Presence of Mycoplasma bovis in Bulk Tank Milk and Associated Risk Factor Analysis in Serbian Dairy Farms.

Mycoplasma bovis (M. bovis) is a significant pathogen responsible for highly transmissible mastitis in cattle globally. It primarily spreads through colostrum, milk, and semen. Cows with persistent infections act as carriers, intermittently releasing the pathogen, making their milk a pivotal factor in infection transmission. Given the limited seroprevalence surveys in Serbia, this study aimed to detect M. bovis presence in bulk tank milk (BTM), determine route shedding, and evaluate infection risks. BTM samples were collected from 115 dairy farms across Serbia, with M. bovis DNA detected in 11 out of the 115 samples by real-time PCR. Additionally, M. bovis was detected in 1.30% of nasal swabs sampled from apparently healthy animals. A univariate analysis of the risk factors associated with M. bovis presence in the BTM samples revealed correlations with factors such as the breed, farm seropositivity, pre-milking and post-milking disinfection practices, farm type, cow population, milk yield, number of cows in the BTM samples, and parity. Seropositive farms exhibited the highest likelihood of M. bovis presence in milk. Moreover, pre- and post-milking disinfection practices and highly productive cows yielding over 8000 L of milk were identified as risk factors for PCR-positive BTM. In a multivariable mixed regression analysis, a risk factor for the presence of M. bovis infection in the BTM sample was the Holstein breed. These findings underscore a relatively high prevalence of M. bovis in BTM within Serbian dairy farms, suggesting a potential risk for M. bovis spreading through milk and oral route of calves' infection.

Genetic analysis reveals multiple intergenic region and central variable region in the African swine fever virus variants circulating in Serbia.

This study provides the first comprehensive report on the molecular characteristics of African swine fever virus (ASFV) variants in Serbia between 2019 and 2022. Since its first observation in July 2019, the disease has been found in wild boar and domestic swine. The study involved the analysis of 95 ASFV-positive samples collected from 12 infected administrative districts in Serbia. Partial four genomic regions were genetically characterized, including B646L, E183L, B602L, and the intergenic region (IGR) between the I73R-I329L genes. The results of the study suggest that multiple ASFV strains belonging to genotype II are circulating in Serbia, as evidenced by the analysis of the IGR between I73R-I329L genes that showed the most differences. Furthermore, the phylogenetic analysis of the B602L gene showed three different clades within the CVR I group of ASFV strains. Regarding the IGR, 98.4% were grouped into IGR II, with only one positive sample grouped into the IGR III group. These findings provide essential insights into the molecular characteristics of ASFV variants in Serbia and contribute to the knowledge of circulating strains of ASFV in Europe. However, further research is necessary to gain a better understanding of ASFV spread and evolution.

First Report of Brucella Seroprevalence in Wild Boar Population in Serbia.

Brucellosis is one of the most important bacterial zoonotic diseases worldwide, characterized in domestic animals by long-term reproductive disorders. As known, wild boars (Sus scrofa) are natural hosts for Brucella suis biovar 2, in which the infection passes in inapparent form, increasing the pathogen transmission risk to domestic pigs, other domestic animals and humans. So far, no studies regarding brucellosis in wild boars in Serbia have been published. During the hunting season 2020/2021, 480 sera of wild boars living in Serbia were collected and tested for the presence of anti-Brucella antibodies. For the serological survey, the Rose Bengal Test (RBT) and competitive enzyme-linked immunosorbent assay (c-ELISA) were used. Of the 480 sera, 45 sera tested positive, indicating the acquired Brucella seroprevalence in wild boars of 9.4%. The greatest numbers of Brucella seropositive animals were detected in the eastern parts of the country and in one of the central districts, i.e., Pomoravski, Branicevski, Borski and Juznobanatski. This study provides the first data regarding brucellosis in the wild boar population in Serbia, revealing the seroprevalence of Brucella, thus indicating that wild boars as natural hosts and/or vectors of Brucella likely present a risk for the infection of other animals.

