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Antimicrobial resistance (AMR) is an increasing global concern in small animal veterinary practices. The overuse and misuse of antimicrobials in companion animals can contribute to the spread of AMR between animals and humans. This study aimed to assess small animal veterinarians’ knowledge, attitudes, and behaviors towards antimicrobial use (AMU) and AMR in North Macedonia. A web-based questionnaire was developed, including questions addressing demographics, owner influence on antibiotic prescription and prescribing practices. The response rate of the small animal veterinarians was 47.50% (57/120), with most of them (84.20%) having 1-15 years of work experience. Pearson’s chi-square test of independence was used for contingency tables that met the chi-square assumption, and Fisher’s exact test was conducted for contingency tables that did not. Most veterinarians responded that they had perceived pressure and direct demands from pet owners to prescribe antimicrobials (35.09% frequently, 45.61% occasionally). In the survey, 50.77% of the veterinarians reported frequently administering antibiotics without conducting bacteriological or antimicrobial susceptibility testing. They typically resorted to these tests in cases with recurrent infections, such as skin infections (19.63%), or when empirical therapy is ineffective (14.72%). Based on the findings, it can be concluded that veterinarians play a crucial role in tackling the AMR in North Macedonia. The lack of stewardship programs or guidelines for responsible antimicrobial use in companion animals is a critical issue that requires urgent attention, emphasizing the vital role of veterinarians in combating AMR.

IntroductionVeterinary clinical microbiology laboratories play a key role in antimicrobial stewardship, surveillance of antimicrobial resistance and prevention of healthcare associated-infections. However, there is a shortage of international harmonized guidelines covering all steps of veterinary bacterial culture from sample receipt to reporting.MethodsIn order to gain insights, the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT) designed an online survey focused on the practices and interpretive criteria used for bacterial culture and identification (C&ID), and antimicrobial susceptibility testing (AST) of animal bacterial pathogens.ResultsA total of 241 microbiology laboratories in 34 European countries completed the survey, representing a mixture of academic (37.6%), governmental (27.4%), and private (26.5%) laboratories. The C&ID turnaround varied from 1 to 2 days (77.8%) to 3–5 days (20%), and 6– 8 days (1.6%), with similar timeframes for AST. Individual biochemical tests and analytical profile index (API) biochemical test kits or similar were the most frequent tools used for bacterial identification (77% and 56.2%, respectively), followed by PCR (46.6%) and MALDI-TOF MS (43.3%). For AST, Kirby-Bauer disk diffusion (DD) and minimum inhibitory concentration (MIC) determination were conducted by 43.8% and 32.6% of laboratories, respectively, with a combination of EUCAST and CLSI clinical breakpoints (CBPs) preferred for interpretation of the DD (41.2%) and MIC (47.6%) results. In the absence of specific CBPs, laboratories used human CBPs (53.3%) or veterinary CBPs representing another body site, organism or animal species (51.5%). Importantly, most laboratories (47.9%) only report the qualitative interpretation of the result (S, R, and I). As regards testing for AMR mechanisms, 48.5% and 46.7% of laboratories routinely screened isolates for methicillin resistance and ESBL production, respectively. Notably, selective reporting of AST results (i.e. excluding highest priority critically important antimicrobials from AST reports) was adopted by 39.5% of laboratories despite a similar proportion not taking any approach (37.6%) to guide clinicians towards narrower-spectrum or first-line antibiotics.DiscussionIn conclusion, we identified a broad variety of methodologies and interpretative criteria used for C&ID and AST in European veterinary microbiological diagnostic laboratories. The observed gaps in veterinary microbiology practices emphasize a need to improve and harmonize professional training, innovation, bacterial culture methods and interpretation, AMR surveillance and reporting strategies.

The global antimicrobial resistance crisis has been the driver of several international strategies on antimicrobial stewardship. For their implementation at the field level, the veterinary sector encounters several specific challenges and in particular: (i) a shortage of experts in key disciplines related to antimicrobial stewardship, (ii) a lack of evidence-based antimicrobial treatment guidelines, and (iii) inferior diagnostic tests available compared to human medicine. The present white paper describes how the COST Action ENOVAT (the European Network for Optimization of Veterinary Antimicrobial Treatment, CA18217), comprising 332 persons from 51 countries, worked towards solutions to these challenges. Initially, surveys were conducted to explore the present state in Europe in terms of existing antimicrobial use guidelines and microbiology practices performed. Concurrently, various research activities were launched to optimize diagnostics, including development of epidemiological cut-offs, clinical breakpoints and matrix-assisted laser desorption ionization time of flight mass spectrometry interpretive criteria. Also, guidelines drafting groups working towards evidence-based antimicrobial treatment guidelines for six conditions in food-producing and companion animals were established. The processes and outcomes, also in terms of capacity building, are summarized in this white paper where emphasis is placed on sustainability of the activities. Although several ENOVAT initiatives and spin-off projects will continue beyond the Action, we recommend that a new European veterinary research agenda is launched focusing on research and funding leading to long-term impacts on veterinary antimicrobial use.

