A Green HPLC Approach to Florfenicol Analysis in Pig Urine.

Florfenicol (FF) is a broad-spectrum antibiotic used to treat gastrointestinal and respiratory infections in domestic animals. Considering FF's rapid elimination via urine after drug treatment, its use increases concerns about environmental contamination. The objective of the study was to establish a sustainable chromatographic method for simple analysis of FF in pig urine to investigate the urinary excretion of FF after a single intramuscular administration of 20 mg FF/kg body weight. The urine sample was prepared using a centrifuge and regenerated cellulose filter, and the diluted sample was analyzed. The method was validated in terms of linearity, the limit of detection (0.005 µg/mL) and quantitation (0.016 µg/mL), repeatability and matrix effect (%RSD ranged up to 2.5), accuracy (varied between 98% and 102%), and stability. The concentration-time profile of pig urine samples collected within 48 h post-drug administration showed that 63% of FF's dose was excreted. The developed method and previously published methods used to qualify FF in the urine of animal origin were evaluated by the National Environmental Method Index (NEMI), Green Analytical Procedure Index (GAPI) and Analytical GREENness Metric Approach (AGREE). The greenness profiles of published methods revealed problems with high solvents and energy consumption, while the established method was shown to be more environmentally friendly.

Effects of immunostimulators of microbial origin on T cells of pigs vaccinated with attenuated vaccine against Aujeszky's disease.

Aujeszky's disease (AD) is a viral infectious disease caused by Suid herpesvirus 1 (SuHV-1). Vaccination and eradication of AD in domestic pigs is possible using marker vaccines with attenuated or inactivated SuHV-1, or subunit vaccines. However, vaccines with attenuated SuHV-1 have shown to be more potent in inducing strong cell-mediated immune response. The studies have shown that Parapoxvirus ovis, as well as Propionibacterium granulosum with lipopolysacharides (LPS) of Escherichia coli have pronounced immunomodulatory effects and that in combination with the vaccines can induce stronger humoral and cellular immune responses than use of vaccines alone. In our study distribution of peripheral blood T cell subpopulations was analysed after administration of vaccine alone (attenuated SuHV-1), immunostimulators (inactivated Parapoxvirus ovis or combination of an inactivated P. granulosum and detoxified LPS of E. coli) and combinations of vaccine with each immunostimulator to the 12-week old piglets. Throughout the study no significant changes were found in the proportions of γδ and most αß T cell subpopulations analysed. However, on the seventh day of the study combination of an inactivated P. granulosum and LPS of E. coli with vaccine induced transient but significant increase of the proportions of CD4+CD8α+ and CD4-CD8α+ αß T cells, that have been strongly associated with early protection of SuHV-1 infected pigs. Our findings indicate that combination of inactivated P. granulosum and detoxified E. coli LPS could be used for enhancement of a cellular immune response induced by vaccines against AD.

Effect of Propolis on Diet-Induced Hyperlipidemia and Atherogenic Indices in Mice.

Obesity, a major health problem worldwide, is associated with increased cardiovascular risk factors, such as dyslipidemia, glucose intolerance, and hypertension. We investigated the antioxidative capacity of the ethanol extract of propolis (EEP) and its effect on the lipid profile, the hepatorenal function, and the atherogenic indices in mice fed with a high-fat diet (HFD). EEP (50 mg/kg) was given orally to mice for 30 days. After the treatments, levels of the serum total triglyceride and cholesterol, the high density lipoprotein (HDL-c) and low density lipoprotein (LDL-c) cholesterols, the serum enzymes, and the metabolites were measured, and atherogenic indices [atherogenic index of plasma (AIP); cardiac risk ratio (CRR); cardioprotective index (CPI); atherogenic coefficient (AC)] were calculated and compared with the antioxidant, the reducing power, the radical-scavenging, and the chelating activity of EEP. The HFD diet with EEP significantly reduced the negative lipid profile and lowered AIP, CRR, and AC and increased CPI in animals on a HFD. In addition, EEP reduced the weight of mice and lipid accumulation in the liver, and it had significant in vitro antioxidative activities. The EEP possesses anti-hyperlipidemic and antioxidant activity and exhibits protective action on the cardiovascular system and hepatorenal functions. Our results contribute towards the validation of the traditional use of propolis as a food supplement in aiding hyperlipidemic disorders.

