Patogenost metacerkarija Clinostomum complanatum (Digenea: Clinostomidae) za klena (Squalius cephalus) i šarana (Cyprinus carpio) u prirodnim uvjetima

Metacercariae of the digenetic trematode Clinostomum complanatum Rudolphi, 1814 were observed in chub (Squalius cephalus) and common carp (Cyprinus carpio) from the Orljava River (Croatia). Both the prevalence and infection intensity were higher in the chub (prevalence 23.4%, average intensity 70.5) than in the common carp (5.9%, 4.5). In general, the metacercariae were located in the wall of the anterior part of the digestive tract and branchial cavity, the supraocular region, and the hypaxial musculature near the paired fins. Histology revealed the obvious affinity of the parasite for striated muscles and connective tissue. In situ, the metacercariae were surrounded by wellvascularized connective tissue capsules composed primarily of collagen fibers, fibroblasts, and fibrocytes. This study provides some insight into the parasite’s pathogenicity and the relationship between host and parasite.Metacerkarije dvodomnog metilja Clinostomum complanatum Rudolphi, 1814 utvrđene su u klena (Squalius cephalus) i šarana (Cyprinus carpio) podrijetlom iz rijeke Orljave (Republika Hrvatska). Prevalencija i intenzitet invazije veći su u klena (prevalencija 23,4% i srednji intenzitet 70,5), nego u šarana (5,9% i 4,5). Općenito, metacerkarije se smještaju u stjenku prednjeg dijela probavne cijevi i škržne šupljine, područje oko oka i hipaksijalno mišićje oko parnih peraja. Histološki nalaz pokazuje afinitet metilja za skeletno mišićno tkivo i vezivno tkivo. In situ, metacerkarije okružuje dobro vaskularizirana vezivnotkivna kapsula građena pretežno od kolagenih vlakana, fibroblasta i fibrocita. Ovo istraživanje doprinosi razumijevanju patogenosti parazita i daje uvid u odnos između domaćina i parazita

Morphological study of larvae of the genus Eustrongylides Jägerskiöld, 1909 (Nematoda: Dioctophymatidae) isolated from perch (Perca fluviatilis) in the Republic of Croatia

Oblići roda Eustrongylides (Dioctophymatidae) imaju složen razvojni ciklus koji uključuje maločetinaše i ribe kao međudomaćine te ribojedne ptice kao konačnog domaćina. U ribama se ličinke četvrtog stupnja razvijaju u tjelesnoj šupljini, ispod seroznih ovojnica i u mišićju, a ljudi se mogu invadirati konzumacijom sirove ili termički nedovoljno obrađene ribe. U Europi je posljednjih godina zabilježen veći broj nalaza vrste Eustrongylides excisus u grgeča. O prisutnosti ovog oblića u Republici Hrvatskoj nema podataka u dostupnoj literaturi. Pregledom grgeča podrijetlom iz rijeke Kupe, jezera Jarun i Lonjskog polja utvrđena je prisutnost ličinaka iz roda Eustrongylides. Ovo istraživanje daje detaljan morfološki i morfometrijski prikaz ličinaka četvrtog stupnja izdvojenih iz grgeča. Dobiveni podaci pridonose dosadašnjim spoznajama o rasprostranjenosti ličinaka roda Eustrongylides u Europi.Nematodes of the genus Eustrongylides (Dioctophymatidae) have a complex life cycle, which includes oligochaetes and fish as intermediate hosts, and piscivorous birds as definitive hosts. In fish, fourth stage larvae can be found in the body cavity, under serous membranes, and in the musculature. The parasite has a zoonotic potential and humans can get infected by consumption of raw or undercooked fish. Recently, there have been several reports on the occurrence of Eustrongylides excisus larvae in perch in Europe. There are no data on the presence of Eustrongylides larvae in fish in Croatia. Examination of perch collected from the Kupa River, Jarun Lake and Lonjsko polje revealed the presence of Eustrongylides sp. larvae. In this work, a detailed morphological and morphometric description of fourth stage larvae from perch is given. These data contribute to the current knowledge about the distribution of Eustrongylides larvae in Europe

