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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 objective of the present study was to determine the prevalence of postpartum dysgalactia syndrome (PDS) and associated clinical signs in farmed sows in the Republic of Macedonia (RM) in the first 12-24 h postpartum. A total of 202 sows of different parity and different genetic lines from 5 pig farms in RM were included in the study. The sows and their litters were clinically examined 12-24 hours after farrowing. Postpartum dysgalactia syndrome was detected in 23.3% of all clinically examined sows, while prevalence between farms ranged from 14.8% to 38.1%. Altered piglet’s behavior was the most frequent clinical pattern observed in 68.1% of the PDS–affected (PDSA) sows. Regarding the clinical signs in PDSA sows detected among farms, significant differences were observed in the altered piglet’s behavior (p

Oxidative stress and inflammation contribute to the pathogenesis and progression of nonalcoholic fatty liver disease (NAFLD), and the control of lipid status by statins may help to stop the progression of NAFLD. We hypothesized that the addition of antioxidant vitamins C and E to atorvastatin therapy is associated with improved serum enzyme antioxidant status. NAFLD-related serum parameters and the activity of antioxidant enzymes, before and after 3 months of treatment, were determined in patients receiving atorvastatin alone or atorvastatin plus antioxidants. Compared to healthy controls, the patients, before receiving therapy, had increased catalase and glutathione reductase, with no significant difference in glutathione peroxidase activity. After the treatment, the levels of all three antioxidant markers were reduced to the same degree in both groups of patients, indicating therapy-induced lower level of reactive oxygen species production and/or improved nonenzymatic antioxidant mechanisms. Both therapies led to the normalization of the serum lipid profile and aminotransferase levels in the patients, but the reduction in CRP, although significant, did not reduce levels to those of the controls. The obtained results favor the notion that therapy with atorvastatin alone is equally efficient during the early stages of NAFLD, regardless of the addition of antioxidant vitamins. This trial is registered withTCTR20180425001.

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.

The aim of this study is to compare the results from the histopathology and the immunohistochemical method in the diagnostic of Porcine circovirus type 2 (PCV2) infection in pigs. The circovirus infection is a pig disease that is caused by a small, spherical, nonenveloped virus with a single stranded DNA genome which is spread throughout the pig industry worldwide. The circovirus is the etiological agent of a several pig diseases which today are thought to be the cause of the greatest economical loses in pig production. The most important of these diseases is the PMWS (post-weaning multisystemic wasting syndrome). In this article we have performed an investigation of four farms on which there had been a previous clinical diagnosis of the Post-weaning multisystemic wasting syndrome. The examination was performed on thirty pigs from these farms, from two to five months old, which had the most severe symptoms of the disease. Necropsy, histopathology and immunohistochemical diagnostic methods were performed. The most significant necropsy findings were the enlarged lymph nodes (especially the inguinal, mediasinal and the mesenteric lymph nodes). The main histopathological changes were located in the lymphatic organs presented by B and T lymphocyte depletion and increase in the number of the macrophages. PCV2 antigen and nucleic acid were detected in almost all of the examined tissues. The examination showed that the histopathological and immunohistochemical methods provide complementary results in diagnosing PCV2 in pigs.

Considering the preexisting influence of the process of natural aging on antioxidant enzymes activity and the level of lipid peroxidation, the age of the rats at which D-galactose (D-gal) treatment is started could strongly impact the development of D-gal induced senescence. To evaluate this, we subjected 1, 3 and 15 months old rats to D-gal treatment in parallel with having appropriate placebos (0.9 % saline). Our results showed elevated glutathione peroxidase (GPx) activity and no significant changes in superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) activity or malondialdehyde (MDA) levels in relation to natural aging. In mature and aged senescent livers we observed positive correlation between increased ratio R=SOD/(GPx+CAT) and increased MDA concentration. MDA levels seemed to correlate positively with the age of the animals at which D-gal treatment had started. In the case of 3 and 15 months old rats there was D-gal induced decrease in SOD and GR activity, but this effect of the treatment was not observed in 1 month old rats. Our results imply that the changes in the antioxidant enzyme activities are not only under the influence of the D-gal overload, but also depend on the developmental stage of the rats. According to our results, with regard to enzymatic antioxidant capacity and the level of lipid peroxidation, the best age for induction of senescence is somewhere after the third month.

BLAD and CVM are Holstein-specific recessive mutations which have had significant economic impact on dairy cattle breeding worldwide. The aim of this study was to optimize a diagnostic test for detection of these two disorders and to obtain a preliminary picture about theire existence in Macedonian HF population. Blood samples were obtained from 84 HF cows from several different farms in Macedonia. Genomic DNA was extracted using DNeasy blood and tissue kit (Qiagen) which was also used for DNA extraction from deeply frozen sperm doses from 6 different HF bulls. Genotyping was performed by PCR-RFLP analysis with TaqI and HaeIII restriction endonucleases for BLAD and with PstI for CVM. The correctness of the genotyping was evaluated by direct sequencing of the amplified products. Among 90 HF cattle tested two cows were identified as carriers of the BLAD allele and one was carrier of the CVM allele. No bulls were found to be carriers of either of the two disorders. This study demonstrates that carriers of BLAD and CVM are present in Macedonia, although at low frequency. PCR followed by RFLP is efficient and inexpensive method for detection of the carriers and should be used as laboratory test in eradication programs. In order to establish a more accurate picture of the prevalence of these disorders, it is necessary to screen a larger number of animals. It is indispensable to test each bull before entering the A.I. program to prevent accidental transmission to a large number of offspring.

