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The influence of different stress parameters in racing pigeon flocks, such as the presence of diseases and environmental conditions at the time of the races, were described. A total of 96 racing pigeons from 4 pigeon flocks were examined, and health monitoring was carried out. No helminth eggs and coccidia were found. Trichomonas sp. was confirmed in subclinical form. Paramyxoviruses and avian influenza viruses were not confirmed, but circovirus infections were confirmed in all flocks. Chlamydia psittaci was confirmed in one flock. Blood samples were collected, and HI antibody titers against paramyxoviruses before and 25 days after vaccination were determined. To improve the conditions during racing and the welfare of the pigeons, critical points were studied with regard to stress factors during the active training season. Serum corticosterone levels were measured in the blood serum of four different categories of pigeons from each flock. Corticosterone levels were almost twice as high in pigeons from the category that were active throughout the racing season, including medium- and long-distance racing, compared to the other three categories that were not racing actively. Within five hours of the finish of a race, the average serum corticosterone level was 59.4 nmol/L in the most physically active category. The average serum corticosterone level in this category remained at 37.5 nmol/L one month after the last race.

Abstract Background Pigs were domesticated independently in Eastern and Western Eurasia early during the agricultural revolution, and have since been transported and traded across the globe. Here, we present a worldwide survey on 60K genome-wide single nucleotide polymorphism (SNP) data for 2093 pigs, including 1839 domestic pigs representing 122 local and commercial breeds, 215 wild boars, and 39 out-group suids, from Asia, Europe, America, Oceania and Africa. The aim of this study was to infer global patterns in pig domestication and diversity related to demography, migration, and selection. Results A deep phylogeographic division reflects the dichotomy between early domestication centers. In the core Eastern and Western domestication regions, Chinese pigs show differentiation between breeds due to geographic isolation, whereas this is less pronounced in European pigs. The inferred European origin of pigs in the Americas, Africa, and Australia reflects European expansion during the sixteenth to nineteenth centuries. Human-mediated introgression, which is due, in particular, to importing Chinese pigs into the UK during the eighteenth and nineteenth centuries, played an important role in the formation of modern pig breeds. Inbreeding levels vary markedly between populations, from almost no runs of homozygosity (ROH) in a number of Asian wild boar populations, to up to 20% of the genome covered by ROH in a number of Southern European breeds. Commercial populations show moderate ROH statistics. For domesticated pigs and wild boars in Asia and Europe, we identified highly differentiated loci that include candidate genes related to muscle and body development, central nervous system, reproduction, and energy balance, which are putatively under artificial selection. Conclusions Key events related to domestication, dispersal, and mixing of pigs from different regions are reflected in the 60K SNP data, including the globalization that has recently become full circle since Chinese pig breeders in the past decades started selecting Western breeds to improve local Chinese pigs. Furthermore, signatures of ongoing and past selection, acting at different times and on different genetic backgrounds, enhance our insight in the mechanism of domestication and selection. The global diversity statistics presented here highlight concerns for maintaining agrodiversity, but also provide a necessary framework for directing genetic conservation.

