Research Assignments Competing for the Prešeren Award for the Year 2020

The topics of the research assignments that will compete for the Prešeren Award for the year 2020 are as follows:

1. The safety and efficacy of metronomic chemotherapy for the treatment of various types of canine cancer

Mentor: Prof. Dr. Nataša Tozon, co-mentor: Assis. Prof. Tanja Švara
 
The research assignment envisages a clinical study, which increasingly uses a current method of metronome therapy.
Twenty clinical studies on the use of metronomic chemotherapy have been published to date Of these, five studies are descriptions of clinical cases, while in 15 studies a larger number of dogs were included (Gaspar TB et al., 2018). In a study involving 30 dogs with soft-tissue sarcomas treated with metronomic chemotherapy (cyclophosphamide 10mg/m2/day and piroxicam 0.3mg/kg/day), the average survival time was 410 days, compared to just 211 days for 55 surgically treated dogs. Mild gastrointestinal signs occurred in 40% of dogs, and cystitis developed in only one dog (Penel N et al., 2012).

Hypotheses:

1. Metronomic chemotherapy with cyclophosphamide alone, as well as in combination with piroxicam/meloxicam or prednisolone/methylprednisolone, does not cause clinically notable adverse effects in the treatment of dogs.
2. With metronomic chemotherapy with cyclophosphamide at a dose of 12.5mg/m2/24 hours for a minimum of six months, an objective response to treatment and a statistically significant prolongation of the survival period of dogs with various types of cancers is achieved.

The purpose of the assignment is to verify the safety of therapy for treating patients and the efficacy in relation to different types of tumours in dogs.

In recent years, metronomic chemotherapy has been an increasingly used method of treatment, especially for soft tissue sarcomas. Therefore, further validation of the protocols of metronomic chemotherapy and its safe use requires further translational potential for human medicine.
The clinical study will include dogs with various histologically confirmed tumours. The method of selection of the metronomic chemotherapy will be agreed with the owner.

Prior to starting treatment a comprehensive clinical examination will be performed to determine the clinical stage of the disease, as well as an analysis of selected laboratory parameters (complete hematological analysis, coagulation profile and selected biochemical parameters in blood serum):

Patients will then be monitored on a monthly basis by assessing local and systemic responses to treatment based on the clinical stage of the disease and changes in the selected laboratory parameters.

Adverse side effects will be monitored according to the applicable criteria of the Veterinary Co-operative Oncology Group (VCOG) (Vet Comp Oncol 2: 194-213, 2004).
 

2. The Impact of Some Pathogens on Haemolymph in Bees

Mentor: Prof. Dr. Vlasta Jenčič, co-mentor: Assist. Dr. Metka Pislak Ocepek
 
In honeybees (Apis mellifera), the cellular elements of the haemolymph and the beneficial gut microbiota are actively involved in defence against infections by various pathogens. Non-native pathogens of European bee diseases, changes in bee grazing, and the environmental burden of various xenobiotics can be affected to varying degrees by these mechanisms. The research assignment will monitor the health and development of ten bee families in two hive colonies and bees will be sampled at regular intervals for withdrawal of haemolymph and other laboratory tests.

We will determine the impact of Varroa mite infestations (Varroa destructor) and infections of nosema spores (Nosema spp.) will be determined, as well as viruses on the cellular composition of haemolymph. Based on the results of these tests, the composition of intestinal microflora will also be analysed in some samples. There is very little information in the world of scientific literature on the composition of cellular structures of haemolymph in insects and almost none in bees.

This will be the first study in which we will open the little-known area of the composition of bee haemolymph in relation to various bee infections. The results will thus bring entirely newfound knowledge and will provide a starting point for further research in this area. The following methods will be used: clinical examinations of bee families, dissection of bees, haemolymph withdrawal, making cytological preparations, microscopy and detection of cellular elements, molecular diagnostic methods for the detection of pathogens, next-generation sequencing methods and statistical processing of results.

3. Gender Differences in the Brain in terms of Stress Axis Regulation  

Mentor: Assist. Prof. Neža Grgurevič  
 
Stress is currently one of the most studied factors in the field of neuroscience. Although gender differences in response to stress have been known for some time, very little is known about the causes of their origin and the impact of genetic factors on the stress response. In this research assignment, using the method of immunohistochemistry, or PCR, we will take a real-time look at whether gender differences exist in the expression of some important proteins in the brain that are involved in regulating stress and anxiety.