The Beneficial Effect of Proanthocyanidins and Icariin on Biochemical Markers of Bone Turnover in Rats.

Nutrition is an important factor that influences bone metabolism, the endocrine and/or paracrine system, and bone-active mineral elements homeostasis. We studied antiosteoporotic effects of grape seed proanthocyanidins extract, icariin or alendronate (ALN) in retinoic acid-induced (13cRA) bone loss in rats. Proanthocyanidins and icariin have beneficial effects on bone health; they have improved the bone weight reduction, the length and the diameter of the bone, calcium, and phosphorus content in bone ash, bone mineral density (BMD), the biochemical markers of bone turnover and uterus atrophy induced by 13cRA. All results suggest that proanthocyanidins and icariin reverse osteoporosis in 13cRA rats by stimulating bone formation or regulating bone resorption by their antioxidative and estrogenic-like activity without toxic side-effects observed in ALN treatment.

Amblyopia in Children: Analysis Among Preschool and School Children in the City of Tuzla, Bosnia and Herzegovina.

AIM: This study measured the prevalence of amblyopia in preschool and school children between 4 and 15 years of age in Tuzla, Bosnia and Herzegovina and as well and to examine its relations with anisometropia and strabismus. METHODS: Children from eight daycare centers and twenty four elementary schools were screened for amblyopia by volunteer personnel (medical students), any child who failed to pass the screening examination, was referred to the ophthalmologist for complete examination at University Clinic Center Tuzla. The examination included VA, stereopsis, cover testing, refractive retinoscopy, and examination of the red reflex and posterior pole. RESULTS: Total of 7415 children, which included 3790 males and 3625 females, in the age range of 3 to 15 years from 24 schools and 8 preschool were screened. Fifty night children (1.9%) were diagnosed with amblyopia, unilateral in 28 and bilateral in 31. CONCLUSION: Prevalence of significant refractive errors is high enough to justify a school eye screening program solely for this purpose. preschool and school screening program in children in critical period of development of amblyopia must be conducted to find out the ametropias and amblyopia in time; and treat them earlier.

EPIDEMIOLOGICAL ANALYSIS OF OCULAR MELANOMA IN UNIVERSITY CLINIC CENTER IN TUZLA, BOSNIA AND HERZEGOVINA.

AIM: Melanoma represents a malignant tumour arising from melanocytes. Uveal melanoma is the most common primary ocular malignancy among the adult population. The aim of the study was to examine epidemiological characteristics of ocular melanoma in University Clinic Centre in Tuzla, Bosnia and Herzegovina from January 2001 till November 2015. METHODS AND MATERIALS: In this retrospective study we used all available medical documentation to investigate the clinical findings which included age, gender, tumour size, histopathological features and the precise anatomic origin of the ocular melanoma. RESULTS: Over the 14 year period of this study, there were 32 patients with microscopically confirmed ocular melanoma at the Department of Pathology. All malignant melanoma were uveal origin. DISCUSSION AND CONCLUSION: For early detection of the disease, regular checkups are necessary, especially in older population. As there is a limited number of reports on the epidemiology of malignant tumors of eye and ocular adnex in our region, this is very important study. We conclude that this is a first study in Bosnia and Herzegovina that document the number of uveal melanomas.

Is Prevention of Glaucoma Possible in Bosnia and Herzegovina?

INTRODUCTION: Glaucoma is the second leading cause of blindness in the world and represents a significant social and health problem. Early detection of glaucoma enables early initiation of treatment and may delay disease progression. The aim of this work is to determine whether it is possible to detect glaucoma in early stages. METHODS: A public awareness campaign was carried out in University Clinic Center (UCC) in Tuzla, Bosnia and Herzegovina (B&H) during 2012, 2013, 2014 and 2015 Glaucoma Week, with one-day, free of charge screening of individuals. This screening program was composed of getting brief medical history, slit-lamp examination including intraocular pressure and anterior chamber dept evaluation and non-mydriatic fundus exam with evaluation of the cup/disk ration. RESULTS: A total of 682 individuals were screened, 277 were male and 405 were female. The youngest individual was 8 years old and the oldest individual was 84 years old. The mean age was 57.6 years. Intraocular pressure higher then 21,9 mmHg was found in 83 patients. CONCLUSION: Glaucoma is a disease that affects visual acuity and gradually leads to blindness. It occurs in all age groups in both sexes and in all races. Early detection of disease and proper treatment can prevent permanent loss of vision. Detection and early treatment of glaucoma must become one of the leading public health programs in B&H.

