Division of Pathomorphology and Veterinary Forensics
1. Evaluation of the remodelling of myocardium in dogs with dilated cardiomyopathy (DCM) and myxomatous mitral valve disease (MMVD).
Team: Izabela Janus, DVM, Marcin Nowak DVM, PhD, Rafał Ciaputa DVM, PhD, Małgorzata Kandefer-Gola, DVM, PhD, prof. Janusz A. Madej, DVM, PhD (in collaboration with the Department of Internal Diseases: Agnieszka Noszczyk-Nowak, DVM, PhD, prof. Urszula Pasławska, DVM, PhD)
Dilated cardiomyopathy (DCM) and myxomytous mitral valve disease (MMVD) are the most common heart diseases in dogs. The first one occurs most often in large- and giant breed dogs and involves progressing loss of contractility and dilation of left ventricle with a secondary mitral regurgitation and left atrial enlargement. Structural changes can be accompanied by rhythm disturbances (mainly atrial fibrillation). MMVD is a disease in which mitral valve undergoes a progressive degeneration with thickening and stiffness of valve leaflets. Those changes cause mitral regurgitation and left atrial enlargement. Both diseases lead to escalation of heart insufficiency and often do animal’s death.
Both differences (breed predisposition, age of disease onset, various pathomechanism) and similarities (heart chamber enlargement) can be noted between those diseases.
Our research aims to a comparison of histopathological changes present in the myocardium of dogs with DCM and MMVD.
The majority of histopathological studies on these diseases concern on specimens obtained from left ventricle, therefore we begun our research from a comparison of specimens from ventricles and from atria in dogs with DCM and MMVD. We noted that myocardial changes seen in ventricles are expressed on the same or higher level in cardiac atria (Janus et al. 2015).
In the second stage of the research, we compared the histopathological changes (fibrosis, fatty infiltration, vascular changes, cardiomyocyte degeneration) in left atrial specimens from dogs with DCM and MMVD. We have noted that, although both diseases are accompanied by left atrial enlargement, the changes in heart tissue have various character (Janus et al. 2016).
In the next part of our research, we performed an immunohistochemical evaluation of left atrial specimens from dogs with either DCM or MMVD, using various cell markers (desmin, vimentin, periostin, caspase-3, sarcomeric actin, smooth muscle actin, SERCA proteins, dystrophin). In that examination we also found significant differences between these diseases (Janus et al. 2016; Janus et al – paper not yet published).
Our research shows that despite similarities, changes occurring in heart muscle in DCM and MMVD have various character. The differences noted in our work can contribute to better understanding of mechanism underlying these two diseases, that result in clinical signs (e.g. rhythm disturbances).
2. An immunohistochemical evaluation of neoplastic lesions in bitch mammary glands using novel cell markers.
Team: Borecka Paulina, DVM, Marcin Nowak, DVM PhD
Mammary gland tumours are one of the most common neoplasms in bitches. Despite wide range of publications in that topic, usefulness of various cell markers used in human medicine, has not been yet examined in dogs. In our research we will evaluate the reaction of novel neoplastic markers of tumour stroma and lymphatic and blood vessels.
The research will be conducted on specimens of mammary gland neoplasms that were collected during surgery or after death from bitches of various age and breed. Both benign and malignant tumours will be subject of our study. Also the age of an animal and tumour diameter will be analysed. Control group will consist of normal mammary glands form bitches of various breed and age that underwent euthanasia or sudden death and in the necropsy did not show neoplastic lesions, especially in mammary gland.
Specimens from mammary gland will be fixed in 7% buffered formalin, dehydratated and placed in paraffin blocks. A preliminary histopathological examination will be performed after standard haematoxylin-eosin staining basing on Goldschmidt classification. Next, an immunohistochemical analysis, using various markers (Ki67, HER2, podoplanin, vimentin) will be performed. Both monoclonal and polyclonal antibodies will be used in the study. The immunohistochemical reaction will be evaluated using IRS (immune-reactive score) scale, that take into consideration both the percentage of antibody-positive cells and the intensity of reaction. Photomicrographs will be taken from the examined specimens, and statistical analysis (using Statistica 12 for Windows, StatSoft, Poland) will be performed on the results.
