Ekspresija antigena ćelijske proliferacije na mastocitima i njihov značaj za gradaciju mastocitoma pasa

Cutaneous mast cell tumours (MCTs) are one of the most common neoplasm in dogs. Originate from the bone marrow, from the mast cells line, but because of its location are being studied within skin tumors. MCTs show a highly variable biologic behavior and therefore several criteria have been proposed for their classification, including histological grading and cell proliferation markers. Cytoplasmic expression of CD117 or KIT protein (tyrosine kinasa receptor) has been found to correlate with higher histological grade and with a worsened prognosis. It has been revealed that there is a conection between the aberrant expression of CD117 and presence of c-kit mutations, although mutations were not present in all mast cell tumors with aberrant expression of KIT protein. Twenty four MCTs and 6 control specimens from 30 dogs of different age, breed and sex were included in this study. DNA isolated from the dog with mast cell tumor which had previously found to has specified mutation was used as a positive control for c-kit mutation. Formalin fixed and paraffin embedded tissue samples were stained with hematoxylin-eosin and toluidin blue and immunohistochemically tested for CD117, Ki-67, PCNA i CD45RA expression. DNA had been extracted from the same paraffin blocks and subsequent polymerase chain reaction amplification was performed using PE1 and PE2 primers. Fourteen of 24 MCTs were histologically high-grade, while ten were classified as a low-grade malignancy. Seven MCTs have more than six mitotic figures in 10 high-power fields. The presence of more than two multinucleated cells in 10 high-power fields was observed in 11 MCTs, while more than two cells with bizarre nuclei in 10 hpf were observed in 10 MCTs. Eight MCTs have more than 10% of neoplastic mast cells with nuclei about 8-10 microns in diameter. Of the total MCTs, 9 tumors are Ki-67 positive (7 mastocytomas of high and 2 mastocytomas of low-grade malignancy), while the expression of PCNA and CD45RA was observed in all 24 MCTs. Nine MCTs of high-grade malignancy showed aberrant cytoplasm labeling for CD117. Mutations of c-kit proto-oncogene were not detected in any of the tested MCTs. The presence of mitotic figures, multinucleated cells, bizarre nuclei and karyomegaly in 10 high-power fields, as well as the pattern of CD117 expression are prerequisite for the new classification and most important prognostic factor in dogs with mast cell tumor.Kutani mastocitomi su jedna od najčešći neoplazmi pasa. Vode poreklo iz kostne srži, iz linije opredeljene za mastocite i pokazuju vrlo promenljivo ponašanje, te je predloženo nekoliko kriterijuma za nji ovu klasifikaciju, uključujući istološku gradaciju i markere proliferacije ćelija Citoplazmatska ekspresija CD117 ili KIT proteina (tirozin kinaza receptor) dovodi se u vezu sa višim stepenima maligniteta mastocitoma i lošijom prognozom Dokazano je da postoji veza između aberantne ekspresije CD117 i postojanja c-kit mutacija, iako mutacije često nisu prisutne kod svih mastocitoma sa aberantnom ekspresijom KIT proteina. Ispitivanjima su obu vaćena 24 kutana mastocitoma i 6 kontrolni uzoraka kože od 30 pasa različitog uzrasta, rase i pola. Kao pozitivna kontrola za c-kit mutaciju korišćen je izolat DNK psa sa mastocitomom za koga je ranije utvrđeno da poseduje navedenu mutaciju Uzorci tkiva fiksiranog u formalinu i ukalupljenog u parafinske blokove, bojeni su hematoksilin-eozinom i toluidin blue metodom, a imunohistohemijski na CD117, Ki-67, PCNA i CD45RA. DNK je izolovana iz istih parafinskih blokova, a primenom PCR metode i korišćenjem specifični prajmera PE1 i PE2 amplifikovan je željeni fragment DNK. Četrnaest od 24 mastocitoma su prema istološkim osobinama svrstana u mastocitome visokog stepena maligniteta, dok su preostalih deset, mastocitomi niskog stepena maligniteta. Kod 7 mastocitoma prisutno je više od šest mitotskih figura na 10 polja velikog uvećanja Prisustvo više od dve multinuklearne ćelije na 10 polja velikog uvećanja uočeno je kod 11 mastocitoma, dok su više od dve ćelije sa bizarnim jedrom na 10 polja velikog uvećanja uočene kod 10 mastocitoma. Osam mastocitoma sadrži više od 10% anaplastični mastocita sa jedrima promera oko 8-10 mikrometara. Od ukupno 24 mastocitoma, devet je Ki-67 pozitivno (7 mastocitomima višeg i 2 mastocitomima nižeg stepena maligniteta), dok se ekspresija PCNA i CD45RA uočava kod svih mastocitoma. Devet mastocitoma višeg stepena maligniteta ispoljava aberantnu citoplazmatsku ekspresiju CD117 Ni kod jednog od ispitanih uzoraka mastocitoma nije detektovano prisustvo c-kit mutacije. Prisustvo mitotskih figura, višejedarni ćelija, bizarnih jedara i kariomegalije, kao i tip ekspresije CD117 predstavljaju preduslov za novu klasifikaciju i ključne prognostičke parametre kod pasa sa mastocitomima

