Parvovax vaccine, against the titre values in sows vaccinated once with the Porcilis Parvo vaccine. In control animals, the average value of the antibody level was many times lower in comparison with the established values in the experimental groups. This provides justification for the implementation of immunoprophylaxis against swine parvovirosis by the vaccination of sows and gilts before mating using inactivated vaccines.

The success of supervizing an infectious disease depends on the ability for speedy detection and characterization of the cause and the forming of a corresponding system for examining the success of control implemented in order to prevent a recurrence of the disease. Since influenza viruses continue to circle, causing significant morbidity and mortality both among the human population and among animals all over the world, it is essential to secure the timely identification and monitoring of the strains that are in circulation. The speedy detection and characterization of new highly-virulent varieties is one of the priorities of the World Health Organization monitoring network. The implementation of molecular methods has an increasingly significant role in diagnostics and the monitoring of the influenza virus. Among a large number of molecular methods, the one particularly in use is the reverse transcription-polimerase chain reaction (PT-PCR). Technological progress in the area of the conducting of molecular methods has enabled that we can prove, in one day, using the RT-PCR method even very small quantities of the infective agent in a sample. In an obtained PCR product, we can relatively easily establish the nucleotide sequence, a detailed analysis and molecular epidemiology of the circulating strains. The molecular diagnostics procedure (RT-PCR) is based on the correct choice or designing of primers depending on the desired knowledge. In order to obtain a specific diagnosis of influenza A, B or C, primers are used which multiply internal genes, such as the nucleoprotein (NP) or matrix gene (M), because these are genes that are highly conserved among the virus types. In the event that we are interested in the subtype of influenza A, after obtaining a positive reaction, primers for genes of surface antigens are selected, such as hemagglutinin. Following the correct detection of the H subtype, it is possible to establish the virus virulence through the direct sequencing of the PCR product. The possibility of typization using molecular methods is based on the big difference at the amino acid and nucleotide levels between different HA subtypes (from 20- 74%), while the differences between strains of the same HA subtype are relatively small (0- 9%). The basic advantage in the detection and typization of influenza viruses using the RTPCR method is that it saves time. Namely, it can be performed directly from the samples taken in the field, and the result can be obtained within the same day, contrary to conventional methods that take 7 to 10 days. The obtained PCR product can also be sequenced immediately, which can provide an answer to the possible virulent potential of the isolate and its further spreading. The establishment of changes in the HA gene sequence can provide us with the information about the direction of the development of the genetic drift. The paper will describe in detail the possibilities for the implementation of molecular methods in diagnostics and typization, in fact, in the molecular epizootiology of avian influenza.

Aujeszky’s disease (Morbus Aujeszky) is an acute viral contagious disease occurring in a large number of domestic and wild animals. This epizootiological disease has been present in our country for quite some time now, and it has been increasingly frequently diagnosed in carnivora in the recent years. The objective of the investigations was to determine and establish the pathways for the transfer and the manner of spreading of the MA virus to carnivora. Epizootiological data on the appearance of MA, collected in the field, as well as an epizootiological report by the Serbian Ministry of Agriculture and of the Federal Ministry for Agriculture were used as material. Detailed epizootiological, clinical, pathomorphological, and laboratory analyses were performed in five critical spots for MA registered in the territory of Vojvodina province. We established the following pathways for the spread of the MA viral infection to carnivora: the consumption of thermally unprocessed swine carcasses, the consumption of thermally unprocessed pork originating from butcher shops or from regular slaughter on private farms, the consumption of viscera from emergency slaughtered sheep, cohabitation and contact with infected swine, and vaccination using a live vaccine contaminated with the MA virus.

The pathomorphological changes established following infection with the virus of classical swine plague in non-immune individuals are well known. However, piglets present a problem from the clinical-pathological aspect, in which this virus can be multiplied in spite of established colostral antibodies, but without the exhibiting of the clinical symptoms characteristic for the disease. The question of the characteristics of the pathomorphological finding is raised in the event of the breakdown of the colostral immunity of the piglets. With the objective of determining the influence of colostral antibodies on the development of pathomorphological changes in classical swine plague, piglets aged 28, 35, 44, and 54 days, originating from sows that had received a vaccine of the K-strain of the classical swine plague virus, were experimentally infected with a virulent variety of this virus (Becker strain). The control group comprised non-vaccinated animals originating from non-vaccinated sows. Following the death and/or sacrificing of the piglets in the experiment, a pathomorphological examination was performed of all organic systems and the presence of classical swine plague virus antigens was established in organs and tissues of piglets using the immunoenzyme (ELISA) test. Even though clinical signs characteristic for this disease were not found in all animals following artificial infection, the pathomorphological findings following death and/or sacrificing indicated a successful experimental infection and was typical for the acute course of classical swine plague. Bleeding was established in most organs and serous membranes (haemorrhagic diathesis). However, certain variations were also established regarding the expression and distribution of the pathomorphological changes in certain animals.

