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Infectious laryngotracheitis (ILT) is a WOAH-listed respiratory disease in poultry caused by Gallid alphaherpesvirus 1, known as ILT virus (ILTV). We monitored two unvaccinated commercial layer flocks of 46- and 64-weeks old birds, more than 3 months after the onset of ILT. For this purpose, tracheal swabs, cloacal swabs, and blood samples were collected. Molecular and serology results were compared with the mortality data. The increased mortality in flocks 1 and 2 lasted 9 and 15 days, reaching 13.0% and 11.3%, respectively. We isolated the virus by inoculation on chicken embryo’s chorioallantoic membrane. Tracheal swabs were positive at each sampling point, but cloacal swabs were negative. Based on the molecular and phylogenetic analysis of the ICP4 gene, the ILTV closely matched vaccine strains. In flock 1, seroconversion was evident at the second sampling (day 15). Thereafter, an increase in antibody titer was observed, eventually achieving levels that were nearly identical to those on day 15 and on 109. During the acute period of the outbreak, seroconversion was already visible in flock 2, and a similar pattern was then seen as in flock 1. Three months after the outbreak, the virus DNA was still persistently detected in tracheal swabs.

Mycoplasma hyopneumoniae (Mhyo) is primary pathogen involved in porcine respiratory diseases complex (PRDC). The objective of this study was to evaluate Mycoplasma hyopneumoniae (Mhyo) infection in five commercial pig farms by using serology and lung lesion scoring at slaughter. Five Macedonian pig herds with a history of respiratory diseases were included in the study. Blood samples were taken from pigs at 6, 10, 14, 18, and 22 weeks of age. Ten animals per age group from each farm were sampled and tested for antibodies to Mhyo. At abattoir, 50 lungs per farm were scored for lesions associated with enzootic pneumonia (EP). All farms were seropositive to Mhyo. Higher seroprevalence to Mhyo was observed in grower and finisher pigs, while significant difference (p10) were found among some of the farms (p

β-lactamases are a diverse class of enzymes produced by bacteria that present a major cause for resistance to β-lactams. In this study we analysed 159 fecal samples from dairy cows, for the presence of presumptive ESBL, AmpC, and carbapenemase-producing E. coli. Phylotyping was done using Clermont phylo-typing method, targeting arpA, ChuA, and YjaA genes, along with the DNA fragment TspE4.C2. Convetional PCR method was used to confirm the presence of bla genes among 39 phenotypically confirmed ESBL producing E. coli. The results showed presence of CTX-M, SHV, TEM and OXA1 bla genes in 28 (71.79%), 1 (2.56%), 29 (74.35%), 2 (5.12%) of isolates, respectively Twenty (51.28%) isolates showed presence of both blaCTX-M and TEM genes. The strain that carried the blaSHV gene was found to carry blaTEM gene as well, while one of the strains that carried blaOXA1 gene was also carrying blaCTX-M and TEM gene. The ration between isolates and phylo-groups was as follows: 9 (23.07%) strains were assigned to phyllo-group D; 14 (35.89%) to phyllo-group B; 16 (41.02%) to phyllo-group A. Out of the 39 strains where bla genes were identified, 29 (74.35%) were categorized as multi drug resistant.

In this study, we report the first outbreak of highly pathogenic avian influenza (HPAI) A H5N8, clade 2.3.4.4b in Kosovo on 19 May 2021. The outbreak consisted of three phases: May–June 2021, September–November 2021, and January–May 2022. In total, 32 backyards and 10 commercial holdings tested positive for the virus. Interestingly, the third and last phase of the outbreak coincided with the massive H5N1 clade 2.3.4.4b epidemic in Europe. Phylogenetic analyses of 28 viral strains from Kosovo revealed that they were closely related to the H5N8 clade 2.3.4.4.b viruses that had been circulating in Albania, Bulgaria, Croatia, Hungary, and Russia in early 2021. Whole genome sequencing of the 25 and partial sequencing of three H5N8 viruses from Kosovo showed high nucleotide identity, forming a distinctive cluster and suggesting a single introduction. The results of the network analysis were in accordance with the three epidemic waves and suggested that the viral diffusion could have been caused by secondary spreads among farms and/or different introductions of the same virus from wild birds. The persistent circulation of the same virus over a one-year period highlights the potential risk of the virus becoming endemic, especially in settings with non-adequate biosecurity.

Poultry red mite (PRM), Dermanyssus gallinae, causes egg drop production, anemia and can be a vector in transmitting diseases. The PRM control mainly focuses on usage of the conventional chemical biocides. The objective of this study was to analyze the farmers’ perception regarding the impact, management and control of PRM in Macedonian layer farms. The data were collected with direct on-site visits using a unified questionnaire. In total, 29 poultry farms (28% of farms in the country), all with conventional cages, were part of this study. The data were analyzed by descriptive statistics, inferential statistics and Naïve Bayes Classifier technique. In 30% of the poultry farms the farmers had observed that the flock was infested with PRM. In total, 32 different treatments against PRM were reported from the farmers, and three of them were non-biocide treatments. The most used biocides (17% of the farms) were crude oil, Formalin, Neopitroid® and disinfectants. The highest agreement regarding biocides application among the farms (38%) was before the production starts. Most of the farmers applied biocides routinely, before the infestation is evident (75%). The median costs for PRM treatment were 175€ per flock, higher in the infested farms 493±677€ compared to non - infested 100±71€, p