Frequency of SARS-CoV-2 infection in dogs and cats: Results of a retrospective serological survey in Sumadija District, Serbia.

It has long been known that coronaviruses cause various infectious diseases in animals. Although SARS-CoV-2 is genetically related to viruses isolated from Rhinolophus bats, the exact origin, mode of transmission, and how the human species has become the epidemiological reservoir of the virus have not yet been established with certainty. Although the main route of transmission is human-to-human, there are considerable numbers of reported cases of infection in animal species, predominantly among pet animals. The aim of this retrospective study was to assess SARS-CoV-2 seropositivity in dogs and cats during the COVID-19 pandemic in Sumadija District, Serbia. We used serology to identify household contacts of pet animals with infected pet owners and the degree of association. The study presented in this paper is also the first study of this type in Serbia. The results of a retrospective serosurvey, which was conducted in dogs and cats with different exposure risk factors, were analyzed to find the possible modes of transmission between humans and animals. The relative frequency of SARS-CoV-2 infection in dogs was 1.45% bounded with a 95% confidence interval (CI) of 0.0007-7.73%, while in cats, it was 5.56% (95% CI: 0.77-4.13%). The relative frequency of SARS-CoV-2 infection in pet owners was 11% (95% CI: 6.25-18.63%). In pets that were in close contact with COVID-19 positive owners, the seropositivity was found to be 9%. Out of a total of five stray dogs and cats tested, seropositivity was observed in two animals. Detected SARS-CoV-2 infection in pets shows that these animals are susceptible to infection and that the most common means of virus transmission to pets is through contact with diseased owners. However, the presence of infection in stray dogs and cats is not clear and needs further research.

Spatiotemporal Analysis of West Nile Virus Epidemic in South Banat District, Serbia, 2017-2019.

West Nile virus (WNV) is an arthropod-born pathogen, which is transmitted from wild birds through mosquitoes to humans and animals. At the end of the 20th century, the first West Nile fever (WNF) outbreaks among humans in urban environments in Eastern Europe and the United States were reported. The disease continued to spread to other parts of the continents. In Serbia, the largest number of WNV-infected people was recorded in 2018. This research used spatial statistics to identify clusters of WNV infection in humans and animals in South Banat County, Serbia. The occurrence of WNV infection and risk factors were analyzed using a negative binomial regression model. Our research indicated that climatic factors were the main determinant of WNV distribution and were predictors of endemicity. Precipitation and water levels of rivers had an important influence on mosquito abundance and affected the habitats of wild birds, which are important for maintaining the virus in nature. We found that the maximum temperature of the warmest part of the year and the annual temperature range; and hydrographic variables, e.g., the presence of rivers and water streams were the best environmental predictors of WNF outbreaks in South Banat County.

Simulation and prediction of spread of COVID-19 in The Republic of Serbia by SEAIHRDS model of disease transmission.