Expression of proliferating cell antigens on mast cells and their significance for mast cell tumor classification

Kutani mastocitomi su jedna od najčešći neoplazmi pasa. Vode poreklo iz kostne srži, iz linije opredeljene za mastocite i pokazuju vrlo promenljivo ponašanje, te je predloženo nekoliko kriterijuma za nji ovu klasifikaciju, uključujući istološku gradaciju i markere proliferacije ćelija Citoplazmatska ekspresija CD117 ili KIT proteina (tirozin kinaza receptor) dovodi se u vezu sa višim stepenima maligniteta mastocitoma i lošijom prognozom Dokazano je da postoji veza između aberantne ekspresije CD117 i postojanja c-kit mutacija, iako mutacije često nisu prisutne kod svih mastocitoma sa aberantnom ekspresijom KIT proteina. Ispitivanjima su obu vaćena 24 kutana mastocitoma i 6 kontrolni uzoraka kože od 30 pasa različitog uzrasta, rase i pola. Kao pozitivna kontrola za c-kit mutaciju korišćen je izolat DNK psa sa mastocitomom za koga je ranije utvrđeno da poseduje navedenu mutaciju Uzorci tkiva fiksiranog u formalinu i ukalupljenog u parafinske blokove, bojeni su hematoksilin-eozinom i toluidin blue metodom, a imunohistohemijski na CD117, Ki-67, PCNA i CD45RA. DNK je izolovana iz istih parafinskih blokova, a primenom PCR metode i korišćenjem specifični prajmera PE1 i PE2 amplifikovan je željeni fragment DNK. Četrnaest od 24 mastocitoma su prema istološkim osobinama svrstana u mastocitome visokog stepena maligniteta, dok su preostalih deset, mastocitomi niskog stepena maligniteta. Kod 7 mastocitoma prisutno je više od šest mitotskih figura na 10 polja velikog uvećanja Prisustvo više od dve multinuklearne ćelije na 10 polja velikog uvećanja uočeno je kod 11 mastocitoma, dok su više od dve ćelije sa bizarnim jedrom na 10 polja velikog uvećanja uočene kod 10 mastocitoma. Osam mastocitoma sadrži više od 10% anaplastični mastocita sa jedrima promera oko 8-10 mikrometara. Od ukupno 24 mastocitoma, devet je Ki-67 pozitivno (7 mastocitomima višeg i 2 mastocitomima nižeg stepena maligniteta), dok se ekspresija PCNA i CD45RA uočava kod svih mastocitoma. Devet mastocitoma višeg stepena maligniteta ispoljava aberantnu citoplazmatsku ekspresiju CD117 Ni kod jednog od ispitanih uzoraka mastocitoma nije detektovano prisustvo c-kit mutacije. Prisustvo mitotskih figura, višejedarni ćelija, bizarnih jedara i kariomegalije, kao i tip ekspresije CD117 predstavljaju preduslov za novu klasifikaciju i ključne prognostičke parametre kod pasa sa mastocitomima.Cutaneous mast cell tumours (MCTs) are one of the most common neoplasm in dogs. Originate from the bone marrow, from the mast cells line, but because of its location are being studied within skin tumors. MCTs show a highly variable biologic behavior and therefore several criteria have been proposed for their classification, including histological grading and cell proliferation markers. Cytoplasmic expression of CD117 or KIT protein (tyrosine kinasa receptor) has been found to correlate with higher histological grade and with a worsened prognosis. It has been revealed that there is a conection between the aberrant expression of CD117 and presence of c-kit mutations, although mutations were not present in all mast cell tumors with aberrant expression of KIT protein. Twenty four MCTs and 6 control specimens from 30 dogs of different age, breed and sex were included in this study. DNA isolated from the dog with mast cell tumor which had previously found to has specified mutation was used as a positive control for c-kit mutation. Formalin fixed and paraffin embedded tissue samples were stained with hematoxylin-eosin and toluidin blue and immunohistochemically tested for CD117, Ki-67, PCNA i CD45RA expression. DNA had been extracted from the same paraffin blocks and subsequent polymerase chain reaction amplification was performed using PE1 and PE2 primers. Fourteen of 24 MCTs were histologically high-grade, while ten were classified as a low-grade malignancy. Seven MCTs have more than six mitotic figures in 10 high-power fields. The presence of more than two multinucleated cells in 10 high-power fields was observed in 11 MCTs, while more than two cells with bizarre nuclei in 10 hpf were observed in 10 MCTs. Eight MCTs have more than 10% of neoplastic mast cells with nuclei about 8-10 microns in diameter. Of the total MCTs, 9 tumors are Ki-67 positive (7 mastocytomas of high and 2 mastocytomas of low-grade malignancy), while the expression of PCNA and CD45RA was observed in all 24 MCTs. Nine MCTs of high-grade malignancy showed aberrant cytoplasm labeling for CD117. Mutations of c-kit proto-oncogene were not detected in any of the tested MCTs. The presence of mitotic figures, multinucleated cells, bizarre nuclei and karyomegaly in 10 high-power fields, as well as the pattern of CD117 expression are prerequisite for the new classification and most important prognostic factor in dogs with mast cell tumor