Causes and laboratory diagnostic of pleural effusions in dogs

Pleuralne izljeve definiramo kao patološko nakupljanje tekućine u pleuralnoj šupljini, a koji u pasa nastaju posljedično različitim upalnim, sistemskim, metaboličkim, kardiovaskularnim, infekcijskim i neoplastičnim bolestima te traumama, a dijelimo ih na transudate, modificirane transudate i eksudate. Uzroci pleuralnog izljeva su, patofiziološki gledano, porast hidrostatskog tlaka (pr. posljedično zastojnom zatajivanju srca), pad onkotskog tlaka (pr. posljedično hipoproteinemiji), povećana propustnost stijenke krvnih žila (najčešće posljedično upalnoj reakciji) i opstrukcija limfnih žila (najčešće posljedično tumorskom rastu). Najčešći klinički znak u pasa s patološkim pleuralnim izljevima je otežano disanje (respiratorni distres, dispneja). Ostali klinički znaci uključuju: skvrčen, ležeći položaj s ispruženim glavom i vratom, disanje otvorenim ustima, ubrzano i naglašeno abdominalno disanje, a u uznapredovalih slučajeva prisutna je i cijanoza. Postupkom torakocenteze, osim što uklanjamo pleuralni izljev, možemo ga i prouzročiti, a daljnja laboratorijska dijagnostička obrada je ključna za postavljanje sumnje (ponekad i konačne dijagnoze) na uzrok ili uzroke koji su doveli do patološkog nakupljanja tekućine u pleuralnoj šupljini. Svrha je ovog preglednog rada prikazati uzroke i specifične vrste izljeva u pleuralnu šupljinu u pasa, kao i dijagnostički pristup istima.Pleural effusions are defined as pathologic accumulation of free fluid in the pleural cavity, and in dogs they arise from a variety of inflammatory, systemic, infectious and neoplastic conditions, metabolic disorders, trauma and cardiovascular diseases. Pleural effusions are typically classified as transudates, modified transudates, and exudates. From a pathophysiologic standpoint the accumulation of free fluid can be caused by (1) an increase in hydrostatic pressure (as is the case with congestive heart failure), (2) a decrease in oncotic pressure (i.e. due to hypoproteinemia), (3) increased permeability of the vessel wall (most often as a result of inflammatory reaction) and lymphatic obstruction (most often due to tumor growth). Dogs with pathologic pleural effusions often show heavy breathing (respiratory distress, dyspnea) as the most common clinical sign. Other clinical signs include a crouched, sternal recumbent position with extension of the head and neck, open - mouth breathing, tachypnea, forceful abdominal respiration and cyanosis. Thoracocentesis is used to drain the free fluid, but also to sample pleural effusion, as further laboratory examination is crucial to establish a suspicion and sometimes a definitive diagnosis of the cause or causes that led to the pathological accumulation of fluid in the pleural cavity. The purpose of this review is to present the causes and specific types of effusions in the pleural cavity in dogs, as well as their diagnostic approach

Honey vs. Mite—a trade-off strategy by applying summer brood interruption for Varroa destructor control in the Mediterranean region

Ectoparasitic mite Varroa destructor with its associated viruses is a common global threat to the health of honey bee colonies. If colonies are not treated, the vast majority die in a 3-year period. Existing acaricides used for treatment are becoming less effective, and new approaches to honey bee protection are required. A reliable method is to create a broodless condition in a colony by preventing the queen from laying eggs, and after 25 days all mites will be exposed to the treatment with organic acids or essential oils. The focus of our study, performed on 178 colonies in six Mediterranean countries, was to compare different periods of queen caging on honey production, colony development, and the effect of treatment. Queen caging had no negative effect on colony strength before the wintering period, while it affected honey production; colonies in which queens were caged two weeks before the main summer nectar flow produced significantly less honey. However, tested colonies ten weeks after the treatment had significantly lower infestation with V. destructor mites. This study shows that caging the queen with subsequent oxalic acid treatment 25 days after caging is an efficient method to control V. destructor infestation, while the starting point of queen caging in relation to the main summer nectar flow affects honey production

