Genotyping of Leptospira spp. in wild rats leads to first time detection of L. kirshneri serovar Mozdok in Serbia

Introduction: This study aimed to investigate the prevalence and molecular characterization of Leptospira species in Belgrade, Serbia, an area where this disease is underexplored. Specifically, the study sought to employ molecular and multilocus sequence typing analyses to fill the gap in understanding the diversity and distribution of Leptospira species within the region. Methods: A comprehensive molecular analysis was conducted on kidney samples obtained from Norway rats (Rattus norvegicus) in the urban environment. The study utilized molecular diagnostic techniques including real-time PCR targeting the lipL32 gene and performing sequence-based typing schemes utilizing adk, icdA, lipL32, lipL41, rrs2, and secY genes. These methodologies were applied to ascertain the presence and characterize different Leptospira species and serovars, respectively. Results: The findings revealed the presence of two Leptospira species and three separate serovars in the Belgrade area. This study identified the presence of L. kirschneri serovar Mozdok in Serbia for the first time, a significant discovery previously undocumented in the region. This pioneering investigation sheds light on the molecular diversity and prevalence of Leptospira species in Serbia. Discussion: The study underscores the importance of employing molecular typing methods to gain insights into the epidemiology and characterization of Leptospira species. These findings significantly contribute to both local and global perspectives on leptospirosis epidemiology, providing vital insights for the development of effective control strategies and interventions. Summary: In our recent study, we explored the presence and performed molecular typing of the Leptospira species, the bacteria responsible for leptospirosis, in wild rats in Serbia. This was the first time such a study was conducted in the region. Leptospirosis is a serious disease that affects both animals and humans, often transmitted through contact with water contaminated by infected animals. Our focus was on understanding which types of Leptospira were present in these animals. Excitingly, we discovered a particular strain of Leptospira, known as L. kirshneri serovar Mozdok, for the first time in Serbia. This finding is significant because it sheds light on the presence and spread of different Leptospira serovars in Serbia. It also raises awareness about the potential health risks associated with this serovar, which was previously unknown in the area. Our work fits into a broader context of disease surveillance and public health. By identifying the types of Leptospira present in a specific region, we can better understand the risks to public health and take steps to prevent and control the spread of leptospirosis. This discovery is not just important for scientists studying infectious diseases; it has real implications for public health officials, veterinarians, and anyone concerned with preventing and treating leptospirosis. Our findings highlight the need for ongoing monitoring of Leptospira in wildlife and synanthropic fauna, to protect both animal and human health

Hypothyroidism in pregnant rats affects proliferation and cell death of growth plate chondrocytes in pups

Overt and subclinical maternal hypothyroidism affects cartilage and bone extracellular matrix formation during fetal endochondral bone development. Less is known about the effect maternal hypothyroidism has on cell proliferation and death of growth plate chondrocytes. This study aimed to evaluate the expression of proliferation (Ki67), autophagy (LC3) and apoptosis (caspase 3) markers in the epiphyseal cartilage of the tibia in pups from subclinical and overt hypothyroid does, in their early postnatal development.Hypothyroidism was induced with propylthiouracil using low (1.5 mg/L) for subclinical (H1) and high dose (150 mg/L) for overt (H2) form. Propylthiouracil was administered through drinking water in pregnant Albino Oxford does from the first day of gravidity and during lactation. Control (C) group was not treated. Six, seven-day-old male pups from each group were euthanized. Histological examinations were performed on paraffin sections of the proximal tibial growth plate. Immunohistochemistry was used to assess the expression of Ki67, LC3 and caspase 3. The number of Ki67 positive chondrocytes was evaluated in the resting and proliferating zone using Image J. The expression of LC3 was performed by counting the number of positive dots in chondrocytes.The number of Ki67 positive chondrocytes was higher in both hypothyroid groups, while the number of LC3 positive dots per cell was lower indicating that the chondrocyte cell cycle in hypothyroid animals is faster and that the basal level of autophagy is reduced due to frequent mitosis. Compared to controls, caspase 3 expression in the terminal hypertrophic chondrocytes was reduced in H1 and completely absent in H2 pups which might indicate impaired differentiation.We conclude that both forms of maternal hypothyroidism in rats lead to accelerated proliferation, slowed autophagy and compromised apoptosis of terminal hypertrophic chondrocytes in the early infantile period and a delay in cartilage to bone transition

Macrophages, the main marker in biocompatibility evaluation of new hydrogels after subcutaneous implantation in rats