The research will focus primarily on the glucocorticoid, mineralocorticoid receptor, corticotropin releasing hormone and some of the enzymes involved in the synthesis of catecholamines. As a model, we will use the brains of mice without the gene for steroidogenic factor 1 (SF-1 KO), which are born without sex and adrenal glands. SF-1 KO mice thus separate by gender only after the presence of the XX and XY sex chromosomes. 

In order to study the stress response, adrenal glands of the same or opposite sex were transplanted into SF-1 KO mice. The mice were then tested in various behavioural tests. We compared them to wild-type mice. The preliminary results have shown an interesting influence of the genetic sex and the influence of the sex of the adrenal glands on the behaviour and concentration of corticosterone in the blood, thus the research assignment of focusing on the brain has a very good starting point for further work.
 

4. Primerjava različnih metod merjenja krvnega tlaka pri mačkah

Mentor: Associate Prof. Aleksandra Domanjko Petrič, co-author: Assist. Dr. Martina Krofič Žel
 
Measurement of blood pressure is very important in the monitoring of many cats with hypertension. Since a veterinary clinic is a stressful environment for cats, blood pressure levels are often falsely elevated. For this reason, performing blood pressure measurements and obtaining realistic blood pressure values is the most challenging part of the diagnosis of hypertension. False increases in blood pressure can result in years of inappropriate treatment of ‘hypertension’, which in turn can actually damage the kidneys in particular. There is currently no fully standard method of blood pressure measurement in cats, and the available oscillometric devices are not entirely reliable.

The purpose of the study is to compare the results of different methods of measuring blood pressure (a Doppler device with, and without, the use of headphones and the oscillometric petMAP device) and to determine whether not using headphones results in higher pressures or how much the use of the Doppler method differs from the aforementioned oscillometric method. The results of this assignment will determine whether the use of headphones when measuring with a Doppler device is appropriate or, by how much, pressure measured using the petMAP oscillometer differs from that measured by the Doppler method. Using the results, we will develop a blood pressure measurement protocol that delivers the most accurate results for cats, and in which stress is minimised.

Eighty adult cats of all breeds and both genders will be included in the study. Cats will not be sedated or anaesthetised when blood pressure measurements are taken. Cats will be clinical patients whose owners agree to participate in the study. For each cat, blood pressure will be measured in three ways in various orders: with a Doppler pressure gauge using headphones, without headphones, and an oscillometric petMAP pressure gauge.
 

5. Parameters of haemostasis in dogs with brachycephalic syndrome before and after surgical treatment

Mentor: Asst. Prof. Vladimira Erjavec, co-mentor: Assoc. Prof. Alenka German Saint
 
Brachycephalic dog breeds (pugs, French and English bulldogs, Boston terriers, Pekinese, shih-tzu, etc.) have become increasingly popular in recent years. Selective mating to achieve a distinctive appearance with a shortened facial area causes problems due to narrowing of the upper respiratory tract. Characteristic clinical signs of brachycephalic syndrome are loud and difficult breathing, digestive problems, difficulty sleeping, snoring, inability to exert physical effort and cyanosis, and collapse. Early recognition of the syndrome is of the utmost importance for the caretakers of animals to be able to decide on surgical treatment in a timely manner. In dogs with brachycephalic syndrome, as well as in healthy dogs of brachycephalic breeds, respiratory problems lead to a lack of oxygen, causing many systemic effects, including hypercoagulability.

The purpose of the study is to evaluate the health status of dogs in various stages of brachycephalic syndrome and to determine whether poor upper airway transience in these patients results in haemostasis disorders, which are manifested in increased platelet count, shortened prothrombin time (PT), and activated partial thromboplastin time (APTT), as well as increased concentration of D-dimers - the breakdown products of cross-linked fibrin. Furthermore, the aim is to determine the impact of surgical treatment on the aforementioned parameters of haemostasis. Given that there is no such information in existing literature, the proposed research represents an original contribution to science.

With the consent of their caregivers, dogs of different breeds and different ages with pronounced brachycephalic syndrome and those that are prone to developing brachycephalic syndrome and healthy dolichocephalic dogs will be included in the research. The latter will represent the control group. For the purposes of the study, blood samples will be taken before and 1 month after surgical treatment to determine haematological and biochemical tests, gas analysis of blood and parameters of haemostasis. This will also evaluate the health status of dogs with pronounced brachycephalic syndrome before and after surgical treatment, and provide important information on possible haemostatic disorders in these dogs and the impact of surgical treatment on selected parameters of haemostasis and on the health status and quality of life of brachycephalic dogs.
 