DIAGNOSTIC AND PROGNOSTIC SIGNIFICANCE OF CLINICAL AND LABORATORY PARAMETERS IN EFFUSIONS IN DOGS

Izljev je patološko nakupljanje tekućine u abdominalnoj, pleuralnoj ili perikardijalnoj šupljini, do kojeg dolazi zbog jedne ili više bolesti, poput trauma, neoplazija, kardiovaskularnih bolesti, metaboličkih poremećaja te infektivnih i upalnih bolesti. Izljevi se tradicionalno klasificiraju prema koncentraciji ukupnih proteina i broju stanica s jezgrom. Prema tom sustavu klasifikacije razlikujemo transudate, modificirane transudate i eksudate. Laboratorijske pretrage izljeva dijagnostički su postupak kojim se može posumnjati na proces koji je uzrokovao patološko nakupljanje tekućine u tjelesnoj šupljini. Modificirano klasificiramo izljeve prema mogućem uzroku na: (1) transudate siromašne proteinima, (2) transudate bogate proteinima, (3) septičke eksudate, (4) neseptičke eksudate, (5) izljeve podrijetlom od rupturiranih krvnih žila i visceralnih organa (hemoragijski, limforagijski ili hilozni izljevi, uroperitoneum i bilijarni peritonitis) i (6) izljeve nastale zbog ljuštenja stanica. Osim istraživanja laboratorijskih pokazatelja (najčešće obuhvaćaju citološke i biokemijske pretrage) provedenih istovremeno u izljevima u tjelesne šupljine i serumima istraživane populacije pasa, dio je rada usmjeren i na proširivanje spoznaja o drugim biomarkerima, kao što su egzosomi. Egzosomi bi postali pomoćni alat u dijagnostički najizazovnijih tumorskih i septičkih bolesti kod pasa, koje se očituju izljevom u njihove tjelesne šupljine. U dosadašnjim istraživanjima malo se pažnje posvećivalo ispitivanjima egzosoma u tjelesnim šupljinama pasa, tako se s ciljem proširivanja tih spoznaja istražilo egzosomne proteine flotilin 1, vaskularnu staničnu adhezijsku molekulu 1, adiponektin i protein CD63 u serumu i izljevu u tjelesne šupljine te ustanovilo njihove međusobne korelacije kako bi se otkrile molekule pomoću kojih je moguće razlikovati uzroke procesa, posebice u razlikovanju tumorskih od septičkih procesa. Cilj je ovog istraživanja bio otkriti praktične dijagnostičke pokazatelje u pasa s izljevima u tjelesne šupljine, koji bi u svakodnevnom kliničkom radu omogućili lakšu etiološku klasifikaciju izljeva, pomogli u postavljanju prognoze primarnog procesa te utvrditi statističku značajnost omjera koncentracije odabranih biokemijskih pokazatelja u serumu i izljevu (tumorske i netumorske etiologije, septičke i neseptičke etiologije). Najboljim laboratorijskim pokazateljima u razlikovanju tumorskih od netumorskih te septičkih od neseptičkih pleuralnih i abdominalnih izljeva pokazao se omjer LDH. Ekspresija odabranih VIII egzosomnih proteina poput flotilina 1, adiponektina i CD63 pridonosi razlikovanju izljeva uzrokovanih tumorskim procesom od izljeva nastalih zbog septičkih procesa. Flotilin 1 i CD63 pojačano su eksprimirani i u tumorskim i septičkim procesima, dok je ekspresija flotilina 1 i adiponektina, znatno veća u serumima pasa koji boluju od tumora, te može poslužiti za razlikovanje tumorskih od septičkih procesa kod pasa.Effusion is a pathological accumulation of fluid in the abdominal, pleural, or pericardial cavity that results from one or more diseases, such as trauma, neoplasia, cardiovascular disease, metabolic disorders, and infectious and inflammatory diseases. Accumulation of fluid in body cavities results from an imbalance in fluid production and removal. Effusions are traditionally classified according to the concentration of total proteins and total nucleated cell count (TNCC). According to the above classification system, we distinguish between transudates (total proteins < 2,5 g/dl, TNCC < 1500 cells/μl), modified transudates (total proteins 2,5 – 7,5 g/dl, TNCC 1000 – 7000 cells/μl) and exudates (total proteins > 3,0 g/dl, TNCC > 7000 cells/μl). Laboratory examinations of effusions represent a diagnostic procedure that can aid in revealing the pathological process responsible for the accumulation of fluid in the body cavity (abdominal, pleural and pericardial). Collection and evaluation of fluid from body cavities may be a therapeutic as well as a diagnostic procedure. A modified classification of effusions on the basis of the possible cause includes the following groups: (1) protein-poor transudates, (2) protein-rich transudates, (3) septic exudates, (4) non-septic exudates, (5) effusions originating from ruptured blood vessels and visceral organs (hemorrhagic, lymphoragic or chylous effusions, uroperitoneum, and biliary peritonitis) and (6) effusions resulting from cell exfoliation. Very few proteomic studies of effusions have been conducted in veterinary medicine. Extracellular vesicles (EV), like exosomes are now recognized as important mediators of cell-cell communication. EV contain cell specific cargo, including specific sets of proteins, lipids, miRNA, mRNA and DNA and are also important regulators of cancer progression. Many studies indicate that exosomes such as flotillins play an important role in many biological processes like cell proliferation, apoptosis, adhesion and cell proliferation. Flotillins are overexpressed in various tumor processes and are closely related to tumor development, grade and metastasis. This study was performed in dogs with clinical signs of effusion in the pleural, pericardial and/or abdominal cavity. Dogs were admitted to the Clinic for Internal Medicine of the Faculty of Veterinary Medicine, University of Zagreb and referred for diagnostic and therapeutic procedures. The study included a total of 134 dogs, with clinical signs indicating the presence of effusions in body cavity or more cavities. Clinical examination, hematological and biochemical blood analysis, as well as electrocardiogram, x-ray and ultrasound findings served in the diagnosis of patients with effusions into body cavities. Dogs with clinical signs were divided into subgroups that consisted of non-tumor transudate, tumor transudate, non-tumor modified transudate, tumor modified transudate, non-septic non-tumor exudate, non-septic tumor exudate, septic non-tumor exudate and septic tumor exudate. Tumor related effusions were identified by finding of malignant cells in the effusion using cytological or histopathological examination. Patients with septic effusions were diagnosed based on the presence of intracellular bacteria in the smear and a positive bacterial culture. Patients with effusions which did not show any systemic disease, but were also negative for tumor, inflammatory or septic processes, were classified as idiopathic effusions. Abdominocentesis, thoracocentesis and pericardiocentesis were diagnostic as well therapeutic procedures for removal of large effusions. All body cavity centesis procedures were performed using aseptic technique, including proper hair removal and disinfection of the skin with chlorhexidine or povidone-iodine scrub before entering the body cavity. Blood for complete blood count was sampled in ethylenediaminetetraacetic acid (EDTA) tubes, and blood for biochemical tests in serum separating tubes containing gel. Additionally, the serum was separated by centrifugation at 1500 x g for 10 minutes, and was further used for biochemical tests. The effusion fluid was sampled in two tubes as well as blood samples (Vacutainer, Becton Dickinson, USA). Complete blood counts were performed on Animal Blood Counter (ABC, Horiba ABX, Diagnostics, Montpellier, France) using the manufacturer’s original solutions with the dog’s blood test device settings. Differential blood count was determined on May-Grünwald-Giemsa stained smears by counting 100 leukocytes under light microscope BX41 (Olympus, Japan). Biochemical parameters were determined in serum and in effusion on Architect c4000 biochemical analyzer (Abbott Laboratories, Illinois, USA) with original Abbott chemicals (Abbott Laboratories, Illinois, USA). Of the XI biochemical parameters, total proteins, albumin, glucose, aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and canine C-reactive protein (canCRP) were determined. For effusion samples, hematocrit and total nucleated cell count were also determined. Total nucleated cell count was determined from the effusion collected in a tube with anticoagulant EDTA, using an automated hematology analyzer (Horiba ABX, Montpellier, France). Within 30 minutes after effusion sampling, smears were made, directly and/or from resuspended sediment of centrifuged effusion (depending on appearance, cell