Ascaridia galli infection in laying hens and the results of in vitro efficacy of levamisole, piperazine and carvacrol, whether is necessary to change the deworming protocols?

We present a case of Ascaridia galli infection in laying hens on a farm in central Serbia. During the rearing period on litter, ascaridosis was diagnosed at 15 weeks of age by routine parasitological fecal examination. Pullets were treated with flubendazole for one week, and two weeks later the hens were moved to battery cages. The production results were within technological standards until the 23rd week and the medical health status was without any clinical symptoms. After that period weight loss began, the egg production dropped to 70% and eggs were of poor quality. Subsequently, severe feather pecking and an increase of mortality were reported. The postmortal examination showed severe anemia and several gross lesions in the liver, intestines, lungs, and kidneys. Different sizes of A. galli were found in the lumen of the duodenum and jejunum. Gross changes of the intestinal mucosa were present, such as a thickened intestinal wall with hemorrhagic spots, inflammation and necrotic patches. Histopathological examination showed marked changes in the intestines, liver and kidneys. All visible live parasites were collected and stored in Earles balanced salts, and females were used for in vitro susceptibility testing. Median lethal concentration (LC50) of piperazine, levamisole and carvacrol for A. galli was 119.7 mu M, 2.71 mu M and 3.26 mu M, were applied, respectively. Based on our results, it is likely that reinfection occurred after completed dehelmintization. In relation to the new circumstances and the regulation for laying hen welfare the deworming protocol should be changed in order to ensure successful dehelmintization. In order to prevent reinfection the treatment must be done at the end of the rearing period and thus be maximally effective

