In the presented study we investigated the development of the humoral immune response against LSDV during the process of re-vaccination of cattle over a time span of 5 months. In addition, the performance of different serological techniques for antibody detection against LSDV was compared. For sample collection, an area without previous LSD outbreak reports in Serbia was selected. Seventy-nine cattle from twenty farms vaccinated in 2016 and re-vaccinated in 2017 were included in the study. Two farms from the same area with good calving management were selected for investigation of passive LSDV antibody transfer from vaccinated mothers to new-borne calves. All investigated cattle were healthy on the day of vaccination and during the whole study. Swelling at the injection site or other side effects of vaccination did not occur after re-vaccination in the study. Detection of LSD-specific antibodies was performed with the standard serological methods VNT and IFAT as well as a commercially available Capripox double antigen multi-species-ELISA. Capripoxvirus-specific antibodies were detected 46 to 47 weeks after vaccination in 2016, with VNT in 35.06% and with IFAT and ELISA in 33.77%. A secondary response was observed in all three tests 1 month after re-vaccination with a significant increase in seropositive animals compared to the results before re-vaccination. With all applied serological methods, the number of animals testing positive was significantly higher at 1 and 5 months post re-vaccination than before re-vaccination. No significant statistical difference (p > 0.05) was observed between the results of all three tests used. The sensitivity and specificity of ELISA was estimated to be Se_ELISA 91% and Sp_ELISA 87% calculated by the results of VNT and Se_ELISA 88% and Sp_ELISA 76% calculated by the results of IFAT. Passive antibody transfer from vaccinated mothers to new-born calves was investigated at 14 days after birth. Discrepancies for the detection of LSDV specific antibodies between cows and newborn calves at the age of 14 days were observed in VNT and IFAT, but not in ELISA. Of all tests used the commercially available ELISA shows to be the most useful for high throughput analysis compared to VNT or IFAT.

Aujeszky’s disease is a viral disease of suids caused by Suid Herpesvirus 1. The disease has worldwide distribution with significant economic impact. In Serbia, there is neither an Aujeszky’s disease eradication nor national vaccination programme of domestic pigs. Since clinical symptoms of Aujeszky’s disease are not specific, it is important to establish a link between clinical signs and presence of ADV active infection in wild boars. The aim of this study was to investigate the possibility of active infection within wild boar showing signs of ADV and also to examine relationship between isolates from domestic pigs and wild boar. Having in mind that virus has not been previously isolated from wild boars in Serbia, we report the first isolation of Suid Herpesvirus 1 from this species in Serbia. Tissue and serum samples from 40 wild boars from eastern Serbia were examined for evidence of Aujeszky’s disease (AD). Suid Herpesvirus 1 (SHV1), the cause of AD was isolated on PK15 cell line from three tissue samples, inducing cytopathic effect (CPE) with syncytia forming, and viral genome was detected by polymerase chain reaction (PCR) in eight samples. Genetic analysis of us4, us9 and ul49.5 partial sequences showed high homology between ADV isolates from wild boars and between isolates from wild boars and domestic animals. Neutralizing antibodies were not detected by virus neutralisation test (VNT) in sera from four out of eight PCR positive wild boars suggesting recent infection in those animals. This is the first demonstration of Aujeszky’s disease virus (ADV) in the wild boar population in Serbia although seroconversion has been detected previously.

Until July 30 ^th , 2019 when the first case of African swine fever (ASF) was confirmed, Serbia was a country free from ASF. After the owner reported atypical illness and death of a sow, the local veterinarian submitted the organ samples to the National Reference Laboratory for Classical Swine Fever (CSF) and African Swine Fever within the Institute of Veterinary Medicine of Serbia in Belgrade. Observed gross lesions included splenomegaly, serous edema of the wall of the gallbladder and hemorrhages in the enlarged portal lymph nodes, petechial hemorrhages on the kidney and epicardium, and petechial and echymotic hemorrhages on the mucosa of the urinary bladder. Results of real-time PCR confirmed that the cause of illness and death of the swine was African swine fever virus. The samples were sent for confirmation to the EU Reference Laboratory where it was confirmed that Serbian domestic pig virus isolates based on p72 belong to genotype II. In total, 270 pigs from 18 affected holdings were killed in the infected zones. According to the on-record data, mortality was 6.89%, whereas lethality reached 64.5%. Currently, an extensive surveillance program is being conducted, aiming to force passive surveillance. ASF in wild boar has not been confirmed so far.

Background: The detection of antibodies against capripoxvirus has become easier with a commercially available ELISA validated for serum and plasma. In order to explore its suitability for immunological investigations on alternative samples, this study targeted milk as sample matrix available through non-invasive sampling. Methods: Samples for this study were collected from dairy cows vaccinated against LSD in an area without reported LSD virus circulation. Paired serum and milk (individual and bulk) samples were tested by ELISA without and with modifications of the sample incubation time for the milk samples. For the evaluation of the test specificity, 352 milk samples from a milk repository in Germany were used as negative control. Receiver operating characteristic analysis was performed for determination of the Youden index and determination of the most suitable cut-off value for maximum specificity. Results: From 154 analyzed serum samples from Serbia, 75 were detected as positive in the ELISA. Sensitivity and specificity of the ELISA test for milk samples reached values of 88 to 91% using Youden criteria. A cut-off of 10 was determined aiming for maximum specificity. This cut-off value was used for further analysis. Using the protocol for serum, out of 154 milk samples, 38 were detected as positive, number of positive detected milk samples increase up to 48 with modified protocol. Milk samples from Germany reacted negative, except two samples that had borderline results using modified protocol. Significant statistical difference (p < 0.05) was observed between two incubation protocols. The detection of LSD-specific antibodies from bulk milk samples (pools of 2–10 individuals) came along with a reduced sensitivity over the sample of individual animals. Conclusions: Results show that the detection of capripoxvirus specific antibodies in milk samples using the commercially available ELISA from IDvet is feasible and can represent a helpful tool for LSDV monitoring programs.