The aim of the study is to try to better understand the essence of cancerogenesis and to try to find new markers that can be used in evaluating the prognosis of the disease.
3. Immunohistochemical diagnostics of gonads in dog and cats with disorders of sex development.
Team: Stanisław Dzimira, DVM, PhD, prof. Janusz Madej, DVM, PhD
Sex differentiation disorders (disorders of sex development - DSD) are associated with its unusual differentiation at the chromosomal, gonadal or somatic level. The current division into three main groups DSD in humans, i.e., DSD sex chromosomes, XY DSD and DSD XX was adopted also for dogs and cats.
The aim of the research is immunohistochemical analysis of the gonads of healthy dogs and cats, and animals affected by the disorders of sex development, using antibody markers cytokeratin, desmin, vimentyn, smooth muscle actin, non-specific enolase, inhibin ?, MCM3. The material for the study are the gonads of ten dogs, phenotypic females surgically removed and sent for histopathological examination. And the testes of four cats with disorders of sex development. The control group are the ovaries and testes of ten healthy dogs and testes from five healthy cats obtained after routine surgery gonadectomy. Material fixed in formalin, embedded in paraffin and cut to 4 microns thick sections, stained by routine hematoxylin and eosin. Immunohistochemistry was performed using mono- or polyclonal antibodies from DAKO, ie. Vimentin, desmin, smooth muscle actin, S100, NSE, inhibin alfa and MCM3 proteins.
Division of Microbiology
Identification and characterization of coagulase-negative staphylococci isolated from intramammary infections (IMI) in cattle. Working team: Jarosław Król, Anna Wanecka
In many countries, coagulase-negative staphylococci (CNS) have been isolated more and more frequently from cases of IMI in cattle (Pyörälä i Taponen, 2009; Lange i wsp. 2015). There is an increasing evidence that these microorganisms represent a heterogeneous population that vary in pathogenicity, specific host-pathogen relationships and resistance to antimicrobials (Zadoks i Watts, 2009; Podkowik i wsp., 2012). Therefore, accurate species identification of CNS associated with mastitis in cows may be of practical importance for the determination of both epidemiologic (e.g. possible sources and modes of transmission of a microorganism), as well as clinical aspects of the infection. Our research is focused on elaboration of the most useful methods of the identification of CNS by various diagnostic methods (sequencing of the 16S rRNA and “housekeeping” genes, MALDI-TOF technique, determining of phenotypic properties) (an article and doctoral dissertation of Anna Wanecka are in preparation).
The knowledge on CNS isolated from intramammary infections in cattle, despite intensive research of many investigators, seems to be still insufficient. It turns out that IMIs may be, to a higher and higher degree, caused by atypical and rarely encountered CNS that are poorly characterized clinically and epidemiologically. An example of such microorganisms may be Staphylococcus microti, detected by us in a group of animals on a dairy farm (Król i wsp. 2016). We found out that all isolates of that species, recovered in the nearly 2-year-period, belonged to the same genotype, thus suggesting a contagious form of mastitis. This is a novel finding because CNS have been so far considered to be environmental pathogens, not spreading from cow to cow easily. A similar situation may apply to a closely related species, Staphylococcus rostri, isolated by us on another farm (an article under preparation).
Epidemiology of fungal infections of the C. neoformans/C. gattii. - Magdalena Florek
Cryptococcosis is an infectious disease of humans and animals occurring worldwide. It is assumed, about 1 mln of cases of human infections is noted annually, among them 62,5% eventually ends with death of a patient. Data concerning frequency of cryptococcosis in animals is lacking, though it was estimated that during the largest outbreak of Cryptococcus gattii infections that took place in Canada, number of animal cases observed was about 75% higher than those of human cases. Cryptococcosis, associated mainly with CNS and respiratory infections, is caused by a complex of fungi occurring in an environment and belonging to the genus Cryptococcus (C. neoformans/C. gattii complex). The main source of infectious propagules of the pathogen are tree and pigeon feces.