Avian avulavirus type 1 (AAvV-1) belongs to the family Paramyxoviridae, genus Avulavirus. Even though belonging to a single serotype, there is great genetic variability between these viruses. On the basis of the available complete fusion (F) gene and whole genome sequences and using the genotype classification system, AAvV-1 viruses are divided in two major groups: class I and class II. Class I viruses are predominantly avirulent viruses and majority of the isolations come from waterfowl and domestic poultry from live bird markets in USA and China although isolations from wild birds are reported globally. In our study we used classical, molecular and phylogenetic tools to characterize an AAvV-1 isolated from a mute swan in Macedonia. Based on the complete F gene sequence, we have concluded that the virus designated as AAvV-1/mute swan/Macedonia/546/2012 (KP123431) belongs to the class I of AAvV-1 with an avirulent cleavage site motif 112ERQER*L117. The virus could not be assigned to any of the four currently existing subgenotypes (1a, 1b, 1c or 1d) of the single genotype 1 of class I viruses. Two distant viruses, isolated from goose in Alaska in 1991 and from goose in Ohio in 1987, shared the highest nucleotide sequence identity of the complete F gene with the isolate in our study: 92.7% and 92.8%, respectively. This is the first report of isolation of class I AAvV-1 in Southeastern Europe. The asymptomatic nature and the underreporting of sequences from the class I viruses impede the understanding of the molecular epidemiology and evolution of this group of viruses.

The highly virulent Newcastle disease virus (NDV) isolates typically result in severe systemic pathological changes and high mortality in Newcastle disease (ND) illness, whereas avirulent or low-virulence NDV strains can cause subclinical disease with no morbidity and even asymptomatic infections in birds. However, understanding the host’s innate immune responses to infection with either a highly virulent strain or an avirulent strain, and how this response may contribute to severe pathological damages and even mortality upon infection with the highly virulent strain, remain limited. Therefore, the differences in epigenetic and pathogenesis mechanisms between the highly virulent and avirulent strains were explored, by transcriptional profiling of chicken embryonic visceral tissues (CEVT), infected with either the highly virulent NA-1 strain or the avirulent vaccine LaSota strain using RNA-seq. In our current paper, severe systemic pathological changes and high mortality were only observed in chicken embryos infected with the highly virulent NA-1 strains, although the propagation of viruses exhibited no differences between NA-1 and LaSota. Furthermore, virulent NA-1 infection caused intense innate immune responses and severe metabolic disorders in chicken EVT at 36 h post-infection (hpi), instead of 24 hpi, based on the bioinformatics analysis results for the differentially expressed genes (DEGs) between NA-1 and LaSota groups. Notably, an acute hyperinflammatory response, characterized by upregulated inflammatory cytokines, an uncontrolled host immune defense with dysregulated innate immune response-related signaling pathways, as well as severe metabolic disorders with the reorganization of host–cell metabolism were involved in the host defense response to the CEVT infected with the highly virulent NA-1 strain compared to the avirulent vaccine LaSota strain. Taken together, these results indicate that not only the host’s uncontrolled immune response itself, but also the metabolic disorders with viruses hijacking host cell metabolism, may contribute to the pathogenesis of the highly virulent strain in ovo.

Bovine tuberculosis is a chronic infectious disease in cattle caused mainly by Mycobacterium bovis and to a lesser extent by Mycobacterium caprae. The other members of the Mycobacterium tuberculosis complex (MTBC) can also cause the disease in domestic and wild animals and all of them have a zoonotic potential. The main purpose of the study was to determine the presence and distribution of the tuberculous lesions in reactor cattle, and to isolate and identify the causative agents of bovine tuberculosis in the Republic of Macedonia. Lymph nodes and affected organs from 188 reactor cattle slaughtered due to a positive intradermal comparative cervical tuberculin test were analyzed by detection of tuberculous lesions, followed by isolation and molecular identification of the isolated mycobacteria. The isolation was performed on selective media – Lowenstein Jensen with glycerol, Lowenstein Jensen without glycerol and Stonebrink medium supplemented with pyruvate. The molecular identification of the MTBC members was performed by analysis of the Regions of difference (RD1, RD9 and RD4) and detection of single nucleotide polymorphisms in the lepA gene for Mycobacterium caprae. Typical tuberculous lesions were detected in 62 animals (33.0%) and the lesions were most prevalent in the mediastinal lymph nodes (47.5%). The isolated mycobacteria in the MTBC were identified as Mycobacterium bovis and Mycobacterium caprae and were found in both animals with visible lesions (82.2%) and animals without visible lesions (27.7%). The slaughterhouse postmortem examinations and laboratory investigations should be included on regular bases in order to improve the National eradication program.

Bacteria from the genus Brucella are causative agents of brucellosis – a zoonotic disease which affects many wild and domestic animal species and humans. Taking into account the significant socio-economic and public health impact of brucellosis, its control is of great importance for endemic areas. The chosen control strategy could be successful only if adapted to the current epidemiological situation. This implies that a choice of appropriate diagnostic procedures for detection and typing of Brucella spp. strains are of essential importance. Significant advancement of molecular techniques and their advantages compared to classical methods, give strong arguments in promotion of these techniques as a powerful tool for comprehensive diagnostics of brucellosis. Considering this, the major tasks of the study were to select and implement molecular tests for detection and genotyping Brucella spp. and evaluate their performances using DNA from cultivated brucellae (islolates) and limited number of tissue samples from seropositive animals. The obtained results confirmed that implemented real time PCR for Brucella spp. detection, as well as MLVA-16 used for genotyping, have excellent analytical sensitivity (4.2 fg of Brucella DNA were successfully detected and genotyped). Furthermore, compared to bacteriological cultivation of Brucella spp., real time PCR and MLVA-16 protocols showed superior diagnostic sensitivity and detected Brucella DNA in tissues from which Brucella could not be cultivated. Based on the summarized study results, we propose a diagnostic algorithm for detection and genotyping of Brucella spp. bacteria. Routine use of proposed diagnostic algorithm will improve the effectiveness of infection confirmation and help for accurate evaluation of epidemiological situation.