Biocompatibility of materials is one of the most important conditions for their successful application in tissue regeneration and repair. Cell-surface interactions stimulate adhesion and activation of macrophages whose acquaintance can assist in designing novel biomaterials that promote favorable macrophage–biomaterial surface interactions for clinical application. This study is designed to determine the distribution and number of macrophages as a means of biocompatibility evaluation of two newly synthesized materials [silver/poly(vinyl alcohol) (Ag/PVA) and silver/poly(vinyl alcohol)/graphene (Ag/PVA/ Gr) nanocomposite hydrogels] in vivo, with approval of the Ethics Committee of the Faculty of Veterinary Medicine, University of Belgrade. Macrophages and giant cells were analyzed in tissue sections stained by routine H&E and im munohistochemical methods (CD68+ ). Statistical relevance was determined in the statistical software package SPSS 20 (IBM corp). The results of the study in terms of the number of giant cells localized around the implant showed that their number was highest on the seventh postoperative day (p.o.d.) in the group implanted with Ag/PVA hydrogels, and on the 30th p.o.d. in the group implanted with Ag/PVA/Gr. Interestingly, the number of macrophages measured in the capsular and pericapsular space was highest in the group implanted with the commercial Suprasorb© material. The increased mac rophage number, registered around the Ag/PVA/Gr implant on 60th p.o.d. indicates that the addition of graphene can, in a specific way, modulate different biological responses of tissues in the process of wound healing, regeneration, and integration

Molecular prevalence of MecA and MecC genеs in coagulasе-positive staphylococci isolated from dogs with dermatitis and otitis in Belgrade, Serbia: a one year study

The escalating global concern of antimicrobial resistance in human and veterinary medicine is exacerbated by the inappropriate prescription of antibiotics for bacterial infections in companion animals. This study aimed to determine the distribution of coagulase-positive staphylococci causing clinical skin and ear infections in dogs and to determine methicillin-resistant isolates. A total of 78 staphylococcal strains were isolated from clinical samples taken from patients at the Dermatology Clinic at the Faculty of Veterinary Medicine in Belgrade, Serbia. Multiplex PCR was used for species-specific identification, and mecA and mecC genes were used to determine methicillin resistance, in addition to phenotypic determination, MIC values and detection of PBP2a. Out of the 78 samples analyzed, 65.8% were identified as Staphylococcus pseudintermedius, 22.4% as S. aureus, 7.9% as S. coagulans, and 3.9% as S. intermedius. Four S. aureus isolates exhibited methicillin resistance confirmed by cefoxitin disk diffusion, while five were confirmed with MIC testing and latex agglutination. MecA gene was detected in 29.4% of S. aureus and 30% of S. pseudintermedius isolates. These isolates were classified as methicillin- resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP), respectively. No isolates carried the mecC gene. This study provides insights into the prevalence of CoPS species and methicillin resistance in isolates from dogs. Continued surveillance is essential to monitor and understand the emergence and dissemination of antimicrobial resistance in veterinary medicine and the results of this study accent the need for establishment of a continuous antimicrobial resistance surveillance program in the Republic of Serbia

Fetlock Joint Cartilage Lesions in Working Horses – A PILOT STUDY

The horse is still used as a working animal, harnessed in a sled or wheeled chariots loaded with cargo several times heavier than the horse itself. There are no data on the extent of cartilage lesions in the fetlock joints of front and hind legs of working horses. The aim of the study was to determine the size and position of gross cartilage lesions in front and hind metacarpo-phalangeal (fetlock) joints of working horses. Front and hind legs were collected from a local abattoir after the working horses were slaughtered for the reasons not related to this study. The study was conducted on eight horses from 5 to 12 years old (mean 8.625 ± 0.91 years), with a body weight of 550 ± 29.46 kg. Thirty two fetlock joints were examined for external gross anatomy changes. After joint dissection, the cartilage of metacarpal and metatarsal bones was stained with blue ink and photos were taken. The cartilage lesions were stained deep blue, in contrast to light blue surface of the undamaged cartilage. The anatomical position of the lesions was recorded. The ImageJ software was used to measure the total surface of the cartilage and the surface of the cartilage under the lesions. The dimension of the lesions was expressed as a percentage of the total cartilage surface and presented as mean ± standard error. Gross anatomy changes like skin lesions, wounds, joint oedema or any type of deformities were not observed. The position of the cartilage lesions was symmetrical and occupied lateral and medial metacarpal and metatarsal condyles next to the sagittal ridge. The lesions on the lateral condyles had the same surface area like those on the medial, 12.3 ± 1.03% versus 10.7 ± 0.99%, respectively. The cartilage lesions on the front legs (23.62 ± 1.68%) were broader than those on the hind legs (22.5 ± 3.35%). The results of this pilot study indicate no difference between metacarpal and metatarsal cartilage surface lesions. Further studies are indicated to determine the severity of cartilage and putative subchondral bone lesions by histologic evaluationBook of Abstract

Application of histological and histochemical methods in kulen composition analysis

Book of Abstract

Pathology associated with highly pathogenic avian influenza H5N1 in naturally infected birds in Serbia in the 2021/2022 epidemiological year