To investigate early events possibly related to the development of heat shock, we examined whether inflammatory-(interleukin-6, tumor necrosis factor α and 15-keto-13,14-dihydro-PGF 2α ) and peroxidative-(8-iso-PGF 2α and malondialdehyde) markers are altered during acute heat exposure and aging. We also studied the relationships between inflammatory and peroxidative markers in these settings. In order to prevent these reactions developed as a consequence of the conditions mentioned above, we tested the effects of α-tocopherol. Our results demonstrated that 15-keto-13,14-dihydro-PGF 2α and malondialdehyde in the liver were altered during acute heat exposure in the young and middle-aged rats and could be predicted by changes in the levels of circulatory cytokines. Regardless of age, the supplementation with α-tocopherol prevented changes in the plasma cytokine levels and 15-keto-13,14-dihydro-PGF 2α and malondialdehyde levels in the liver, during acute heat exposure. This study notably emphasized the ability of α-tocopherol to prevent different heat induced mechanisms, involved in induction of inflammatory or peroxidative reactions.

Background Oxidative stress and inflammation are involved in the pathogenesis of paracetamol-induced renal damage. This study examines the relationship between 8-iso-prostaglandin F2α (8-iso-PGF2α) and platelet activation as well as the relative contribution of the pro-inflammatory markers interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) in enhanced 8-iso-PGF2α biosynthesis, as a complementary onset during analgesic nephropathy induced by chronic treatment with paracetamol. The protective effects of vitamin C on the aforementioned settings are also investigated. Methods Analgesic nephropathy was induced in Wistar rats. Renal function markers and the activity of antioxidant enzymes were determined spectrophotometrically. Immunoassays were used to measure the pro-inflammatory markers and the markers of lipid peroxidation and platelet activation. Results The chronic treatment with paracetamol led to renal dysfunction, represented by the elevation of plasma urea and creatinine and the decline in the enzymatic antioxidant status, but did not cause a significant increase in TNF-α and IL-1β. The paracetamol-induced lipid peroxidation and enhanced production of 8-iso-PGF2α was not sufficient to cause changes in platelet activation represented by the level of 11-dehydro thromboxane B2. Conclusions Our results suggest that oxidative stress cannot circumvent the need of stimulation by circulatory cytokines in order to induce inflammatory response and changes in platelet activation during analgesic nephropathy. Vitamin C proved to be beneficial in restoring the renal function markers to normal, increasing the renal enzymatic antioxidant potential, inhibiting lipid peroxidation, and lowering cytokine production and 11-dehydro thromboxane B2 excretion. The observed effects of vitamin C offer support for its potential use as protective treatment in cases of chronic paracetamol overdose.

Results. Starting from 2005 until 2009 a total of 559.476 cattle serum samples, from different villages across Macedonia, were collected and sent to the Faculty of Veterinary Medicine – Skopje, for testing for brucellosis. Seroprevalence in some epizootiological areas ranges from 0.1% up to 22.2%. According to the results, obtained with this study, the mean seroprevalence of cattle brucellosis in Macedonia for the last 5 years was estimated at 0.83% (ranging from 0.17% to 2.78%).

To investigate the impact of acute heat exposure on maintenance of redox homeostasis and antioxidant balance related to aging, we have determined the GSH levels in the liver and kidney, and the activity of antioxidant enzymes in the same organs from Wistar rats at two different ages, 35 days and 18 months. The animals were housed individually in a special heated chamber maintaining a constant temperature of 40±0.5 °C. The results showed that the level of endogenous GSH was significantly lower in aged than in young animals. In general, the activity of antioxidant enzymes in investigated tissues displayed an age-dependent decline. Indeed, we found unchanged CAT activity and decreased GPx activity with age. On the other hand acute heat exposure led to disproportion between peroxide metabolizing enzymes (CAT, GPx) and GR, thus promoting H 2 O 2 accumulation and prooxidative state in the liver of young animals. The results for the impact of l -2-oxothiazolidine-4-carboxylate in combined stress model suggested that in spite of restore levels of GSH, the restoration of oxido-reductive balance might have only been partial due to irreversible alterations in antioxidant enzymes set by acute heat exposure and aging. Interestingly, young animals appeared to be more sensitive to the supplementation of the l -2-oxothiazolidine-4-carboxylate, likely because of the more extensive increase of GSH observed in young l -2-oxothiazolidine-4-carboxylate treated animals.