Incidence of endophthalmitis after intravitreal application of anti VEGF therapy at the University Clinical Center in Tuzla, Bosnia and Herzegovina.

AIM: To report the incidence of endophthalmitis following the use of intravitreal injection of anti- vascular endothelial growth factor (anti VEGF) therapy. METHODS: In this retrospective study a total of 986 intravitreal bevacizumab injections were applied between January 2008 and April 2015 at the University Clinical Center Tuzla, Bosnia and Herzegovina (BiH). Since January 2012, a total of 55 intravitreal ranibizumab injections were applied and since October 2014, 60 intravitreal aflibercept injections were applied to patients. RESULTS: Two cases of endophthalmitis following intravitreal injection of bevaciuzumab occurred and none after ranibizumab or aflibercept. The overall incidence of clinical endophtahlmitis was 0.2%. CONCLUSION: The results suggest that a low rate of endophthalmitis can be achieved by means of a protocol. This is a very important study as it is the first of this kind in BiH that documents the incidence of endophthalmitis after intravitreal application. Currently, bevacizumab in BiH is most frequently used intravitreal anti-vascular endothelial growth factor due to very low price.

Comparison of accumulation of clenbuterol and salbutamol residues in animal internal tissues, non-pigmented eyes and hair.

The aim of the study was to compare the accumulation of ß-adrenergic agonist residues clenbuterol (CLB) and salbutamol (SAL) in internal tissues, non-pigmented eyes and hair of laboratory animals repeatedly administered with CLB and SAL during 7 days. Experimental albino guinea pigs (n = 20) were treated with CLB (n = 10) and SAL (n = 10) in anabolic doses of 0.25 and 2.5 mg/kg, whereas the control animal group (n = 10) was left untreated. Methodology validation showed that the ELISA assay to be suitable for ß-agonists' semiquantitative determination. The results revealed a significantly higher (P < 0.05) accumulation potential of CLB in comparison with SAL in all investigated tissues. Despite of their lack of pigmentation and the applied dose, the highest residual CLB concentrations were determined in the eyes of the studied animals, followed by their hair, liver, lungs, kidney, heart and adipose and muscle tissue, whereas residual SAL concentrations found in the eyes and hair of the administered animals did not significantly differ (P > 0.05) from those obtained in their internal tissues.

Detection and genetic characterization of tick-borne encephalitis virus (TBEV) derived from ticks removed from red foxes (Vulpes vulpes) and isolated from spleen samples of red deer (Cervus elaphus) in Croatia.

Tick-borne encephalitis (TBE) is a growing public health concern in central and northern European countries. Even though TBE is a notifiable disease in Croatia, there is a significant lack of information in regard to vector tick identification, distribution as well as TBE virus prevalence in ticks or animals. The aim of our study was to identify and to investigate the viral prevalence of TBE virus in ticks removed from red fox (Vulpes vulpes) carcasses hunted in endemic areas in northern Croatia and to gain a better insight in the role of wild ungulates, especially red deer (Cervus elaphus) in the maintenance of the TBE virus in the natural cycle. We identified 5 tick species (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis punctata, Dermacentor reticulatus, Rhipicephalus sanguineus) removed from 40 red foxes. However, TBE virus was isolated only from adult I. ricinus and I. hexagonus ticks showing a viral prevalence (1.6%) similar to or higher than reported in endemic areas of other European countries. Furthermore, 2 positive spleen samples from 182 red deer (1.1%) were found. Croatian TBE virus isolates were genetically analyzed, and they were shown to be closely related, all belonging to the European TBE virus subgroup. However, on the basis of nucleotide and amino acid sequence analysis, 2 clusters were identified. Our results show that further investigation is needed to understand the clustering of isolates and to identify the most common TBE virus reservoir hosts in Croatia. Sentinel surveys based on wild animal species would give a better insight in defining TBE virus-endemic and possible risk areas in Croatia.

Accumulation of ractopamine residues in hair and ocular tissues of animals during and after treatment.