The global antimicrobial resistance crisis has been the driver of several international strategies on antimicrobial stewardship. For their implementation on field level, the veterinary sector encounters several specific challenges and in particular: (i) a shortage of experts in key disciplines related to antimicrobial stewardship, (ii) a lack of evidence-based antimicrobial treatment guidelines, and (iii) inferior diagnostic tests available compared to human medicine. The present white paper describes how the COST Action ENOVAT (the European Network for Optimization of Veterinary Antimicrobial Treatment, CA18217), comprising 332 persons from 51 countries, worked towards solutions to these challenges. Initially, surveys were conducted to explore the present state in Europe in terms of existing antimicrobial use guidelines and microbiology practices performed. Concurrently, various research activities were launched to optimize diagnostics, including development of epidemiological cut-offs, clinical breakpoints and matrix-assisted laser desorption ionization time of flight mass spectrometry interpretive criteria. Also, guidelines drafting groups working towards evidence-based antimicrobial treatment guidelines for six conditions in food-producing and companion animals were established. The processes and outcomes, also in terms of capacity building, are summarized in this white paper where emphasis is placed on sustainability of the activities. Although several ENOVAT initiatives and spin-off projects will continue beyond the Action, we recommend that a new European veterinary research agenda is launched focusing on research and funding leading to long-term impacts on veterinary antimicrobial use.

Coenurosis is a cystic parasitic disease affecting ruminants and other mammals worldwide. The disease is caused by Coenurus cerebralis, the larval stage of the tapeworm Taenia multiceps. Sheep and goats are frequently infected, while cases in cattle are rare. Here we describe the first recorded case of bovine coenurosis in North Macedonia. The diagnosis was based on the neurological clinical signs and the postmortem findings of a 1-year-old bull with symptoms typical for coenurosis, i.e. ataxia, circling movements, mild depression and impaired vision. Postmortem, a cyst was found in the left cerebral hemisphere and was confirmed as fertile Coenurus cerebralis by parasitological analysis. These findings suggest that coenurosis should be considered part of the differential diagnosis in cattle with neurological symptoms. This highlights the need for routine surveillance of this disease in livestock and proactive tracking of the parasite in the final hosts to enhance disease management.

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.

The incidence of clinical mastitis (CM) in small and large-scale dairy cow farms can be highly variable and can be affected by age, parity, post-calving status, and atmospheric conditions. The current study aimed to assess the CM-incidence and its association with the post-calving days, number of lactations, season, and number of affected udder quarters in dairy cows in small-scale dairy farms. The study was conducted within one calendar year in 177 small-scale farms with 864 dairy cows. Clinically confirmed CM cows (n=72) were sampled from each udder quarter and processed for bacteriology examination. The CM-positive samples were grouped according to the season (spring, summer, autumn, and winter), the number of days after calving (180), the number of lactations (1-st, 2-5-th, and >6-th), and the number of affected udder quarters (one, two, three, and four). The CM-positive samples (n=72, 8.3%) were confirmed on bacteriology examination in significantly lower count (n=56, 6.5%). The 2-5-th lactations cows (n=35, 68.6%) were significantly more compared to the first lactation (n=6, 11.8%), and >6-th lactation cows (n=10, 19.6%). CM cows with infection of one udder quarter (n=40, 78.4%) were significantly more than the cows with two (n=6, 11.8%), and four infected quarters (n=5, 9.8%). The CM-incidence in small-scale dairy cow farms in North Macedonia was 8.3% and 6.5% by clinical and bacteriology examination, respectively. The highest CM susceptibility was observed in the cows between the second and fifth lactations. One udder quarter was most frequently affected in CM-positive cases.