A new group of pathogenic agents has appeared in the last 20 years -verotoxin-producing Escherichiae coli (VTEC), which cause increasing numbers of intestinal diseases in humans and animals. The reservoir for VTEC is the intestinal tract of domestic animals, primarily ruminants. It is believed that cattle are the principal reservoir for VTEC, because the biggest number of epidemics caused by these bacteria occurred through ground beef (hamburgers). Swine are also a significant factor of VTEC infections in humans, but these pathogenic agents are more frequently isolated from diseased animals than healthy ones, and most often from swine with a clinical picture of an endemic disease. Investigations in our geographic region have also demonstrated that domestic animals are a significant VTEC reservoir. In spite of this, sporadic diseases in humans caused by these agents are rare, and no epidemics have been registered so far. Such a characteristic of VTEC infections in our country imposes the need for further epidemiological and epizootiological research.

The bovine viral diarrhea (BVD) virus is a significant health-economic pathogen in cattle which can be excreted and spread also through sperm of persistently or acutely infected bulls. Native sperm of 6 bulls, found to be negative to the BVD virus by isolating the virus and using the RT-PCR method, was experimentally infected with a tenfold dilution of the non-cytopathogen 22146 strain of the BVD virus with a titer of 105,5. This way, dilutions of the BVD virus from 10-1 to 10-6 (5 x 104 TCID/50 do 0,5 TCID/50 in 0.1 ml native sperm were obtained. From sperm infected in this way, the virus was reisolated on FTB cell culture in a microtiter plate with 96 pools in which each sample of the infected sperm was set up in three samples, and each of them was titrated to a dilution of 1:2 to 1:256. The presence of the BVD virus was proven using the technique of fluorescent antibodies in a second blind passage on FTB culture cells. For cell culture an extremely toxic effect of native sperm to a dilution of 1:64 was established. The BVD virus was reisolated from sperm in all three sperm samples with 5 x 104, 5 x 103 i 5 x 102 TCID/50, and it was not reisolated from sperm with 50, with 5, and with 0.5 TCID/50 BVD virus in 0.1 ml native sperm. At the same time, the presence of the BVD viral genome was proved using the RT-PCR method in the same samples of artificially infected native sperm of bulls. A positive re suit was established in native sperm with 5 x 104, 5 x 103, 5 x 102 and 50 TCID/50 BVD virus n 0.1 ml native sperm. The experiment proved that the RT-PCR method has advantages over the isolation of the BVD virus from samples of native sperm of bulls. These are: shortterm investigations (1 to 2 days) and greater sensitivity (10 times bigger than the isolation of the virus). The isolation of the virus takes at least 10 days, and its greater sensitivity is primarily a result of the cyrotoxic effect of native sperm of bulls on cell culture.

In this paper, the antibody titre to Salmonella enteritidis (SE) was examined by the ELISA method in two flocks of laying hens, where during routine bacteriological investigations Salmonellae was never isolated, and in one flock where Colysepticemia was diagnosed and Salmonella isolated accidentally. In the flocks were Salmonellae were not isolated, a titre with a high level of specific antibodies to SE was discovered (15 and 45%), while the flock with accidental findings of SE was poorly positive (5%). These results point to the necessity of introducing serological monitoring to SE so that the infection of salmonella may be discovered early and the prevalence in the flock determined, and also for the purpose of applying adequate measures that could reduce the possibility of secretion of SE through eggs.

It is possible to conduct an analysis of the incidence of viral diseases in calves if these diseases are divided into two basic groups. One group comprises diseases of respiratory organs which are manifested by symptoms of a respiratory syndrome, and the second group comprises diseases of digestive tract organs in the form of a gastrointestinal syndrome. It is considered that viruses have the dominant role in the complex etiology of the respiratory syndrome, primarily the IBR virus or the Bovine Herpes Virus-1 (BHV-1), followed by the parainfluenza 3 virus (RSV), the Bovine Viral Diahrrea Virus (BVDV), the bovine Respiratory Syncytial Virus (RSV), but also other viruses, such as adenoviruses, rhinoviruses, coronaviruses, can also influence the appearance of the respiratory syndrome. The respiratory syndrome is rarely caused by a single viral agent, but most frequently by mixed viruses, but also by bacterial infections. Mixed viral infections often have a lethal outcome. Investigations of the etiology of the gastrointestinal syndrome so far indicate that, in addition to bacteria, viruses can also be a significant etiological factor. Rotaviruses, coronaviruses, adenoviruses parvoviruses, herpesviruses (the IBR virus), pestiviruses (BVDV), can be the causes of a gastrointestinal syndrome. It is believed that viruses can be the cause in about 10% cases in the ethiopathogenesis of this syndrome. The paper describes the etiopathogenesis of calf diseases of viral etiology which are most often found in the local conditions of industrial breeding of calves.

The ELISA technique is one of the most commonly used laboratory-diagnostical methods for diagnosing many infective diseases in humans and animals today. Due to its high specificity and sensitivity this technique is considered a method of choice in diagnosing many viral diseases. The phases and components of the ELISA technique are constantly improved because of its mass use, as well as the high demands of laboratory diagnostics. Therefore it is necessary to continually inform the expert public of all the innovations, forms and possibilities of the ELISA technique. This paper describes the factors that caused the genesis of the ELISA technique and shows its forms. The methodological and practical aspects of the ELISA technique, when applied in laboratory diagnostics of veterinary medicine are also presented.

Advantages and deficiency of different types of viral vaccines currently in use or in development in veterinary medicine are described. We emphasize the role of adjuvants in inactivated and genetically engineered vaccines as well as the role of marker vaccines in detection of infected animals in the herd and eradication of certain viral diseases.