number and effusion density). The smear was stained by May-Grünwald-Giemsa method. In this study, a semiquantitative characterization of exosomal marker proteins flotillin 1, adiponectin, VCAM1 and CD63 was performed on selected serum and effusion samples of dogs with septic and tumor effusions. The exosomal proteins were detected by the Western blot method on the VMR Mini Vertical PAGE System (VWR International Ltd., Northern Ireland) and on the Electroblotting System (Mini Electroblotting System, Biovit, UK). Samples were loaded onto size exclusion chromatography columns (qEV, IZON, Oxford) to separate extracellular vesicles from soluble proteins. In addition to testing hypothesis of the existence of differences in the obtained values between the types of effusions, an analysis of other factors on the subject properties was performed. The statistical program R (version R 3.6.1., R DEVELOPMENT CORE TEAM, 2017) was used to check the significance and to include other factors that influenced the variability of properties in the model by the least squares method and the car package for analysis of variance. A corrected mean value (Least Square Means, LSM) was calculated for each property due to the unequal number of observations per impact classes using the emmeans package. The same package also tested the existence of statistically significant differences between the individual factors included in the model. Statistical significance of differences between arithmetic means in the studied dogs was tested by Bonferoni test. Of the study dogs 108 were males (81%) and 26 females (19%). As can be seen, a significant representation of males in relation to females is evident. Looking at the representation by body weight and size, dogs in the study group were divided into three breed groups according to the European Kennel Club and American Kennel Club classification, so that the population structure was: 20 dogs of small breeds (15%), 37 medium-sized (28%) ) and 77 large dog breeds (57%). The study included dogs with effusions in the body cavity, of which 81 (60,5%) were effusions in the abdominal cavity, 42 (31,3%) effusions in the pleural and 11 (8,2%) effusions in the pericardial cavity. The most common clinical signs in dogs with pleural effusions included dyspnea, respiratory distress, shrunken laying position with outstretched head and neck, open mouth breathing, rapid and pronounced abdominal breathing and in advanced cases, cyanosis was present. Dogs with abdominal effusion most often showed signs of lethargy, weakness, and abdominal tension, and in cases of peritonitis, abdominal pain was present. Clinical signs in dogs with pericardial effusion included weakness, lethargy, tachypnea, physical intolerance, collapse and cough. Modified transudates were the most common effusion in the body cavities of examined dogs (57,5 %). Neoplasia was a common cause of effusions in studied dogs. Adenocarcinoma and carcinoma of lungs and other visceral organs, lymphoma, hemangiosarcoma, histiocytic sarcoma and mesotelioma were the most common causes of tumor-related transudates. Non-tumor modified transudates were preceded by heart diseases, such as idiopathic dilated cardiomyopathy and endoventricular valve endocardiosis, while the remaining cases involved idiopathic effusions and trauma. The aim of this study was to discover practical diagnostic indicators in dogs with effusions in their body cavities, which would allow easier etiological classification of effusions in everyday clinical work. This could also help to predict the primary process outcome and determine the significance level of the ratio of selected biochemical parameters in serum and effusion (tumor and non-tumor etiologies, septic and non-septic etiologies). The application of an established algorithm for the classification of effusions based on the total nucleated cell count and total proteins in the effusion is a useful initial step in the diagnosis, which must be supplemented by additional biochemical and cytological parameters. The combined application of diagnostic procedures (biochemical analysis of effusion and serum, cytological analysis of effusion and advanced laboratory methods) was shown to be useful in determining the cause of effusion. The investigated laboratory indicators are of greater diagnostic importance in effusions than in the serum. TNCC showed the highest values in non-septic tumor exudates, both in dogs with pleural and abdominal effusions. The lowest TNCC was found in transudates. Total proteins showed similar values in sera of dogs from all investigated groups of effusions. Differences found in the values of total proteins in effusion samples were significant only for abdominal effusions. Glucose concentration in dogs with abdominal effusions is a a useful indicator for differentiation of septic tumor from septic non-tumor exudates, as well as non-tumor modified transudates from tumor modified transudates. Determination of lactate dehydrogenase (LDH) concentration in dogs with pleural and XIII abdominal effusions facilitates the etiological classification of effusions. LDH ratio (effusion/serum) showed to be the best laboratory indicator for distinguishing tumor from non-tumor and septic from non-septic causes both in pleural and abdominal effusions. Glucose ratio (effusion/serum) and CRP ratio (effusion/serum) in abdominal effusion have shown to be good indicators for distinguishing the causes of individual effusions (tumor and non-tumor etiology; septic and non-septic etiology). Cytology was shown to be a valuable diagnostic procedure in the investigated population of dogs with effusions, especially for detection of malignant cells, inflammatory cells and microorganisms (mostly intracellular bacteria). In this work, sensitivity of cytology for identification of septic peritonitis was 95%. Most of the pericardial effusions were hemorrhagic and were linked to the presence of hemangiosarcoma, idiopathic pericardial effusion and mesotelioma. The most common cell types found in pericardial effusions were macrophages, neutrophils and reactive mesotelial cells. Cytological examination was hampered by the proliferation of mesotelial cells which can mimic malignant cells. In addition to the research of laboratory indicators (most often including cytological and biochemical tests) in effusions and sera of the entire studied population of dogs, part of the work is focused on expanding knowledge about other biomarkers, such as exosomes. Their potential as an auxiliary tool in the diagnosis of the most common and diagnostically most challenging tumor and septic diseases in dogs with effusions into their body cavities was investigated. In previous studies, little attention has been paid to the study of exosomes in the body cavities of dogs, so in order to expand this knowledge, exosomal proteins such as flotillin 1, vascular cell adhesion molecule 1, adiponectin and CD63 protein were investigated in serum and effusions to discover the molecules by which it is possible to distinguish the causes of processes, especially in distinguishing tumor from septic processes. Expression of selected exosomal proteins such as flotillin 1, adiponectin and CD63 was found useful for distinguishing tumor-related effusions from septic effusions. Flotillin 1 and CD63 are overexpressed in both tumor and septic processes. The expression of flotillin 1 and adiponectin are significantly higher in the serum of dogs with tumors and may serve to distinguish tumor from septic processes in dogs. VCAM1 as a molecule which expression is closely related to tumor angiogenesis, metastases, severe inflammation and chronic conditions of some diseases, was not found as a marker of increased expression in septic and tumor conditions that cause effusions in the body cavities of dogs. Exosomes play a significant role as mediators in sepsis and in the spread of cancer cells. Early diagnosis and access to treatment can prevent serious complications in dogs with existing effusions in body cavities, regardless of various etiological factors. Therefore, it is crucial to identify biomarkers that can be applied in clinical practice. Using selected laboratory parameters and their ratios and increased expression of exosomal proteins in serum and effusions into the body cavities of dogs, effusions caused by the tumor process can be distinguished from effusions caused by septic causes

