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Canine monocytic ehrlichiosis (CME) is a widespread, tick-borne, canine disease, caused by an obligate intracellular bacterium, Ehrlichia canis. The main vector, a brown-dog tick, Rhipicephalus sanguineus, is widely distributed, especially in areas with tropic, subtropic, or Mediterranean climates (Central and South America, Eastern and Western Asia, Africa, Australia and Southern Europe). The study performed in 2012, by Stefanovska et al., determined a seroprevalence of 18.7% of E. canis among the Macedonian dog population. Up to date, the presence of E. canis, using molecular diagnostic methods, has not been investigated in Macedonia. Therefore, this study aimed to confirm the presence of E. canis, in the pet-dog population on the territory of the city of Skopje, North Macedonia, using a highly sensitive multiplex Real-Time PCR method (qPCR). Whole blood samples from 80 dogs of different breeds and ages, with clinical symptoms of CME and positive serology result for the presence of antibodies against E.canis, were collected for analyses. Out of 80 dogs, 36 (45%) were found as positive. The present work reports the first molecular detection of E. canis in pet dogs on the territory of the city of Skopje, Macedonia.

Infectious hematopoietic necrosis (IHN) is a common disease in the intensive production of salmonids. The IHN virus (IHNV) was isolated for the first time in North Macedonia in 2018. This study aimed to investigate the distribution and genotype of IHN in farmed rainbow trout and autochthonous salmonid fish in North Macedonia following the first detection. The samples were collected from 47 trout farms. Trout fry with or without clinical signs of IHN were selected as individual samples. Kidney, spleen, and heart were taken from each fish during the dissection. Three pooled samples were collected from each farm. A total of 141 pooled samples were collected: Rainbow trout (Oncorhynchus mykiss) n=127, Macedonian trout (Salmo macedonicus) n=11, and Ohrid trout (Salmo letnica) n=3. The virus was detected in 43 samples (30.50%): rainbow trout (n=40), Macedonian trout (n=2), and Ohrid trout (n=1). There were 18 (38.30%) positive fish farms. The MAKIHNV1 isolate from 2018 (MN641902) and the newly isolated virus shared a similarity of >99 and were placed in clade E-1 of European genogroup E. The IHN has spread throughout the country and is also present in the autochthonous salmonids. [ABSTRACT FROM AUTHOR]

The genus Bartonella are fastidious, Gram-negative intracellular vector-borne bacteria with zoonotic potential. Cats have a crucial epidemiological role in the transmission of Bartonella henselae, the primary causal agent of cat-scratch disease in humans. Here we describe the first report of B. henselae infection in a cat in North Macedonia after a suspected Bartonella infection in a 7-year-old boy. A combination of cultural and molecular methods for detecting B. henselae in the asymptomatic cat was used. A blood sample was cultured on a blood agar plate, where colonies became visible after nine days of incubation. The DNA of a single colony was extracted, and the isolate was confirmed as B. henselae by sequencing the bacterial 16S ribosomal RNA gene and performing a Bartonella - specific PCR. The phylogenetic analysis demonstrated 100% similarity with B. henselae sequences, clustering on a single branch and establishing a distinct group. This finding indicates that B. henselae infection in cats circulates in North Macedonia and may affect feline and human health. [ABSTRACT FROM AUTHOR]

Aedes albopictus is an invasive mosquito species spreading throughout Europe and its presence in North Macedonia was recorded in 2016. Following the first detection in September 2018, we conducted a two-week mosquito monitoring by ovitraps in order to determine if there were established populations of Aedes albopictus in Skopje, the capital of North Macedonia. Ninety-four Ae. albopictus eggs (0 to 18 eggs per ovitrap per week) were collected from 7 (14%) ovitraps in 3 (30%) municipalities. Thirty-eight eggs (40.4%) successfully hatched and the adult mosquitoes were identified by morphology and PCR. No other potentially invasive species were identified during the monitoring period. Ae. albopictus distribution is expanding and poses a risk for an Aedes-borne disease transmission in North Macedonia. The available data highlight the need for a regular monitoring for tiger mosquitoes to plan adequate control measures. [ABSTRACT FROM AUTHOR]