The highly pathogenic avian influenza (HPAI) epidemic in the 2021/2022 epidemiological year was the most widespread in Europe, and HPAI H5N1 was by far the predominant virus type reported. In Serbia, since early 2021 and 2022, multiple outbreaks of HPAI have occurred. The H5N1 subtype was also dominant during this epizootic, although a few cases of H5N8 subtype infection were reported in mute swans. This epizootic affected backyard chickens (3 outbreaks), and the virus was also detected in wild birds, mostly in mute swans (4 outbreaks). All cases were reported in the north of Serbia. Here we describe the pathological findings of natural HPAI H5N1 infection in poultry species (chickens and turkeys) and wild birds (mute swans) that died during this epizootic. Routine necropsies were carried out on the carcasses of 15 mute swans, 15 chickens, and four turkeys. The external body and internal organs were examined grossly, and the gross pathology was recorded and photographed. Body condition was estimated based on the amount of body fat and musculature. Tissue samples of the brain, pancreas, spleen, and lungs were collected, and the supernatants of the tissue homogenates were used for molecular diagnosis by RT-qPCR method. The affected birds showed nervous manifestations (abnormal head position, tremors, leg paralysis) and all birds were in good condition. The presence of influenza virus was detected in tissue samples of all tested animals. The external macroscopic changes included cyanosis and necrosis in the crest and wattle, and these lesions were more pronounced in chickens. In mute swans, there were no external lesions. The H5N1 HPAI virus produced several consistent gross lesions among the species investigated. Foremost among these lesions was: multifocal pancreatic necrosis and hemorrhages, petechial hemorrhages in coelomic and epicardial fat and epicardial petechiae. The lungs showed moderate (turkeys) to severe (chickens and mute swans) diffuse congestion and oedema. In most cases, mild splenomegaly and spleen necrosis were noted. Additionally, gizzard or proventricular lesions were not observed in any bird. The natural HPAI H5N1 infection in poultry and mute swans showed similar clinical disease, including neurological disorders, as well as similar pathologic presentation involving necrotic lesions and vascular damage, primarily affecting the pancreas and myocardium. Evaluating the pathological presentation of natural disease is particularly important in emerging infectious diseases such as influenza A virus, in which different strains can have different pathogenicity and clinical presentations.Book of abstract

A Nocardia cyriacigeorgica strain causing severe bovine mastitis

Nocardia cyriacigeorgica је узрочник који се све чешће изолује у случајевима нокардиозе људи, међутим његова идентификација до нивоа врсте представља изазов за већину микробиолошких лабораторија. Инфекције изазване овом актиномицетом се релативно ретко сусрећу у ветеринарској клиничкој пракси при чему доступни литературни подаци обухватају мали број описаних случајева пиогрануломатозних лезија код животиња. Приказана студија описује случај тешког маститиса говеда на малом газдинству у Босни и Херцеговини изазваног узрочником N. cyriacigeorgica који је безуспешно третиран применом стандардне антибиотске терапије. С обзиром да изолат није идентификован применом конвенционалних микробиолошких техника, извршена је MALDI-ToF MS анализа чији су резултати били недовољно поуздани. У циљу утврђивања врсте анализирана је секвенца гена 16S субјединице рибозомалне рибонуклеинске киселине (16S rRNK). Наведени налаз је додатно потврђен применом технике секвенцирања више генских локуса (MLST) при чему су коришћене секвенце gyrB, 16S rRNK, secA1 и hsp65. Метода диск дифузије за испитивање осетљивости изолата N. cyriacigeorgica на антибиотике је примењена у складу са препорученим публикованим упутствима. Колико је ауторима познато, ово представља први случај маститиса говеда изазван узрочником N. cyriacigeorgica у Европи, али и први изолат Nocardia spp. пореклом од животиња за чију је идентификацију примењен MLST.Although Nocardia cyriacigeorgica is recognized as a cause of human nocardiosis and is regarded as an emerging pathogen, many clinical laboratories find it difficult to identify this organism at the species level. The infections caused by this actinomycete are relatively uncommon in veterinary clinical practice, and the limited information currently available mostly relates to several cases of pyogranulomatous lesions in animals. We describe a case of severe bovine mastitis in a small holding in Bosnia and Herzegovina caused by N. cyriacigeorgica that did not improve after standard antibiotic therapy. The causative agent could not be identified using standard microbiological techniques and MALDI-ToF MS analysis was performed, yielding unclear results. Subsequently, the species N. cyriacigeorgica was confirmed by 16S rRNA sequence analysis, which was further validated by multilocus sequence analysis (MLSA) using the gyrB, 16S rRNA, secA1, and hsp65 sequences. The disc diffusion method was used to test for antibiotic susceptibility in accordance with published guidelines. To the best of our knowledge, this is the first report of N. cyriacigeorgica isolation from a clinical case of bovine mastitis in Europe, as well as the first time a strain of Nocardia spp. isolated from animals has been distinguished using the MLSA method.Zbornik kratkih sadržaj