Effectiveness of the HACCP system in schools of Ptuj and Ormož area

V specialističnem delu smo želeli predstaviti pomen varne hrane za šoloobveznega otroka, opisati pomen HACCP sistema, njegove prednosti in pomanjkljivosti, ter ugotoviti učinkovitost HACCP sistema v osnovnih šolah ptujskega in ormoškega območja. Z namenom ugotovitve učinkovitosti HACCP sistema smo analizirali poročila o mikrobioloških preskusih odvzetih vzorcev živil in brisov na snažnost, v obdobjih pred in po uvedbo HACCP sistema, ter jih med seboj primerjali. Raziskovalni obdobji sta bili torej ločeni na obdobje od leta 1998 do 2002 in 2004 do 2008. Leto 2003 v raziskavo ni bilo vključeno saj smo smatrali to leto kot uvajalno, s predpostavko, da HACCP sistem v obratih še ni zaživel. Metodologija raziskovanja. Raziskava temelji na obdelavi podatkov, mikrobiološko analiziranih vzorcev živil in brisov na snažnost, odvzetih v osnovnih šolah ptujskega in ormoškega območja, pridobljenih od Zavoda za zdravstveno varstvo Maribor. Rezultati. Ugotovili smo, da se je sicer delež neustreznih brisov v petletnem obdobju po uvedbi HACCP sistema znižal v primerjavi s petletnim obdobjem pred uvedbo HACCP sistema in sicer za 1,8 % (iz 7,5 % na 5,7 %), vendar razlika ni statistično pomenljiva (p=0,067). Zaskrbljujoče je dejstvo, da se je zvišal odstotek neustreznih živil in sicer za 6,4 %. Vzrok neustreznih živil je sicer v največji meri pripisati higienski oporečnosti, kar pomeni, da so živila neustrezna zaradi preseženega skupnega števila mikroorganizmov, vendar pa sta bili dve živili tudi zdravstveno oporečni, torej s prisotnostjo patogenih bakterij. Sklep. Ugotovitve v okviru specialističnega dela nakazujejo, da v šolah ptujskega in ormoškega območja HACCP sistem ni v celoti obvladovani in v primerjavi s prejšnjo ureditvijo ni doprinesel k večji varnosti hrane.In the specialist work we wanted to present the importance of safe food for schoolchildren, describe the importance of the HACCP system, its strengths and weaknesses and to determine the effectiveness of HACCP system in elementary schools in Ptuj and Ormož area. With the intention to establish the effectiveness of the HACCP system, we analyzed the microbiological tests of food samples and swabs of cleanliness, taken in the periods before and after the introduction of HACCP system, and compared them with each other. Research periods were divided on the periods from 1998 to 2002 and from 2004 to 2008. The 2003 survey was not included, because we considered this year as an introductory phase, assuming that HACCP system in plants has not yet been fully realized. Research Methodology. The research is based on data processing, microbiologically analyzed food samples and swabs of cleanliness, taken in elementary schools in Ptuj and Ormož area, which were acquired from the Institute of Public Health Maribor. Results. We have found out that the proportion of inadequate swabs during the five years after the introduction of HACCP system have decreased in comparison with the five-year period before the introduction of HACCP system, namely by 1.8% (from 7,5% to 5,7%), but the difference is not statistically significant (p=0,067). Disturbing fact is, that the percentage of inadequate food increased, namely by 6.4%. The main cause of inadequate food is unsuitable hygiene practice, which means that the food is inadequate due to excess of micro-organisms, but there were also two foodstuffs, which presented a risk for human health because of the presence of pathogenic bacteria. Conclusion. The findings in the context of specialist work suggests that the HACCP system in schools in Ptuj and Ormož area is not fully controlled and compared with the previous regime, it has not contributed to greater food security