Using several genotyping methods, eight molecular types were distinguish among the complex, which differ in presence of virulence factors, course of infection they cause, epidemiology, geographic extent, ecological niche and mating type. There are reports showing that the most virulent molecular types display different resistance patterns against antimycotic drugs. The molecular types also correspond with the new classification of C. neoformans/C. gattii complex, which was introduced in 2015.
In Poland, cryptococcosis is not notifiable disease, neither animal nor humans cases, epidemiological investigation considering an environmental contamination with above mentioned fungi have never been carried on, moreover Cryptococcus strains occurring in the country are usually not identified to the species/molecular type level.
In order to evaluate the occurrence of fungi belonging to the C. neoformans/C. gattii complex, the isolation is carried out from environmental samples (tree swabs, samples of soil, pigeon feces). Obtained environmental isolates as well as clinical strains originating from animals and humans (cooperation with Wrocław Medical University) are analyzed using MLST method and their mating type is determined.
Research planned will allow to assess the occurrence of fungi of C. neoformans/C. gattii complex in the environment of Lower Silesia area as well as to compare frequency of occurrence of particular molecular types among clinical and environmental isolates.
Abortogenic viruses in horses - virological and epizootiological studies. Working team: Barbara Bażanów, Łukasz Gadzała
Abortogenic viruses are one of the main reason of abortion in horses. These includes equine arteritis virus (EAV), equine herpesvirus type 1(EHV-1) and sporadically equine herpesvirus type 4 (EHV-4). Infection of these pathogens causes huge economic losses and problems with international trade of horses. These infections are dangerous, because often there are not limited to individual animals, and often spread within the herd. Additionally, these viruses cause many respiratory symptoms such as dyspnea, serous, mucous or purulent disharge from the nose or coughing. Similar symptoms are observed during infection of other, widely widespread in Poland viruses of horses, which influence on a health status and productivity of these animals: equine influenza virus (Orthomyxoviridae) H7N7 and H3N8 and horses rhinoviruses ERhV type A, B1 and B2 (Picornaviridae). West Nile virus (WNV) and USUTU virus begin to appear in horses in the country, that in addition to the general symptoms cause inflammation of the brain and spinal cord. The purpose of the research carried out by the laboratory is monitoring of the epizootic situation of abortion and respiratory diseases in horses (EAV, EHV, ERhV and equine influenza virus), and determination of the WNV infection level by using: virus isolation method in cell cultures and hemagglutination and hemagglutination inhibition tests. Seroconversion of WNV is tested in cooperation with National Health Laboratory Service in Johannesburg (South Africa) using ELISA test. blood serum, semen, organs of aborted fetuses, nasal swabs and archival materials from horses are used in the investigation. In addition, genetic studies are carried out on isolated viruses. Other activities include work on newly synthesized chemicals as potential disinfectants. Experiments on the cytotoxicity and virucidal properties of these compounds are carried out in cooperation with the University of Wrocław, and substances meeting the criteria of disinfectants may be used to disinfection of stables or skin and mucous membranes in horse. In addition to the standard methods, techniques based on luminescence and fluorescence are used. A similar series of studies on the substances of plant origin is carried out in cooperation with the Department of Phytopathologyof UP in Wrocław.
Virological, serological and molecular diagnosis of selected viral infections in birds living in the wild. - Barbara Bażanów
Viruses affecting wild birds in Polish are poorly know yet. The overal goal of this study is isolation and identification of viruses from sick or dead birds naturally occurring in the wild. Research includes a wide range of tests: isolation of infectious agents in embryonated chicken eggs, continuous cell lines RK-13, BHK and Vero, and in the primary chicken embryo fibroblast cell culture. Further experiments are based on the TEM and molecular biology techniques ,in which isolated an cytophatogenic agent is checked for belonging to one of a few selected families of viruses, e.g. herpesviruses, paramyxoviruses, poxviruses and filoviruses.
