Molekularna detekcija ehrlichia canis u populaciji pasa -kućnih ljubimaca u Severnoj Makedoniji

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.Monocitna erlihioza pasa (CME) je široko rasprostranjena bolest pasa, koja se prenosi krpeljima, a uzrokuje je obligatno-intracelularna bakterija Ehrlichia canis. Glavni vektor, smeđi krpelj pasa, Rhipicephalus sanguineus, široko je rasprostranjen, posebno u oblastima sa tropskom, subtropskom ili mediteranskom klimom (Centralna i Južna Amerika, Istočna i Zapadna Azija, Afrika, Australija i Južna Evropa). U Studiji izvedenoj 2012. godine, Stefanovska i saradnici su utvrdili da među severnomakedonskom populacijom pasa, seroprevalencija E. canis iznosi 18,7%. Do danas, prisustvo E. canis, koristeć i molekularne dijagnostičke metode, nije istraženo u Severnoj Makedoniji. Stoga je ova studija imala za cilj da potvrdi prisustvo E. canis u populaciji kuć nih ljubimaca na teritoriji grada Skoplja u Severnoj Makedoniji, koristeć i visoko osetljivu multiplex Real-Time PCR (qPCR). Za analize su prikupljeni uzorci pune krvi od 80 pasa različitih rasa i uzrasta, sa kliničkim simptomima CME i pozitivnim serološkim rezultatom na prisustvo antitela protiv E.canis. Od 80 pasa, 36 (45%) je ocenjeno kao pozitivno. Ovaj rad izveštava o prvom molekularnom otkrivanju E. canis kod pasa kuć nih ljubimaca na teritoriji grada Skoplja, Severna Makedonija

External quality assessment of Rift Valley fever diagnosis in 17 veterinary laboratories of the Mediterranean and Black Sea regions

Rift Valley fever (RVF) is an arboviral zoonosis that primarily affects ruminants but can also cause illness in humans. The increasing impact of RVF in Africa and Middle East and the risk of expansion to other areas such as Europe, where competent mosquitos are already established, require the implementation of efficient surveillance programs in animal populations. For that, it is pivotal to regularly assess the performance of existing diagnostic tests and to evaluate the capacity of veterinary labs of endemic and non-endemic countries to detect the infection in an accurate and timely manner. In this context, the animal virology network of the MediLabSecure project organized between October 2016 and March 2017 an external quality assessment (EQA) to evaluate the RVF diagnostic capacities of beneficiary veterinary labs. This EQA was conceived as the last step of a training curriculum that included 2 diagnostic workshops that were organized by INIA-CISA (Spain) in 2015 and 2016. Seventeen veterinary diagnostic labs from 17 countries in the Mediterranean and Black Sea regions participated in this EQA. The exercise consisted of two panels of samples for molecular and serological detection of the virus. The laboratories were also provided with positive controls and all the kits and reagents necessary to perform the recommended diagnostic techniques. All the labs were able to apply the different protocols and to provide the results on time. The performance was good in the molecular panel with 70.6% of participants reporting 100% correct results, and excellent in the serological panel with 100% correct results reported by 94.1% of the labs. This EQA provided a good overview of the RVFV diagnostic capacities of the involved labs and demonstrated that most of them were able to correctly identify the virus genome and antibodies in different animal samples

A multi gene-approach genotyping method identifies 24 genetic clusters within the genotype II-European African swine fever viruses circulating from 2007 to 2022

IntroductionAfrican swine fever (ASF) is a contagious viral disease of pigs and wild boar that poses a major threat to the global swine industry. The genotype II African swine fever virus (ASFV) entered the European Union (EU) in 2014 and since then fourteen countries have been affected, Italy and North Macedonia being the last in 2022. While whole genome sequencing remains the gold standard for the identification of new genetic markers, sequencing of multiple loci with significant variations could be used as a rapid and cost-effective alternative to track outbreaks and study disease evolution in endemic areas.Materials and methodsTo further our understanding of the epidemiology and spread of ASFV in Europe, 382 isolates collected during 2007 to 2022 were sequenced. The study was initially performed by sequencing the central variable region (CVR), the intergenic region (IGR) between the I73R and I329L genes and the O174L and K145R genes. For further discrimination, two new PCRs were designed to amplify the IGR between the 9R and 10R genes of the multigene family 505 (MGF505) and the IGR between the I329L and I215L genes. The sequences obtained were compared with genotype II isolates from Europe and Asia.ResultsThe combination of the results obtained by sequencing these variable regions allowed to differentiate the European II-ASFV genotypes into 24 different groups. In addition, the SNP identified in the IGR I329L-I215L region, not previously described, grouped the viruses from North Macedonia that caused the 2022 outbreaks with viruses from Romania, Bulgaria, Serbia and Greece, differentiating from other genotype II isolates present in Europe and Asia. Furthermore, tandem repeat sequence (TRS) within the 9R-10R genes of the multigene family 505 (MGF505) revealed eight different variants circulating.DiscussionThese findings describe a new multi-gene approach sequencing method that can be used in routine genotyping to determine the origin of new introductions in ASF-free areas and track infection dynamics in endemic areas