DIAGNOSTIC AND PROGNOSTIC SIGNIFICANCE OF CLINICAL AND LABORATORY PARAMETERS IN EFFUSIONS IN DOGS

Izljev je patološko nakupljanje tekućine u abdominalnoj, pleuralnoj ili perikardijalnoj šupljini, do kojeg dolazi zbog jedne ili više bolesti, poput trauma, neoplazija, kardiovaskularnih bolesti, metaboličkih poremećaja te infektivnih i upalnih bolesti. Izljevi se tradicionalno klasificiraju prema koncentraciji ukupnih proteina i broju stanica s jezgrom. Prema tom sustavu klasifikacije razlikujemo transudate, modificirane transudate i eksudate. Laboratorijske pretrage izljeva dijagnostički su postupak kojim se može posumnjati na proces koji je uzrokovao patološko nakupljanje tekućine u tjelesnoj šupljini. Modificirano klasificiramo izljeve prema mogućem uzroku na: (1) transudate siromašne proteinima, (2) transudate bogate proteinima, (3) septičke eksudate, (4) neseptičke eksudate, (5) izljeve podrijetlom od rupturiranih krvnih žila i visceralnih organa (hemoragijski, limforagijski ili hilozni izljevi, uroperitoneum i bilijarni peritonitis) i (6) izljeve nastale zbog ljuštenja stanica. Osim istraživanja laboratorijskih pokazatelja (najčešće obuhvaćaju citološke i biokemijske pretrage) provedenih istovremeno u izljevima u tjelesne šupljine i serumima istraživane populacije pasa, dio je rada usmjeren i na proširivanje spoznaja o drugim biomarkerima, kao što su egzosomi. Egzosomi bi postali pomoćni alat u dijagnostički najizazovnijih tumorskih i septičkih bolesti kod pasa, koje se očituju izljevom u njihove tjelesne šupljine. U dosadašnjim istraživanjima malo se pažnje posvećivalo ispitivanjima egzosoma u tjelesnim šupljinama pasa, tako se s ciljem proširivanja tih spoznaja istražilo egzosomne proteine flotilin 1, vaskularnu staničnu adhezijsku molekulu 1, adiponektin i protein CD63 u serumu i izljevu u tjelesne šupljine te ustanovilo njihove međusobne korelacije kako bi se otkrile molekule pomoću kojih je moguće razlikovati uzroke procesa, posebice u razlikovanju tumorskih od septičkih procesa. Cilj je ovog istraživanja bio otkriti praktične dijagnostičke pokazatelje u pasa s izljevima u tjelesne šupljine, koji bi u svakodnevnom kliničkom radu omogućili lakšu etiološku klasifikaciju izljeva, pomogli u postavljanju prognoze primarnog procesa te utvrditi statističku značajnost omjera koncentracije odabranih biokemijskih pokazatelja u serumu i izljevu (tumorske i netumorske etiologije, septičke i neseptičke etiologije). Najboljim laboratorijskim pokazateljima u razlikovanju tumorskih od netumorskih te septičkih od neseptičkih pleuralnih i abdominalnih izljeva pokazao se omjer LDH. Ekspresija odabranih VIII egzosomnih proteina poput flotilina 1, adiponektina i CD63 pridonosi razlikovanju izljeva uzrokovanih tumorskim procesom od izljeva nastalih zbog septičkih procesa. Flotilin 1 i CD63 pojačano su eksprimirani i u tumorskim i septičkim procesima, dok je ekspresija flotilina 1 i adiponektina, znatno veća u serumima pasa koji boluju od tumora, te može poslužiti za razlikovanje tumorskih od septičkih procesa kod pasa.Effusion is a pathological accumulation of fluid in the abdominal, pleural, or pericardial cavity that results from one or more diseases, such as trauma, neoplasia, cardiovascular disease, metabolic disorders, and infectious and inflammatory diseases. Accumulation of fluid in body cavities results from an imbalance in fluid production and removal. Effusions are traditionally classified according to the concentration of total proteins and total nucleated cell count (TNCC). According to the above classification system, we distinguish between transudates (total proteins < 2,5 g/dl, TNCC < 1500 cells/μl), modified transudates (total proteins 2,5 – 7,5 g/dl, TNCC 1000 – 7000 cells/μl) and exudates (total proteins > 3,0 g/dl, TNCC > 7000 cells/μl). Laboratory examinations of effusions represent a diagnostic procedure that can aid in revealing the pathological process responsible for the accumulation of fluid in the body cavity (abdominal, pleural and pericardial). Collection and evaluation of fluid from body cavities may be a therapeutic as well as a diagnostic procedure. A modified classification of effusions on the basis of the possible cause includes the following groups: (1) protein-poor transudates, (2) protein-rich transudates, (3) septic exudates, (4) non-septic exudates, (5) effusions originating from ruptured blood vessels and visceral organs (hemorrhagic, lymphoragic or chylous effusions, uroperitoneum, and biliary peritonitis) and (6) effusions resulting from cell exfoliation. Very few proteomic studies of effusions have been conducted in veterinary medicine. Extracellular vesicles (EV), like exosomes are now recognized as important mediators of cell-cell communication. EV contain cell specific cargo, including specific sets of proteins, lipids, miRNA, mRNA and DNA and are also important regulators of cancer progression. Many studies indicate that exosomes such as flotillins play an important role in many biological processes like cell proliferation, apoptosis, adhesion and cell proliferation. Flotillins are overexpressed in various tumor processes and are closely related to tumor development, grade and metastasis. This study was performed in dogs with clinical signs of effusion in the pleural, pericardial and/or abdominal cavity. Dogs were admitted to the Clinic for Internal Medicine of the Faculty of Veterinary Medicine, University of Zagreb and referred for diagnostic and therapeutic procedures. The study included a total of 134 dogs, with clinical signs indicating the presence of effusions in body cavity or more cavities. Clinical examination, hematological and biochemical blood analysis, as well as electrocardiogram, x-ray and ultrasound findings served in the diagnosis of patients with effusions into body cavities. Dogs with clinical signs were divided into subgroups that consisted of non-tumor transudate, tumor transudate, non-tumor modified transudate, tumor modified transudate, non-septic non-tumor exudate, non-septic tumor exudate, septic non-tumor exudate and septic tumor exudate. Tumor related effusions were identified by finding of malignant cells in the effusion using cytological or histopathological examination. Patients with septic effusions were diagnosed based on the presence of intracellular bacteria in the smear and a positive bacterial culture. Patients with effusions which did not show any systemic disease, but were also negative for tumor, inflammatory or septic processes, were classified as idiopathic effusions. Abdominocentesis, thoracocentesis and pericardiocentesis were diagnostic as well therapeutic procedures for removal of large effusions. All body cavity centesis procedures were performed using aseptic technique, including proper hair removal and disinfection of the skin with chlorhexidine or povidone-iodine scrub before entering the body cavity. Blood for complete blood count was sampled in ethylenediaminetetraacetic acid (EDTA) tubes, and blood for biochemical tests in serum separating tubes containing gel. Additionally, the serum was separated by centrifugation at 1500 x g for 10 minutes, and was further used for biochemical tests. The effusion fluid was sampled in two tubes as well as blood samples (Vacutainer, Becton Dickinson, USA). Complete blood counts were performed on Animal Blood Counter (ABC, Horiba ABX, Diagnostics, Montpellier, France) using the manufacturer’s original solutions with the dog’s blood test device settings. Differential blood count was determined on May-Grünwald-Giemsa stained smears by counting 100 leukocytes under light microscope BX41 (Olympus, Japan). Biochemical parameters were determined in serum and in effusion on Architect c4000 biochemical analyzer (Abbott Laboratories, Illinois, USA) with original Abbott chemicals (Abbott Laboratories, Illinois, USA). Of the XI biochemical parameters, total proteins, albumin, glucose, aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and canine C-reactive protein (canCRP) were determined. For effusion samples, hematocrit and total nucleated cell count were also determined. Total nucleated cell count was determined from the effusion collected in a tube with anticoagulant EDTA, using an automated hematology analyzer (Horiba ABX, Montpellier, France). Within 30 minutes after effusion sampling, smears were made, directly and/or from resuspended sediment of centrifuged effusion (depending on appearance, cell