As a response to the pandemic caused by SARS-Cov-2 virus, on 15 March 2020, the Republic of Serbia introduced comprehensive anti-epidemic measures to curb COVID-19. After a slowdown in the epidemic, on 6 May 2020, the regulatory authorities decided to relax the implemented measures. However, the epidemiological situation soon worsened again. As of 7 February 2021, a total of 406,352 cases of SARSCov-2 infection have been reported in Serbia, 4,112 deaths caused by COVID-19. In order to better understand the epidemic dynamics and predict possible outcomes, we have developed an adaptive mathematical model SEAIHRDS (S-susceptible, E-exposed, A-asymptomatic, I-infected, H-hospitalized, R-recovered, d-dead due to COVID-19 infection, S-susceptible). The model can be used to simulate various scenarios of the implemented intervention measures and calculate possible epidemic outcomes, including the necessary hospital capacities. Considering promising results regarding the development of a vaccine against COVID-19, the model is extended to simulate vaccination among different population strata. The findings from various simulation scenarios have shown that, with implementation of strict measures of contact reduction, it is possible to control COVID-19 and reduce number of deaths. The findings also show that limiting effective contacts within the most susceptible population strata merits a special attention. However, the findings also show that the disease has a potential to remain in the population for a long time, likely with a seasonal pattern. If a vaccine, with efficacy equal or higher than 65%, becomes available it could help to significantly slow down or completely stop circulation of the virus in human population. The effects of vaccination depend primarily on: 1. Efficacy of available vaccine(s), 2. Prioritization of the population categories for vaccination, and 3. Overall vaccination coverage of the population, assuming that the vaccine(s) develop solid immunity in vaccinated individuals. With expected basic reproduction number of Ro=2.46 and vaccine efficacy of 68%, an 87% coverage would be sufficient to stop the virus circulation.

Suitability of individual and bulk milk samples to investigate the humoral immune response to lumpy skin disease vaccination by ELISA.

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.

Twenty-five-year study of Nosema spp. in honey bees (Apis mellifera) in Serbia.

A total of 7386 samples of adult honey bees from different areas of Serbia (fifteen regions and 79 municipalities) were selected for light microscopy analysis for Nosema species during 1992-2017. A selection of honey bee samples from colonies positive for microsporidian spores during 2009-2011, 2015 and 2017 were then subjected to molecular diagnosis by multiplex PCR using specific primers for a region of the 16S rRNA gene of Nosema species. The prevalence of microsporidian spore-positive bee colonies ranged between 14.4% in 2013 and 65.4% in 1992. PCR results show that Nosema ceranae is not the only Nosema species to infect honey bees in Serbia. Mixed N. apis/N. ceranae infections were detected in the two honey bee samples examined by mPCR during 2017. The beekeeping management of disease prevention, such as replacement of combs and queens and hygienic handling of colonies are useful in the prevention of Nosema infection.

Humoral immune response to repeated lumpy skin disease virus vaccination and performance of serological tests.

BACKGROUND: 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. RESULTS: 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 SeELISA 91% and SpELISA 87% calculated by the results of VNT and SeELISA 88% and SpELISA 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. CONCLUSION: Of all tests used the commercially available ELISA shows to be the most useful for high throughput analysis compared to VNT or IFAT.

Molecular detection of black queen cell virus and Kashmir bee virus in honey.

Considering the intensive trading nowadays, the honey from the local market was tested for the presence of the six most common bee viruses. To prove the suitability of honey as a sample for the bee viruses detection, the set of different sample types taken directly from the hives we comparatively tested. The study included 30 samples of domestic and 5 samples of imported honey. Additionally, we tested 40 sets of samples including live bees, dead bees, and the honey taken from four apiaries for the evaluation of honey suitability for the virus detection, Two out of the six most common bee viruses were detected in the samples of honey from the market. Black queen cell virus (BQCV) genome was found in 24 domestic honey samples and Kashmir bee virus (KBV) genome was detected in one sample of imported honey. The nucleotide sequences of 24 BQCV isolates showed the highest identity (86.4%) with strains from Europe at the polyprotein gene, whilst the Serbian isolates between each other showed 98.5% similarity. By comparative testing of the different type of samples, in three out of four apiaries BQCV genome was detected in both bees and honey. Evaluating the suitability of honey for the detection of the viral disease by simultaneous testing of live, dead bees, and honey from the same hive, it was shown that the honey can be successfully used for the detection of BQCV. Since, as of yet, there has been no evidence of KBV circulation in Serbia, after its detection in imported honey, there is a substantial risk of its introduction and consequently the need for its surveillance. Therefore, the programs of bee diseases screening should be included in the regular control procedures for the international trade. In addition to this benefit, honey gives an opportunity to beekeepers for continuous monitoring of bees' health status.