Ehrlichia canis, the etiologic agent of canine monocytic ehrlichiosis (CME), is mainly transmitted by the brown dog tick Rhipicephalus sanguineus. Clinical signs of the disease can be various, depending on the stage of the disease. Typical changes in hematological and blood biochemical parameters are: severe thrombocytopenia, mild to marked non regenerative anaemia and hypoalbuminemia. In order to present the effects of the treatment protocol on several hematological and biochemistry parameters, 34 Ehrlichia canis positive dogs were compared before and after treatment with doxycycline 10mg/kg/day, in duration of four weeks. Besides the clinical sings and laboratory findings, diagnosis was confirmed by antibody tests (Bionote, Korea, AGROLABO S.p.A., Italy). The most common clinical sings were depression, lethargy, pyrexia, vomiting and anorexia. Hematological analyses were performed on the automatic hematology analyzer Exigo EsoVet (Sweden), while biochemistry analyses (alanine aminotransferase, aspartate aminotransferase, urea, creatinine, albumin, total protein, globulin and alkaline phosphatase) were performed using the automatic analyzer ChemWell 2910 (Awareness Technology, INC, USA). Statistically significant difference (p<0.05) in hematology changes was present regarding the red blood cells count, platelet count, hematocrit and hemoglobin before and after treatment. Hypoalbuminaemia (Mean 19.21 ±4.96 g/l) was the only serum biochemistry parameter with significant change before and after treatment, as well. Treatment with doxycycline in patients with E.canis resulted in significant increase of hematology parameters (red blood cells, hemoglobin, haematocrit and platelets), as well as albumins in serum. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The study investigated the antimicrobial activity of 13 enterococcal strains (E. faecalis -8, E. faecium-2, E. hirae-2, E. spp.-1) isolated from our traditional cheeses against pathogen microorganisms. Also, it includes the detection of the following enterocin structural genes: enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin Q, enterocin EJ97 and cytolysin by using PCR method. All isolates inhibited growth of L. monocytogenes and L.innocua. One isolate had a broader antimicrobial activity. None of the isolates showed inhibitory activity against S. enteritidis, E. coli and Y. enterocolitica. The genes enterocin P, cytolysin and enterocin A were the most frequently detected structural genes among the PCR positive strains. No amplification was obtained in two strains E. faecalis-25 and E. faecalis-86. Three different genes were identified in some strains. With the exclusion of strains possessing a virulence factor, such as cytolysin, producers of more than one enterocins could be of a great technological potential as protective cultures in the cheese industry. [ABSTRACT FROM AUTHOR]

Aim of this study was to characterize pigeon variant of Newcastle disease virus (NDV) isolated from backyard poultry using classical and molecular methods. In standard hemagglutination inhibition (HI) test both polyclonal NDV antiserum and monoclonal antibodies 161/617 specific for pigeon variants of NDV showed inhibition of heamagglutination of the isolated virus. Intracerebral pathogenicity index (ICPI) has shown that the isolate is mesogenic virus (ICPI = 0.81). One-step RT-qPCR for detection of M gene was performed indicating a presence of NDV and RT-qPCR for discrimination between lentogenic and velogenic strains based on F gene was also performed indicating a presence of virulent NDV. A portion of the F gene was amplified and sequenced for determination of virulence and phylogenetic characterization. The F protein cleavage site sequence of the isolate had multiple basic amino acids at residues 112-116 and a phenyl alanine at residue 117 (112RRQKR*F117) which is typical for velogenic strains. The nucleotide sequence of 374 bp was aligned to begin at nt 47 and finish at 420 immediately after the cleavage site and compared with other reference strains from the region and worldwide. In the phylogenetic tree, the isolate clustered into genotype VIb, typical for PPMV-1. This strain is phylogenetically very similar to other PPMV-1 isolated from pigeons in Macedonia. Poultry infected with PPMV-1 can spread the virus in the absence of clinical signs, thus PPMV-1's are constant threat to domestic poultry. This is the first report of evidenced spillover of PPMV-1 into poultry in Macedonia. [ABSTRACT FROM AUTHOR]

Canine monocytic ehrlichiosis (CME) is a widespread, tick-borne, canine disease, caused by an obligate intracellular bacterium, Ehrlichia canis. The main vector, a brown-dog tick, Rhipicephalus sanguineus, is widely distributed, especially in areas with tropic, subtropic, or Mediterranean climates (Central and South America, Eastern and Western Asia, Africa, Australia and Southern Europe). The study performed in 2012, by Stefanovska et al., determined a seroprevalence of 18.7% of E. canis among the Macedonian dog population. Up to date, the presence of E. canis, using molecular diagnostic methods, has not been investigated in Macedonia. Therefore, this study aimed to confirm the presence of E. canis, in the pet-dog population on the territory of the city of Skopje, North Macedonia, using a highly sensitive multiplex Real-Time PCR method (qPCR). Whole blood samples from 80 dogs of different breeds and ages, with clinical symptoms of CME and positive serology result for the presence of antibodies against E.canis, were collected for analyses. Out of 80 dogs, 36 (45%) were found as positive. The present work reports the first molecular detection of E. canis in pet dogs on the territory of the city of Skopje, Macedonia. [ABSTRACT FROM AUTHOR]