number and effusion density). The smear was stained by May-Grünwald-Giemsa method. In this study, a semiquantitative characterization of exosomal marker proteins flotillin 1, adiponectin, VCAM1 and CD63 was performed on selected serum and effusion samples of dogs with septic and tumor effusions. The exosomal proteins were detected by the Western blot method on the VMR Mini Vertical PAGE System (VWR International Ltd., Northern Ireland) and on the Electroblotting System (Mini Electroblotting System, Biovit, UK). Samples were loaded onto size exclusion chromatography columns (qEV, IZON, Oxford) to separate extracellular vesicles from soluble proteins. In addition to testing hypothesis of the existence of differences in the obtained values between the types of effusions, an analysis of other factors on the subject properties was performed. The statistical program R (version R 3.6.1., R DEVELOPMENT CORE TEAM, 2017) was used to check the significance and to include other factors that influenced the variability of properties in the model by the least squares method and the car package for analysis of variance. A corrected mean value (Least Square Means, LSM) was calculated for each property due to the unequal number of observations per impact classes using the emmeans package. The same package also tested the existence of statistically significant differences between the individual factors included in the model. Statistical significance of differences between arithmetic means in the studied dogs was tested by Bonferoni test. Of the study dogs 108 were males (81%) and 26 females (19%). As can be seen, a significant representation of males in relation to females is evident. Looking at the representation by body weight and size, dogs in the study group were divided into three breed groups according to the European Kennel Club and American Kennel Club classification, so that the population structure was: 20 dogs of small breeds (15%), 37 medium-sized (28%) ) and 77 large dog breeds (57%). The study included dogs with effusions in the body cavity, of which 81 (60,5%) were effusions in the abdominal cavity, 42 (31,3%) effusions in the pleural and 11 (8,2%) effusions in the pericardial cavity. The most common clinical signs in dogs with pleural effusions included dyspnea, respiratory distress, shrunken laying position with outstretched head and neck, open mouth breathing, rapid and pronounced abdominal breathing and in advanced cases, cyanosis was present. Dogs with abdominal effusion most often showed signs of lethargy, weakness, and abdominal tension, and in cases of peritonitis, abdominal pain was present. Clinical signs in dogs with pericardial effusion included weakness, lethargy, tachypnea, physical intolerance, collapse and cough. Modified transudates were the most common effusion in the body cavities of examined dogs (57,5 %). Neoplasia was a common cause of effusions in studied dogs. Adenocarcinoma and carcinoma of lungs and other visceral organs, lymphoma, hemangiosarcoma, histiocytic sarcoma and mesotelioma were the most common causes of tumor-related transudates. Non-tumor modified transudates were preceded by heart diseases, such as idiopathic dilated cardiomyopathy and endoventricular valve endocardiosis, while the remaining cases involved idiopathic effusions and trauma. The aim of this study was to discover practical diagnostic indicators in dogs with effusions in their body cavities, which would allow easier etiological classification of effusions in everyday clinical work. This could also help to predict the primary process outcome and determine the significance level of the ratio of selected biochemical parameters in serum and effusion (tumor and non-tumor etiologies, septic and non-septic etiologies). The application of an established algorithm for the classification of effusions based on the total nucleated cell count and total proteins in the effusion is a useful initial step in the diagnosis, which must be supplemented by additional biochemical and cytological parameters. The combined application of diagnostic procedures (biochemical analysis of effusion and serum, cytological analysis of effusion and advanced laboratory methods) was shown to be useful in determining the cause of effusion. The investigated laboratory indicators are of greater diagnostic importance in effusions than in the serum. TNCC showed the highest values in non-septic tumor exudates, both in dogs with pleural and abdominal effusions. The lowest TNCC was found in transudates. Total proteins showed similar values in sera of dogs from all investigated groups of effusions. Differences found in the values of total proteins in effusion samples were significant only for abdominal effusions. Glucose concentration in dogs with abdominal effusions is a a useful indicator for differentiation of septic tumor from septic non-tumor exudates, as well as non-tumor modified transudates from tumor modified transudates. Determination of lactate dehydrogenase (LDH) concentration in dogs with pleural and XIII abdominal effusions facilitates the etiological classification of effusions. LDH ratio (effusion/serum) showed to be the best laboratory indicator for distinguishing tumor from non-tumor and septic from non-septic causes both in pleural and abdominal effusions. Glucose ratio (effusion/serum) and CRP ratio (effusion/serum) in abdominal effusion have shown to be good indicators for distinguishing the causes of individual effusions (tumor and non-tumor etiology; septic and non-septic etiology). Cytology was shown to be a valuable diagnostic procedure in the investigated population of dogs with effusions, especially for detection of malignant cells, inflammatory cells and microorganisms (mostly intracellular bacteria). In this work, sensitivity of cytology for identification of septic peritonitis was 95%. Most of the pericardial effusions were hemorrhagic and were linked to the presence of hemangiosarcoma, idiopathic pericardial effusion and mesotelioma. The most common cell types found in pericardial effusions were macrophages, neutrophils and reactive mesotelial cells. Cytological examination was hampered by the proliferation of mesotelial cells which can mimic malignant cells. In addition to the research of laboratory indicators (most often including cytological and biochemical tests) in effusions and sera of the entire studied population of dogs, part of the work is focused on expanding knowledge about other biomarkers, such as exosomes. Their potential as an auxiliary tool in the diagnosis of the most common and diagnostically most challenging tumor and septic diseases in dogs with effusions into their body cavities was investigated. In previous studies, little attention has been paid to the study of exosomes in the body cavities of dogs, so in order to expand this knowledge, exosomal proteins such as flotillin 1, vascular cell adhesion molecule 1, adiponectin and CD63 protein were investigated in serum and effusions to discover the molecules by which it is possible to distinguish the causes of processes, especially in distinguishing tumor from septic processes. Expression of selected exosomal proteins such as flotillin 1, adiponectin and CD63 was found useful for distinguishing tumor-related effusions from septic effusions. Flotillin 1 and CD63 are overexpressed in both tumor and septic processes. The expression of flotillin 1 and adiponectin are significantly higher in the serum of dogs with tumors and may serve to distinguish tumor from septic processes in dogs. VCAM1 as a molecule which expression is closely related to tumor angiogenesis, metastases, severe inflammation and chronic conditions of some diseases, was not found as a marker of increased expression in septic and tumor conditions that cause effusions in the body cavities of dogs. Exosomes play a significant role as mediators in sepsis and in the spread of cancer cells. Early diagnosis and access to treatment can prevent serious complications in dogs with existing effusions in body cavities, regardless of various etiological factors. Therefore, it is crucial to identify biomarkers that can be applied in clinical practice. Using selected laboratory parameters and their ratios and increased expression of exosomal proteins in serum and effusions into the body cavities of dogs, effusions caused by the tumor process can be distinguished from effusions caused by septic causes