6. Assessment of the sedative action of cannabidiol during administration under general anaesthesia and its anxiolytic effect in rats

Mentor: Assoc. Prof. Tomaž Snoj, co-mentor: Assist. Prof. Matjaž Uršič
 
Cannabidiol is an active substance in the group of phytocannabinoids found in some cannabis plants. In human medicine, cannabidiol is used to treat pain and epilepsy. Above all, in the lay public, many medicinal effects have been attributed to cannabinoids based on different observations by individuals, however, these have often not been confirmed in the planned research. In the interest of the public, relevant information on the functioning of cannabinoids is important not only for the profession but for society as a whole.

The aim of the research is to:

• identify the possibility of using cannabidiol as a sedative to reduce the induction time of general anaesthesia
• evaluate the anxiolytic action of cannabidiol in stressed animals

In determining the sedative effect of cannabidiol, one hour prior to administration of general anaesthesia, animals in the four groups will be orally administered different doses of cannabidiol, and control animals in the fifth group will not receive cannabidiol. General anaesthesia will be performed using isoflurane. During the experiment, the induction time of general anaesthesia and the time of awakening for each animal will be determined.

When studying the anxiolytic action of cannabidiol, different doses of cannabidiol will be administered to the animals in four groups, while the fifth group will serve as a control. An hour after the application, movement will be completely restricted for one hour for each individual animal following the standard rodent stress procedure. Therefore, the behaviour of the animals will first be recorded in the open field and the recording will later be analysed. At the end of the behavioural test, blood will be taken from the animals to determine adrenocorticotropic hormone (ACTH), corticosterone, glucose and lactate levels.

Preclinical testing in laboratory rats will provide basic information on indications for cannabidiol use.

7. Efficacy of paromomycin for the treatment of bacterial enteritis and in reducing cryptosporidiosis in nursing calves

Mentor: Assoc. Prof. Jože Starič, co-mentor: Assoc. Prof. Irena Zdovc

 Enteritisi zaradi okužbe z Escherichio coli in invazijo s protozojem Cryptosporidium parvum so najpogostejše obolenje pri sesnih teletih. Klinično se kažejo z drisko, ki privede do dehidracije, acidoze, elektrolitskega neravnotežja, vročino in lahko poginom. Paromomicin je aminoglikozidni antibiotik za per os uporabo, ki se praktično ne resorbira sistemsko in je registriran za zdravljenje enteritisov povzročenih z E. coli pri sesnih teletih. V Združenem kraljestvu je registriran tudi za zdravljenje kriptosporidioze telet.

Enteritis due to Escherichio coli infection (E. coli) and invasion by the protozoa Cryptosporidium parvum are the most common disease in nursing calves. Clinically they manifest with diarrhoea, which leads to dehydration, acidosis, electrolyte imbalance, fever and even death. Paromomycin is an aminoglycoside antibiotic for oral administration (per os), which practically is not systemically resorbed and is registered for the treatment of E. coli-induced enteritis in nursing calves. It is also registered in the UK for the treatment of cryptosporidiosis in calves.

The study will treat a group of calves with enteritis due to E. coli infection with paromomycin according to the manufacturer's instructions, and will monitor the effectiveness of the treatment. In addition, the enteritis agent will be isolated prior to initiation of treatment and the susceptibility of the isolates to paromomycin will be tested. Since co-infections with bacteria and cryptosporidia are common, the efficacy of paromomycin on the secretion of cryptosporidium oocysts will also be checked by counting oocysts before and the day after completion of treatment. The study will, for the first time, test the effectiveness of the antibiotic paromomycin against E. coli bacterial isolates in calf enteritis in Slovenia. In parallel, for the first time in Slovenia, the efficacy of paromomycin against cryptosporidiosis, will be tested, for which there are very few publications in the world.
The research will mix clinical work, sampling and laboratory work.
 

8. Effect of pumpkin seeds and cloves (syzygium aromaticum) on the secretion of internal parasite eggs in small cattle

Mentor: Assist. Prof. Jožica Ježek, co-mentor: Assist. Prof. Aleksandra Vergles Rataj
 
In recent years, the occurrence of antiparasitic resistance has been reported in various parts of the world and in Europe (Waller, 1997; Sargison et al., 2001), mainly due to the frequent and inappropriate use of antiparasitics. Due to restrictions on the use of these drugs and the appearance of resistance, there is increasing interest and a need to find other ways to suppress parasites. Plants that are not only a source of nutrients but also contain bioactive substances with potential antiparasitic action are one option. Cucumber and pumpkin seeds are thought to accelerate the elimination of gastrointestinal tracts (Guarrera, 1999). Pumpkin seed extract reduced the survival potential of nematode larvae in vitro and reduced the excretion of nematode eggs in animals and, according to the authors, would be useful for controlling parasites (Grzybek et al., 2016).