Ekspresija KIT receptora na kutanim mastocitomima pasa bez c-kit mutacija

Histopathological examination, grading, immunohistochemical staining and molecular genetic examinations are the proposed criteria that should be used for cutaneous mast cell tumors (CMCTs) classification. The presence of aberrant CD117 expression and mutations of the c-kit proto-oncogene could be an indicative parameter for final histological grading. Determination of the connection between the localization of KIT receptor expression and the histological grade of CMCTs without c-kit proto-oncogene mutations was the main goal of this study. The study included twenty four CMCTs and six control skin samples from 30 dogs of different ages, breed and sex. Formalinfixed and paraffin-embedded tissue samples were stained with hematoxylin-eosin and toluidine blue and immunohistochemically tested for CD117 expression. DNA was extracted from the same paraffin blocks and subsequent polymerase chain reaction amplification was performed using PE1 and PE2 primers. Degree of malignancy was determined based on the presence of mitotic figures, multinucleated cells, bizarre nuclei and karyomegaly in 10 high power fields. Based on histological features, fourteen of 24 CMCTs were of a high histological grade, while ten were classified as a lowgrade malignancy. CD117 cytoplasmic expression was observed in nine of fourteen high-grade malignancy CMCTs, which confirms the link between the aberrant CD117 expression and increased cell proliferation.Histopatološko ispitivanje, gradacija, imunohistohemijsko bojenje i molekularno genetska ispitivanja su predloženi kriterijumi za klasifikaciju kutanih mastocitoma pasa. Prisustvo aberantne ekspresije CD117 i mutacija c-kit protoonkogena moglo bi da bude indikativni parameter za finalnu histopatološku klasifikaciju mastocitoma. Glavni cilj ispitivanja je određivanje povezanosti mesta ekspresije KIT receptora i histološkog gradusa mastocitoma bez c-kit mutacija. Ispitivanjima su obuhvaćena 24 mastocitoma i 6 kontrolnih isečaka kože uzorkovanih od ukupno 30 odraslih pasa različitog uzrasta, rase i pola. Uzorci fiksirani u formalinu i ukalupljeni u parafinske kalupe su bojeni hematoksilin-eozinom, toluidin plavo metodom i imunohistohemijski primenom CD117 antitela. DNK je izolovana iz istih parafinskih kalupa, a PCR amplifikacija je izvršena primenom specifičnih prajmera PE1 i PE2. Stepen malignosti je utvrđen na osnovu prisustva mitotskih figura, multinuklearnih ćelija, ćelija sa bizarnim jedrom i kariomegalije na 10 polja velikog uveličanja. Na osnovu histopatoloških osobina, 14 od 24 mastocitoma su svrstani u visoki stepen maligniteta, dok je 10 mastocitoma niskog stepena maligniteta. Citoplazmatska ekspresija CD117 je uočena kod 9 od 14 mastocitoma visokog stepena maligniteta, što potvrđuje vezu između aberantne ekspresije CD117 i povećane ćelijske proliferacije

Salmonellosis in poultry breeding in view of introducing vaccination

With the increased demand of consumers for safe poultry products, efficient control of pathogenic microorganisms that cause diseases in humans is becoming the chief challenge in contemporary poultry production. Poultry meat, eggs and related products present the main source of salmonellosis in humans that occurs as a result of the consumption of contaminated food. Any serotype of Salmonella spp. that is not strictly linked to the host is capable of causing gastrointestinal disorders of various degrees in humans. However, according to the World Health Organization (WHO), Salmonella enterica serotype enteritidis has been the leading cause of salmonellosis in humans over the past 10 years. One of the available prophylactic measures is resorting to the vaccination of poultry against salmonellosis. Several vaccines against poultry salmonellosis, produced by different manufacturers, have been registered in Serbia. In order for these vaccines to become widely used in our country, it is necessary that the factors in primary poultry production are aquainted with the mechanisms of action of these vaccines, the effects achieved by their implementation, the economic feasibility, and the possibilities for monitoring in keeping with the valid legal regulations. Certainly, vaccination should not be taken as the only method in the prophylaxis of salmonellosis, but as one of the very important methods in a series of biosafety and other measures that should be applied on poultry farms for the successful curbing of salmonellosis infections