Multiplex Assay for Simultaneous Detection of Antibodies against Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein and Glycoproteins in Ruminants.

peer reviewedCrimean-Congo hemorrhagic fever virus (CCHFV) is a widespread tick-borne zoonotic virus that causes Crimean-Congo hemorrhagic fever (CCHF). CCHF is asymptomatic in infected animals but can develop into severe illness in humans, with high case-fatality rates. Due to complex environmental and socio-economic factors, the distribution of CCHFV vectors is changing, leading to disease occurrence in previously unaffected countries. Neither an effective treatment nor a vaccine has been developed against CCHFV; thus, surveillance programs are essential to limit and control the spread of the virus. Furthermore, the WHO highlighted the need of assays that can cover a range of CCHFV antigenic targets, DIVA (differentiating infected from vaccinated animals) assays, or assays for future vaccine evaluation. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies in ruminants to three recombinantly produced CCHFV proteins: the nucleocapsid (N) protein and two glycoproteins, GN ectodomain (GNe), and GP38. This triplex assay was used to assess the antibody response in naturally infected animals. Out of the 29 positive field sera to the N protein, 40% showed antibodies against GNe or GP38, with 11 out of these 12 samples being positive to both glycoproteins. To determine the diagnostic specificity of the test, a total of 147 sera from Spanish farms free of CCHFV were included in the study. This multiplex assay could be useful to detect antibodies to different proteins of CCHFV as vaccine target candidates and to study the immune response to CCHFV in infected animals and for surveillance programs to prevent the further spread of the virus. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever, which is one of the most important tick-borne viral diseases of humans and has recently been found in previously unaffected countries such as Spain. The disease is asymptomatic in infected animals but can develop into severe illness in humans. As neither an effective treatment nor a vaccine has been developed against CCHFV, surveillance programs are essential to limit and control the spread of the virus. In this study, a multiplex assay detecting antibodies against different CCHFV antigens in a single sample and independent of the ruminant species has been developed. This assay could be very useful in surveillance studies, to control the spread of CCHFV and prevent future outbreaks, and to better understand the immune response induced by CCHFV

Bovine tuberculosis in the Republic of Macedonia: postmortem, microbiological and molecular study in slaughtered reactor cattle

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

Application of fluorescence based molecular assays for improved detection and typing of Brucella strains in clinical samples

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

Distribution and Genotyping of Infectious Hematopoietic Necrosis Virus in Farmed Rainbow Trout and Autochthonous Salmonids in North 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

Development of a multiplex assay for antibody detection in serum against pathogens affecting ruminants.

peer reviewedNumerous infectious diseases impacting livestock impose an important economic burden and in some cases also represent a threat to humans and are classified as zoonoses. Some zoonotic diseases are transmitted by vectors and, due to complex environmental and socio-economic factors, the distribution of many of these pathogens is changing, with increasing numbers being found in previously unaffected countries. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies to five important pathogens of livestock (three of them zoonotic) that are currently emerging in new geographical locations: Rift Valley fever virus (RVFV), Crimean-Congo haemorrhagic fever virus (CCHFV), Schmallenberg virus (SBV), Bluetongue virus (BTV) and the bacteria complex Mycobacterium tuberculosis. Using the Luminex platform, polystyrene microspheres were coated with recombinant proteins from each of the five pathogens. The mix of microspheres was used for the simultaneous detection of antibodies against the five corresponding diseases affecting ruminants. The following panel of sera was included in the study: 50 sera from sheep experimentally infected with RVFV, 74 sera from calves and lambs vaccinated with SBV, 26 sera from cattle vaccinated with Mycobacterium bovis, 30 field sera from different species of ruminants infected with CCHFV and 88 calf sera infected with BTV. Finally, to determine its diagnostic specificity 220 field sera from Spanish farms free of the five diseases were assessed. All the sera were classified using commercial ELISAs specific for each disease, used in this study as the reference technique. The results showed the multiplex assay exhibited good performance characteristics with values of sensitivity ranging from 93% to 100% and of specificity ranging from 96% to 99% depending on the pathogen. This new tool allows the simultaneous detection of antibodies against five important pathogens, reducing the volume of sample needed and the time of analysis where these pathogens are usually tested individually

Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera

International audienceSheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade

Circulation of Crimean-Congo Hemorrhagic Fever Virus in the Former Yugoslav Republic of Macedonia Revealed by Screening of Cattle Sera Using a Novel Enzyme-linked Immunosorbent Assay

<div><p>Background</p><p>There are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia.</p><p>Principal Findings</p><p>A highly sensitive and specific ELISA was developed to detect CCHFV-specific IgG antibodies in cattle. The assay was validated by using 503 negative serum samples from a country where CCHFV has never been detected until now, and by using 54 positive serum samples. The positive sera were verified by using two commercially available assays (for testing human serum) which we have adapted for use in animals. The sensitivity of the novel ELISA was 98% and its specificity 99%. The presence of Hyalomma ticks was demonstrated in the Former Yugoslav Republic of Macedonia and depending on the region antibody prevalence rates up to 80% were detected in the cattle population.</p><p>Conclusion</p><p>This article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia.</p></div