The prevalence and impact of Babesia canis and Theileria sp. in free-ranging grey wolf (Canis lupus) populations in Croatia

Abstract Background Babesia spp. and Theileria spp. are important emerging causes of disease in dogs. Alongside these domesticated hosts, there is increasing recognition that these piroplasms can also be found in a range of wild animals with isolated reports describing the presence of these pathogen in foxes (Vulpes vulpes) and captive grey wolves (Canis lupus). The prevalence and impact of these infections in free-ranging populations of canids are unknown. To gain a better insight into the epidemiology and pathogenesis of piroplasm infections in free-ranging grey wolves, pathological and molecular investigations into captive and free-ranging grey wolves in Croatia were performed. Results The carcasses of 107 free-ranging wolves and one captive wolf were the subjects of post-mortem investigations and sampling for molecular studies. A blood sample from one live captured wolf for telemetric tracking was also used for molecular analysis. PCR amplification targeting the 18S RNA gene revealed that 21 of 108 free-ranging wolves and one captive animal were positive for Theileria/Babesia DNA. Subsequent sequencing of a fragment of the 18S RNA gene revealed that 7/22 animals were positive for Babesia canis while the other amplified sequence were found to be identical with corresponding 18S rDNA sequences of Theileria capreoli isolated from wild deer (15/22). Haematological and cytological analysis revealed the presence of signet-ring shaped or pear-shaped piroplasms in several animals with the overall parasite burden in all positive animals assessed to be very low. Pathological investigation of the captive animal revealed fatal septicemia as a likely outcome of hemolytic anaemia. There was little or no evidence of hemolytic disease consistent with babesiosis in other animals. Conclusion Importantly, the presence of B. canis in free-ranging grey wolves has not been described before but has been reported in a single fox and domestic dogs only. That B. canis infections cause disease in dogs but have little impact on wolf health possibly suggests that the wolf is the natural and the domestic dog is a secondary host. Surprisingly, the frequent finding of Theileria capreoli in wolves suggests that this Theileria species is not restricted to ungulates (cervids) but commonly infects also this carnivore species. Nevertheless, the potential role that these asymptomatically infected animals may play in the dispersal of these pathogens to susceptible sympatric species such as domesticated dogs requires further investigation