Effect of zeolite on health condition of canines

The paper shows the results of investigations of elementary hematological and biochemical blood parameters of German Shepherd dogs administered zeolite through feed daily for a period of 50 days. No undesired effects were established during the period of observation, and in fact there was a full normalization of the condition of the dogs that previously exhibited transitory symptoms of gastrointestinal tract disorders. No significant digressions were observed in the examined biochemical parameters of blood, either, that could indicate any harmful effects of zeolite, with the exception of the tendency toward a reduction in the concentrations of phosphorus and calcium, which was not manifest clinically as well during this period

Distribucija zvezdastih ćelija jetre i njihova uloga u nastanku parazitske fibroze i ciroze jetre kod domaćih životinja

Increasing of the extracellular matrix in rats, as well as in humans, occurs as a consequence of hepatic stellate cells (HSCs) activity. The objective of this work was to investigation the role of these cells in the development of fibrosis and liver cirrhosis which occurs as a consequence of infection of sheep and goats with large (Fasciola hepatica) and small (Dicrocoelium dendriticum) fluke. Liver samples taken from 12 cattle and 10 sheep infected under natural conditions with large and small fluke were fixed in formalin and embedded in paraffin. Paraffin clips were stained with hematoxylin- eosin and masson trichrome method, and immunohistochemical method for α-smooth muscle actin (α-SMA). All tested samples were divided into three groups according to histological criteria: livers of infected animals with the first degree of fibrosis, livers of infected animals with the second degree of fibrosis, and livers of infected animals with cirrhosis. Distribution of HSCs depended on the degree of liver fibrosis. Immunohistochemically reactive HSCs were predominantly placed in perisinusoidal space. In liver samples with cirrhosis, HSCs were placed on the periphery of pseudolobulus. Cells of a different shape and size were positive to α-SMA. HSCs play an important role in synthesis of components of extracellular matrix during the development of parasitic fibrosis and liver cirrhosis in domestic animals.Povećanje ekstracelularnog matriksa kod pacova, kao i kod ljudi, nastaje kao posledica aktivacije zvezdastih ćelija jetre (HSCs). Cilj ovog rada je izučavanje uloge ovih ćelija u razvoju fibroze i ciroze jetre koje nastaju kao posledice infekcija ovaca i goveda velikim (Fasciola hepatica) i malim (Dicrocoelium dendriticum) metiljem. Uzorci jetri 12 goveda i 10 ovaca koje su u prirodinim uslovima inficirane velikim i malim metiljem su fiksirani u formalinu i uklopljeni u parafin. Parafinski isečci su bojeni hematoksilin-eozinom i Mason-trihromnom metodom, kao i imunohistohemijskom metodom za α-glatko-mišićni aktin (α-SMA). Svi ispitani uzorci su prema histološkim kriterijumima podeljeni u tri grupe: jetre inficiranih životinja sa I stepenom fibroze, jetre inficiranih životinja sa II stepenom fibroze i jetre inficiranih životinja sa cirozom. Distribucija HSCs je zavisila od stepena fibroze jetre. Imunohistohemijski reaktivne HSCs su bile predominantno smeštene u perisinusoidnim prostorima. U uzorcima jetre sa cirozom HSCs su bile smeštene na periferiji pseudolobulusa. Ćelije različitog oblika i veličine su bile pozitivne na α-SMA. HSCs igraju važnu ulogu u sintezi komponenti ekstracelularnog matriksa tokom razvoja parazitske fibroze i ciroze jetre kod domaćih životinja

Current swine respiratory diseases morphology in intensive swine production in Serbia

Swine respiratory diseases represent one of the most frequent health issues in pig production worldwide. Despite the great progress that has been made in the field of diagnostics, control and prophylaxis, respiratory diseases still remain the most challenging health problem in modern commercial pig production. The list of infectious agents that cause respiratory diseases in swine is extensive and includes both, bacterial and viral pathogens. In Serbia, more than fifteen years after the introduction of modern vaccines, the list of bacterial pathogens related to swine respiratory infections still include Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida. On the other hand, most commonly involved viral pathogens are Porcine Reproductive and Respiratory Syndrome Virus, Swine influenza virus, Porcine circovirus type 2 and Pseudorabies virus. The morphological features of pneumonia where several agents are involved, depend on the predominant etiological agent. Expanding knowledge of the main pathogens associated with swine respiratory diseases and the effects of their interactions on the disease outcome is important for further investigations of lung diseases and implementation of control strategies in commercial pig populations in Serbia. This review discusses the latest findings on swine respiratory disease and current trends in Serbian pig production