Animal models in bicompatibility assessments of implants in soft and hard tissues

The ethical dilemmas of using animals as in vivo models in preclinical and clinical examinations have been increasingly present in recent decades. Small laboratory animals (rats, rabbits) will continue to be used because they are cost-effective and permit the formation of statistically testable cohort groups; a task that, for financial, maintenance and care reasons, is almost prohibitive for larger animals. Technological advances in the production of new biomaterials for clinical use are enormous, but screening tests and methods used to assess biocompatibility lag behind these advances. The assessment of biological responses is slow and based on millennial recovery mechanisms in eukaryotic organisms. Therefore, the goal of researchers in this field is to re-evaluate old methods of biocompatibility assessment and introduce new methods of evaluation, especially for in vivo testing. In that sense, a revision of the ISO standards was planned and conducted in 2017, which insisted on cytotoxicity testing in cell lines and produced concrete proposals on how biocompatibility should be quantified. In vivo biocompatibility evaluation of biomaterials used for soft tissue recovery commonly utilises rats. Rabbits are recommended for implants used for hard tissues, because of the rabbit’s size, the possibility of implanting the biomaterials on a larger bone surface, and because of the peculiarities of rabbit bone tissue that favours rapid recovery after bone defects and enables easy reading of the results

Procena regenerativnih procesa mekih tkiva nakon supkutane implantacije srebro / poli (vinil alkohol) i novog srebro / poli (vinil alkohol) / grafenskog hidrogela na animalnom modelu

A newly produced biomaterial is necessarily subject of standards, which are performed in vivo on animal models. For the evaluation of soft tissue regenerative possibilities after subcutaneous implantation of biomaterials – silver/poly(vinyl alcohol) (Ag/PVA) and novel silver/poly(vinyl alcohol)/graphene (Ag/PVA/Gr) provided for clinical use, sixteen rats were used, according to the instructions of international standards, ISO 10993-6, 2007. Histological sections were observed 7, 15, 30 and 60 days after grafting. These hydrogels were produced by in situ electrochemical synthesis of silver nanoparticles in the polymer matrices, which enabled obtaining completely safe and biocompatible materials, free from any additional toxic chemical reducing agents. Surgical implantation of hydrogels was done according to the permission of the Ethical Committee of the Faculty of Veterinary Medicine, University of Belgrade. Immunohistochemical (IHC) studies included the assessment of smooth muscle expression actin in blood vessels (α-SMA), the expression of laminin and type I and type III collagen in the skin structures, and, the determination of cell proliferation marker expression (Ki-67) keratinocytes. The results were assessed in a semiquantitative manner. The data were analyzed in the statistical software package IBM SPSS 20. The conclusions indicated that Ag/PVA/Gr might be used as wound dressings to enhance the tissueBilo koji novoproizvedeni biomaterijal nužno podleže standardima, po čijim se protokolima sprovode in vivo istraživanja na životinjskim modelima. Za procenu mogućnosti regeneracije mekog tkiva nakon potkožne implantacije biomaterijala - srebra/poli(vinil alkohola) (Ag/PVA) i novog materijala srebra/poli(vinil alkohola)/grafena (Ag/PVA/ Gr) predviđenog za kliničku upotrebu, šesnaest pacova je korišćeno, po uputstvima međunarodnog standarda, ISO 10993-6 2007. Histološki uzorci su sakupljeni 7, 15, 30 i 60 dana nakon implantacije. Ovi hidrogelovi su proizvedeni in situ elektrohemijskom sintezom nanočestica srebra u polimernim matricama, što je omogućilo dobijanje potpuno sigurnih i biokompatibilnih materijala, bez ikakvih dodatnih toksičnih hemijskih sredstava za redukciju. Hirurška implantacija hidrogela urađena je prema dozvoli Etičkog komiteta Fakulteta veterinarske medicine Univerziteta u Beogradu. Imunohistohemijske (IHC) studije uključivale su procenu ekspresije aktina glatkih mišića u krvnim sudovima (α-SMA), ekspresiju laminina i kolagena tipa I i III u strukturama kože i određivanje ekspresije markera proliferacije (Ki-67) keratinocita. Rezultati su procenjeni semikvantitativnom analizom. Podaci su analizirani u statističkom softverskom paketu IBM SPSS 20. Zaključci su ukazali da se Ag/PVA/Gr može koristiti kao obloga za rane kako bi se povećao potencijal zarastanja tkiva i uspostavila brža integracija i kraće zadržavanje u tkivu