Aim To identify why “test and slaughter policy” for eradication of brucellosis did not significantly reduce the prevalence in sheep and goats in Macedonia. Method Coverage of sampled vs expected number of sheep and goats, absolute number of positive animals, prevalence, frequency distribution, and classes of disease prevalence were retrospectively analyzed at the village level for 2004-2006. A comparative analysis of the disease prevalence in the investigated villages was also performed. The percentage of slaughtered animals was analyzed for 2000-2006. Results We found differences between the expected and actual number of sampled animals, which were related to the type of livestock breeding. Traditionally maintained flocks and migratory flocks were considered to be responsible for the transmission of the disease. The absolute number of positive animals and the number of infected vs non-infected holdings did not decrease over the study period. Most of the villages had between 1 and 10 positive animals. Between 2000 and 2006, 55% of the positive animals were slaughtered, 41% in 2001 and up to 79% in 2002. Moreover, in 2005 and 2006, 34% and 53% of sheep and goats were found to be positive at the slaughter line, respectively, demonstrating that only 21%-23% of the infected animals were correctly removed from the herds. Conclusion Based on the findings of this study, Macedonia changed its control strategy from “test and slaughter” to a vaccination policy for sheep and goats in 2008.

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., Author Summary Crimean-Congo hemorrhagic fever virus (CCHFV) infections in humans can cause a deadly hemorrhagic fever with high fatality rates. Most humans are infected by ticks or by contact with blood. No vaccine prophylaxis or therapeutic interventions are available. Ticks of the genus Hyalomma function as vector of CCHFV. Domestic ruminants play a crucial role in the transmission and amplification of the virus. Since infected ruminants do not develop clinical signs, CCHFV infections can in most cases only be detected by serology. There are only few assays available for the detection of CCHFV-specific antibodies in animals. In general, the real distribution of CCHFV is fairly unknown. Here we present a novel, highly sensitive and specific ELISA, and the adaptations for two commercially available assays for the detection of CCHFV-specific antibodies in cattle. With these assays, we detected CCHFV-specific antibodies in cattle in the Former Yugoslav Republic of Macedonia. This is the first indication of an active circulation of CCHFV in this country. This knowledge will assist decision makers and public health authorities in deciding on effective countermeasures and can help to prevent outbreaks.

THE Republic of Macedonia is located in the southern region of the Balkan Peninsula. The country is bordered by Kosovo to the northwest, Serbia to the north, Bulgaria to the east, Greece to the south and Albania to the west. The area of the country is 25,713 km2, including around 80 per cent mountains and 20 per cent plains. The majority of Macedonia has a moderate continental climate with warm and dry summers and autumns, and relatively cold and wet winters. There had been no rabies cases reported in the country since 2000. Fig 1 shows the total number of rabies cases reported in 2010 in neighbouring countries. In those areas, the vector and reservoir of the disease is the red fox ( Vulpes vulpes ) which is responsible for more than 88 per cent of the reported cases (data extracted from Rabies Bulletin Europe). All countries (except Albania and Greece which are reported as rabies-free) are currently enrolled in oral vaccination programmes which started in Kosovo in spring 2010. In Bulgaria, the first oral vaccination campaign was implemented in spring 2009 as well. Fig 1 Map of Macedonia and neighbouring countries with distribution of rabies cases in 2010 and in …

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus, which causes a serious illness with case-fatality rates of up to 80 % in humans. CCHFV is endemic in many countries of Africa, Asia and Southeastern Europe. Next to the countries with endemic areas, the distribution of CCHFV is unknown in Southeastern Europe. As the antibody prevalence in animals is a good indicator for the presence or absence of the virus in a region, seroepidemiological studies can be used for the definition of risk areas for CCHFV. The aim of the present study was to reveal which ruminant species is best suited as indicator for the detection of a CCHFV circulation in an area. Therefore, the prevalence rates in sheep, goats and cattle in different regions of Albania and Former Yugoslav Republic of Macedonia were investigated. As there are no commercial tests available for the detection of CCHFV-specific antibodies in animals, two commercial tests for testing human sera were adapted for the investigation of sera from sheep and goats, and new in-house ELISAs were developed. The investigation of serum samples with these highly sensitive and specific assays (94–100 %) resulted in an overall prevalence rate of 23 % for Albania and of 49 % for Former Yugoslav Republic of Macedonia. Significant lower seroprevalence rates for CCHFV were found in cattle than in small ruminants in given areas. These results indicate that small ruminants are more suitable indicator animals for CCHFV infections and should therefore be tested preferentially, when risk areas are to be identified.

To investigate the role of mitochondrial antioxidant capacity during increased susceptibility to heat accompanied by the aging, young and aged Wistar rats were exposed on heat for 60 min. After heat exposure, hepatic and brain mitochondria were isolated. Our results revealed changes in antioxidant enzyme activities in liver and brain mitochondria from young and to a greater extent in aged rats. Our measurements of MnSOD, GPx and GR activity indicate greater reactive oxygen species production from the mitochondria of aged heat exposed in comparison to young heat exposed rats. Also in the aged rats, the effect of α-tocopherol treatment in the prevention of oxidative stress occurred as a result of heat exposure, is less pronounced. Taken together, our data suggest that mitochondria in aged rats are more vulnerable and less able to prevent oxidative changes that occur in response to acute heat exposure.