On the devil\u27s curve

U članku se najprije opisuje veza krivulje s igrom diabolo prema kojoj je krivulja dobila i svoje originalno ime. Ukratko se izlaže povijest proučavanja krivulje, kao i osnovne definicije i pojmovi iz algebarske geometrije koji se koriste u daljnjem razmatranju. Potom se navodi definicija te razmatraju osnovna svojstva i osnovni oblici zapisa jednadžbe vražje krivulje, pri čemu se posebno izvodi parametarski oblik dotične jednadžbe. Kao tipičan primjer, zasebno se razmatra tzv. elektromotorna krivulja i njezina svojstva. Na kraju se daje jedna od mogućih elementarnih konstrukcija vražje krivulje pomoću jednakostrane hiperbole.In this article, we firstly describe the connection between the devil\u27s curve and the game of "diabolo" the curve is named after. We shortly describe the history of studying the curve, as well as basic definitions and terms of algebraic geometry used in the article. Then we give the definition, basic properties and basic forms of the equation of the curve, whereby we especially derive the parametric equation of the curve. As a typical example of the devil\u27s curve, we consider the electric motor curve and its properties. Finally, we consider one of the possible constructions of the devil\u27s curve using the equilateral hyperbola

Patohistološki i imunohistohemijski parametri značajni za prognozu i terapiju tumora pasa i mačaka

The development of tumours implies a disproportion between proliferation, growth and differentiation, which is accompanied by significant genotypic and phenotypic variations in the diseased. The scenario of multiple oncogenic effects on the inhibition of cell growth, apoptosis and maturation, on the stimulation of proliferation, on cell migration and tissue invasion, is responsible for the occurrence of tumours both in humans and in ani­mals. The qualitative and quantitative determination of biological prognostic factors in the tissue of affected cats and dogs directly indicates a possible tumour metastasizing, and consequently also the prognosis of neoplastic disease. Monitoring of clinical and biological prognostic factors is important for therapy and prediction of tumours in humans and animals. The determination of the proliferative potential of tumour cells by immunohistochemical detection of Ki-67 and PCNA is used in grading certain tumours in cats and dogs. It is also possible immunohistochemically to prove factors of angiogenesis and numerous activating and inhibiting proteins in the tumour tissue. In addition to other histological, clinical and biological parameters, they are significant for the further prognosis and therapy of neoplastic diseases.Razvoj tumora podrazumeva nesklad između proliferacije, rasta i diferencijacije, što je praćeno značajnim genotipskim i fenotipskim varijacijama kod obolele jedinke. Brojni onkogeni efekti inhibišu starenje ćelije, apoptozu i maturaciju, a stimulišu proliferaciju, ćelijsku migraciju i tkivnu invaziju kod ljudi i životinja. Kvalitativno i kvantitativno određivanje bioloških prognostičkih faktora u tkivu obolelih pasa i mačaka govori direktno o sposobnosti metastaziranja tumora, a time i o prognozi neoplastične bolesti. Praćenje kliničkih i bioloških prognostičkih faktora značajno je za terapiju i predikciju tumora kod ljudi i životinja. Određivanje proliferativnog potencijala tumorskih ćelija, imunohistohemijskom detekcijom Ki-67 i PCNA koristi se u gradiranju nekih tumora kod pasa i mačaka. Faktore angiogeneze i brojne aktivirajuće i inhibitorne proteine, moguće je takođe imunohistohemijski dokazati u tkivu tumora. Oni su uz ostale histološke, kliničke i biološke parametre značajni za dalju prognozu i terapiju neoplastičnih bolesti