Various medicinal effects (antioxidant, antimicrobial, larvicidal) have been attributed to cloves (Syzygium aromaticum). The most important active substance is eugenol, which also contains some other substances (Cortes-Rojas et al., 2014). Syzygium aromaticum extract (0.5 mg/ml) inhibited succinate dehydrogenase (SDH) activity in adult specimens of Haemochus contortus (H. contortus) nematodes in vitro, which reduces production of the ATP parasite and results in its death (Veerakumari et al., 2017). In another study, syzygium aromaticum extract acted ovicidally on H. contortus and caused the death of adult specimens (Charitha et al., 2017). In an in vitro experiment, syzygium aromaticum extract resulted in the death of the parasitic flatwork Fasciola gigantica. According to the authors, ground cloves are effective as antihelmintics (Kumar and Singh, 2014).

The research team would like to investigate the effect of pumpkin seeds and cloves on the secretion of eggs by internal parasites in small animals.

Suppression of parasitosis is crucial to the success of small cattle production. Different methods and strategies of parasite control have been investigated in numerous studies. In addition to their nutritional role, plants are also a source of bioactive substances with potential antiparasitic action. There is limited scientific evidence on the antiparasitic efficacy of herbal products, thus there is increasing interest in research in this field.

The research will include sheep or goats naturally invaded with internal parasites. Prior to the start of the research, parasitological examinations will investigate the excrement patterns of all animals and determine the degree of invasion by internal parasites. The colour of the mucous membranes and the condition of the animals will be evaluated. The animals will be randomly divided into three groups (10 animals each). One group will receive pumpkin seeds, the second group will be given ground cloves and the third group will serve as a control. Seven to 10 days after the end of treatment, a parasitological examination of the faeces of all animals will be performed and the efficacy of treatment will be evaluated by calculating the percentage reduction in the number of eggs/grams of faeces compared to pre-treatment values and the health status of the animals will be assessed.

9. Ubiquitous agents of mammary gland inflammation in dairy cows

Mentor: Prof. Dr. Andrej Pengov
 
S. dysgalactiae and S. uberis are of particular importance in the etiology of this type of udder inflammation. Streptococcus infections belonging to serological groups L and G are less common, with group C streptococci and species (faecium, faecalis) classified as enterococcus. A causal link between infection with these agents and damage to mammals, deficiencies when milking and poor hygiene in barns has been demonstrated. S. uberis is often isolated from the skin, oral cavity and tonsils in cows in infected herds. Individual animals can excrete S. uberis in faeces in large numbers. In barns where this type of mastitis is common, we can usually demonstrate a high concentration of streptococci and enterococci in animal bedding.
The aim is to identify the relationship between the individual mastitis pathogens using the methods described below and to determine the causes that affect the incidence of mammary gland infections of ubiquitous species. Mastitis caused by ubiquitous Sreptococcus species is common in herds where infections of Staphylococcus aureus and Streptococcus agalactiae have been eradicated or at least significantly reduced in recent years.

The following methods will be used: inspection of the barn (breeding method, milking, hygiene ...), sampling (milk, skin swab, bedding), bacteriological examination of the milk, including cell counts and antibiograms and analysis of results.

10. Comparison of different methods of isolation of C. difficile in meat and meat products

Mentor: Assist. Prof. Urška Jamnikar Ciglenečki

Clostridium difficile (C. difficile) is a gram-positive, sporogenic, anaerobic bacterium that can cause various diseases in humans and animals. The infection can be present without clinical signs, or it can be expressed as diarrhoea or colitis - in severe cases it can even lead to bowel perforation, sepsis and death. Numerous studies have been published comparing strains isolated in humans, animals and food, which highlight the possibility of transmitting these bacteria from animals to humans via food. C. difficile bacteria were isolated from different types of foods, mostly from foods of animal origin. In complex samples, such as meat and meat products, C. difficile is often present in the form of spores, which cause poor performance of the isolation of nucleic acid (DNA).

In order to determine the optimal DNA isolation process, three commercial kits will be used and, according to three different procedures, DNA will be isolated from meat samples and meat products, which will be inoculated with different C. difficile strains and known bacterial counts. A real-time PCR method will be performed to prove the DNA and compare the results of different procedures and the most appropriate one will be chosen.

The data obtained from the research will have a significant impact on the design of further research to be used in our laboratory, and will be published in a scientific journal to make it available to other researchers around the world.