The aim of the present study was to assess the accumulation of ractopamine residues in the hair and ocular tissues of guinea pigs during repeated ractopamine administration and after treatment. The experiment was conducted in 38 guinea pigs (30 treated and eight controls). Treated animals were orally administered ractopamine hydrochloride in a dose of 3.5 mg/kg body mass per day using probes for seven consecutive days. Ractopamine concentration was determined in hair during the treatment (Days 1, 3 and 7) with ractopamine hydrochloride and in ocular tissues and hair on defined days after exposure (Days 1, 10, 20 and 30). Residues were present in hair in high concentrations as early as Day 3 (86.15 ± 87.71 ng/g) and Day 7 (85.25 ± 56.97 ng/g). After exposure, residues were found to persist, having depleted from 68.06 ± 30.54 ng/g on Day 1 to 8.01 ± 2.22 ng/g on Day 30, with a significantly higher concentration in hair in contrast to low residue levels in ocular tissues (1.20-0.34 ng/g). The results of the study pointed to high ractopamine accumulation, even in non-pigmented hair, suggesting hair to be used as a matrix in the control of ractopamine abuse in farm animals because of its many advantages over ocular tissues.

Biochemical changes in pig serum after ochratoxin A exposure.

The objective of this study was to investigate the effect of ochratoxin A (OTA) on serum biochemical parameters of pigs during subchronic treatment with 300 µg OTA/kg of feed for 30 days. OTA treatment resulted in significantly higher (p < 0.05) serum levels of creatinine, urea, potassium and alkaline phosphatase, and significantly lower levels of glucose and total protein. These changes in serum biochemical parameters in treated pigs were indicative of impaired liver and kidney function caused by OTA exposure.

Prevalence of antibodies to selected viral pathogens in wild boars (Sus scrofa) in Croatia in 2005-06 and 2009-10.

We determined prevalence of antibody to selected viral pathogens important for domestic pigs and livestock in 556 wild boar (Sus scrofa) sera collected during 2005-06 and 2009-10 in four counties in Croatia. These counties account for an important part of the Croatian commercial pig production and have a high density of wild boars. Samples were tested for antibodies to porcine parvovirus (PPV), Aujeszky's disease virus (ADV), porcine circovirus type 2 (PCV2), swine influenza virus, porcine respiratory and reproductive syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus, and swine vesicular disease virus (SVDV). Antibodies to all of the infectious pathogens except SVDV were detected. There was a statistically significant difference in prevalence between the two periods for PPV, ADV, PCV2, PRRSV, and PRCV, with a higher prevalence of PPV and ADV in the 2009-10 period (P<0.05). During the same period, the prevalence of PCV2, PRRSV, and PRCV was lower (P<0.05). Our results provide information on the current disease exposure and health status of wild boars in Croatia and suggest that wild boars may act as a reservoir for several pathogens and a source of infection for domestic pigs and other livestock as well as humans, especially for ADV.

A practical validation approach for virus titer testing of avian infectious bursal disease live vaccine according to current regulatory guidelines.

The method for virus titer determination of avian infectious bursal disease (IBD) live vaccine, developed long before regulatory validation guidelines is a cell culture based biological assay intended for use in vaccine release testing. The aim of our study was to perform a validation, based on fit-for-purpose principle, of an old 50% tissue culture infectious dose (TCID(50)) method according to Guidelines of the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH). This paper addresses challenges and discusses some key aspects that should be considered when validating biological methods. A different statistical approach and non-parametric statistics was introduced in validation protocol in order to derive useful information from experimental data. This approach is applicable for a wide range of methods. In conclusion, the previous virus titration method had showed to be precise, accurate, linear, robust and in accordance with current regulatory standards, which indicates that there is no need for additional re-development or upgrades of the method for its suitability for intended use.

Effects of local administration of propolis and its polyphenolic compounds on tumor formation and growth.