Cystic echinococcosis is a cosmopolitan zoonotic infection, commonly seen in areas where livestock and dogs are kept together. The objective of this study was to investigate the prevalence of echinococcosis in slaughtered cattle and sheep in North Macedonia and to evaluate the location and fertility/sterility rates of the cysts. A total of 302 slaughtered animals were examined with visual inspection, palpation, and incision of the internal organs in order to detect cysts. Microscopic examination was carried out to determine the presence of protoscoleces. The overall prevalence of echinococcosis in slaughtered cattle and sheep was 60%. The presence of cysts increased with age in cattle. In both cattle and sheep, the most common affected organs were the liver and lungs infected with more than one cyst. The findings showed higher fertility rate in sheep which confirmed their role as the most important intermediate host. This study shows that North Macedonia is an endemic region, and serious control strategies should be implemented, with special emphasis on safety disposal of infected organs and anthelmintic treatment of the dogs.

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.

Subclinical mastitis is an asymptomatic udder infection distributed worldwide with enormous losses in the dairy industry. The study’s objective was to determine the presence of this pathological condition in small dairy farms in the R. of N. Macedonia and to identify the most common associated bacteria. Milk samples were obtained from 96 dairy cows (378 udder quarters) in seven dairy farms, in 3 consecutive samplings 24-72 hours apart. The samples were cultured on routine bacteriological growth media and incubated for 24-48 hours. The isolates were identified by AximaiD Plus MALDITOF MS Platform. Subclinical mastitis was found in 49 animals (51%) and 104 infected quarters (27%). The most frequent isolated bacteria on cow level were Streptococcus uberis (19.4%), Staphylococcus haemolyticus (13.4%), Staphylococcus aureus (7.4%) and Staphylococcu ssimulans (7.4%). On quarter level, the most isolated pathogen was Streptococcus uberis (35.6%) followed by Staphylococcu shaemolyticus and Staphylococcus aureus (10.3% and 9.2% respectively). Subclinical mastitis was found to be highly present in the selected small dairy farms. The most prevalent bacteria identified in the dairy farms (Streptococcus uberis, Staphylococcus aureus and coagulase–negative staphylococci) indicate that poor management and udder health practices, inadequate milking procedures and lack of mastitis control strategies greatly contribute to occurrence and persistence of subclinical mastitis.

The COST action “Standardising output-based surveillance to control non-regulated diseases of cattle in the European Union (SOUND control),” aims to harmonise the results of surveillance and control programmes (CPs) for selected cattle diseases to facilitate safe trade and improve overall control of cattle infectious diseases. In this paper we aimed to provide an overview on the diversity of control for these diseases in Europe. A selected cattle disease was defined as an infectious disease of cattle with no or limited control at EU level, which is not included in the European Union Animal health law Categories A or B under Commission Implementing Regulation (EU) 2020/2002. A CP was defined as surveillance and/or intervention strategies designed to lower the incidence, prevalence, mortality or prove freedom from a specific disease in a region or country. Passive surveillance, and active surveillance of breeding bulls under Council Directive 88/407/EEC were not considered as CPs. A questionnaire was designed to obtain country-specific information about CPs for each disease. Animal health experts from 33 European countries completed the questionnaire. Overall, there are 23 diseases for which a CP exists in one or more of the countries studied. The diseases for which CPs exist in the highest number of countries are enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis, bovine viral diarrhoea and anthrax (CPs reported by between 16 and 31 countries). Every participating country has on average, 6 CPs (min–max: 1–13) in place. Most programmes are implemented at a national level (86%) and are applied to both dairy and non-dairy cattle (75%). Approximately one-third of the CPs are voluntary, and the funding structure is divided between government and private resources. Countries that have eradicated diseases like enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis and bovine viral diarrhoea have implemented CPs for other diseases to further improve the health status of cattle in their country. The control of the selected cattle diseases is very heterogenous in Europe. Therefore, the standardising of the outputs of these programmes to enable comparison represents a challenge.

A six-month-old Pomeranian male dog was referred due to a month long history of unformed, soft faeces and mild weight loss. Stool analyses by direct faecal smear, Zinc sulphate flotation and the Baermann concentration method revealed an infection with Strongyloides stercoralis. The dog was initially treated once with a combination drug of praziquantel, pyrantel and febantel (½ Drontal® Plus Tablets for puppies and small dogs; Bayer; i.e. 31.5 mg/kg bodyweight of febantel). The treatment was repeated after 12 days with the same dosage for 3 consecutive days. The stool analyses performed 14 days and 3 months after the second treatment were negative for S. stercoralis larvae. The results suggest that a repeated treatment with Drontal® Plus Tablets is effective against S. stercoralis in dogs and has no adverse effects.

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.

BACKGROUND: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. PRINCIPAL FINDINGS: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. CONCLUSION: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.