Many dietary constituents are chemopreventive in animal models, and experiments with cultured cells are revealing various potential mechanisms of action. Compounds classified as blocking agents can prevent, or greatly reduce, initiation of carcinogenesis, or suppressing agents can act on cell proliferation. Caffeic acid (CA) and caffeic acid phenethyl ester (CAPE), members of the polyphenolic compounds, are present in high concentrations in medicinal plants and propolis, a natural beehive product. A water-soluble extract of propolis (WSDP) and two components of propolis, CA and CAPE were investigated for direct antitumor activity in vivo and in vitro. The local presence of CA and CAPE in the tissue caused a significant delay in tumor formation and increased life span 29.30 to 51.73%, respectively. CA and CAPE, but not WSDP, significantly suppressed human HeLa cervical carcinoma cell proliferation in vitro. Based on these results, we postulate that the antitumor activity of polyphenolic compounds includes direct cytotoxic effects on tumor cells.

Immunomodulatory and antimetastatic action of propolis and related polyphenolic compounds.

The effect of polyphenolic compounds isolated from propolis and propolis itself was investigated on the growth and metastatic potential of a transplantable mammary carcinoma (MCa) of CBA mouse. Metastases in the lung were generated by intravenous injection of tumor cells (2 x 10(5)). A water-soluble derivative of proplis (WSDP), caffeic acid (CA), caffeic acid phenethyl ester (CAPE) and quercetin (QU) were given to mice per os before tumor cells inoculation. Tested compounds significantly decreased the number of tumor nodules in the lung. According to the results obtained the antitumor activity of tested compounds can be related to the immunomodulatory properties of the compounds, their cytotoxicity to tumor cells, and their capacity to induce apoptosis and necrosis. The experimental data support that WSDP, CA, CAPE and QU could be potentially useful in the control of tumor growth in experimental models.

Leukocyte subsets and specific antibodies in pigs vaccinated with a classical swine fever subunit (E2) vaccine and the attenuated ORF virus strain D1701.

Total white blood cell (WBC) counts and percentages of CD4a+, CD8a+, CD5a+, CD45RA+, CD45RC+, wCD21+ and SWC3a+ cells in the peripheral blood of pigs were analysed in this study. Blood samples were collected before and on days 4, 10, 21 and 28 after vaccination. Group 1 pigs were vaccinated with a subunit E2 vaccine (gp E2 32 microg/dose), and Group 2 received a subunit vaccine combined with an attenuated ORF virus strain D1701 10(6.45) TCID50/dose. Control pigs received a placebo. The total WBC count and percentage of particular cell types were within the normal range in vaccinated and control pigs. Although the mechanism of attenuated ORF virus activity is not clear, changes were observed in CD4a+, CD5a+, CD8a+, CD45RA+ and CD45RC+ cells in pigs that received the combination of a subunit vaccine and ORF virus. However, the percentage of wCD21+ and SWC3a+ did not differ significantly from that recorded in pigs given only the subunit vaccine. At days 4 and 10 the number of pigs positive to E2 antibodies was higher in the group that received the subunit vaccine and ORF virus than in pigs vaccinated with the subunit vaccine only. A higher percentage of memory cells (CD45RC+) as well as Th and Tc lymphocytes in pigs that received the ORF virus and the subunit vaccine could be ascribed to a nonspecific influence of the ORF virus on the development (through cognate interactions between T and B cells) and the duration (presumed according to the finding of the clonal expression of memory cells) of humoral immunity (assessed by a higher number of seropositive pigs in this group). This seems likely since the proportion of these cells was found to be lower in the pigs that received E2 vaccine only.

Wild boars (Sus scrofa) as reservoirs of Brucella suis biovar 2 in Croatia.

This work presents the results of findings for brucellosis in wild boars and domestic swine in two regions of Croatia. In the region of Djakovo the blood samples of 211 wild boars were analysed and in 29.4% of the samples serologically positive reactions were established. In the same region the blood samples of 1080 domestic swine on pastures were also analysed and positive serological reactions were established in 12.3%. In the regions around Lonjsko Polje the blood samples of 53 wild boars were analysed and in 22.6% of them positive serological reactions were established. On several locations around Lonjsko Polje the blood samples of 901 domestic swine were serologically analysed and 13.5% of the swine were found to be seropositive. Bacteriological analyses of submitted materials from 24 wild boars resulted in isolation of Brucella from seven (29.2%) samples, and from 43 samples originating from domestic swine that had aborted and had been serologically positive, Brucella were isolated from 25 (58.1%) swine, as well as from 10 (62.5%) out of 16 aborted piglets. In all the isolates Brucella suis biovar 2 was identified. Wild boars are carriers and reservoirs of Brucella suis biovar 2 in Croatia.