Mycobacteria potentially pathogenic for humans are commonly present in aquarium fish sold in Slovenian pet shops. Mycobacterium marinum is the most important causative agent of skin mycobacteriosis in humans. The purpose of the present study was to evaluate, by means of a questionnaire, aquarium hobbyists’ and pet shop salespersons’ awareness of the health risks related to aquarium fish handling. A total of 198 participants took part in the study, 76.3% of these were aquarium hobbyists, and 23.7% were pet shop salespersons with up to 15 years of fish handling experience. About one third (35.8%) of all participants recognized that fish may be a source of infection for humans and that fish may contract tuberculosis. Fewer (24.4%) were aware of the fact that fish tuberculosis is a zoonotic disease. The vast majority of respondents were unfamiliar with the clinical manifestations of either fish tuberculosis or cutaneous mycobacteriosis in humans. Despite the generally acknowledged belief that aquarium water may pose a risk to human health, the respondents’ aquarium handling practices were surprising as the majority never used waterproof gloves. Several differences were revealed between the two groups of respondents. Pet shop salespersons were better educated on fish handling, and more were aware of the health risks linked to aquarium fish than aquarium hobbyists. The latter often perceived pet shop salespersons as relevant sources of information. However, overall awareness of the zoonotic potential of fish mycobacteria proved to be fairly low. Therefore, more effort should be made to increase the awareness of the role of mycobacteria in infections associated with exposure to aquarium fish. Key words: fish tank granuloma; fish tuberculosis; mycobacteria; questionnaire; zoonosis MIKOBAKTERIJE PRI AKVARIJSKIH RIBAH: ALI SE AKVARISTI ZAVEDAJO NJIHOVEGA ZOONOTSKGA POTENCIALA? Povzetek: Za ljudi potencialno patogene vrste mikobakterij so v Sloveniji splošno prisotne pri akvarijskih ribah, ki so naprodaj v trgovinah z domačimi ljubljenčki, med drugimi tudi vrsta Mycobacterium marinum, ki je najpomembnejša povzročiteljica kožne mikobakterioze pri ljudeh. Namen raziskave je bil, s pomočjo vprašalnika, oceniti zavedanje ljubiteljskih akvaristov in prodajalcev v trgovinah z akvarističnim programom o zdravstvenih tveganjih, povezanih z rokovanjem z ribami. V raziskavi je sodelovalo 198 oseb, 76,3 % ljubiteljskih akvaristov in 23,7 % prodajalcev v trgovinah, ki so v povprečju imeli do 15 let izkušenj pri rokovanju z akvarijskimi ribami. Približno tretjina (35,8 %) sodelujočih se je strinjala s trditvijo, da so ribe lahko vir okužbe za ljudi in da ribe lahko zbolijo za tuberkulozo. V manjšem deležu (24,4 %) so se sodelujoči strinjali s trditvijo, da je ribja tuberkuloza zoonoza. Velika večina anketirancev ni poznala kliničnih znakov ribje tuberkuloze in kožne mikobakterioze pri ljudeh. Kljub splošno izraženemu prepričanju, da lahko voda iz akvarija predstavlja tveganje za zdravje ljudi, je bila splošna praksa rokovanja z ribami presenetljiva, saj velika večina sodelujočih pri rokovanju z ribami nikoli ne uporablja vodoodpornih rokavic. Med obema skupinama anketirancev smo odkrili več razlik. Prodajalci v trgovinah so bolj poučeni o rokovanju z ribami in bolj ozaveščeni o zdravstvenih tveganjih povezanih z akvarijskimi ribami, kot ljubiteljski akvaristi. Slednji prodajalce dojemajo kot zaupanja vreden vir informacij. V splošnem je ozaveščenost o zoonotskem potencialu mikobakterij pri akvarijskih ribah precej slaba, zato bi morali nameniti več pozornosti ozaveščanju o vlogi mikobakterij pri okužbah, povezanih z akvarijskimi ribami. Ključne besede: kožna mikobakterioza; ribja tuberkuloza; mikobakterije; vprašalnik; zoonoza

Pathogens invading the mammary gland are recognized through a range of pattern recognition receptors (PRRs), residing on the plasma membrane of mammary epithelial cells. Toll-like receptor 2 (TLR2) signalling is responsible for recognition of Gram-positive bacteria, which are the most common mastitis-causing pathogens in goats. Somatic cell counts (SCC) in milk are routinely determined in goat dairy flocks and serve as an indicator of milk quality, which is highly correlated to intramammary infections. Recently, a single nucleotide polymorphism of the TLR2 was suggested to be associated with SCC in goat milk. To further test the suggested association, we genotyped 61 Slovenian Alpine goats included in the dataset. The effect of the genotype was analysed using the general linear model (GLM) procedure of SAS/STAT software. We found the TLR2 genotypes significantly (p = 0.0007) associated with milk SCC. Animals with the A/G genotype had significantly (p ≤ 0.05) lower SCC value in milk compared to the G/G genotype. Our data suggest that the A allele is the minor one and is associated with lower milk SCC. In the current study, we provide a validated PCR-RFLP based genotyping assay for the TLR2 SNP (rs650082970) and confirm its association with milk SCC. Further studies to confirm the association on a larger number of animals of different breeds and to explain functional consequences of the polymorphism in relation to SCC are encouraged.

Infectious laryngotracheitis (ILT) is an acute, highly contagious infectious disease of the upper respiratory tract in chickens and other poultry species that causes significant economic losses in countries worldwide. Between 2017 and 2019, seven outbreaks of mild to severe respiratory disorders with high suspicion of ILT occurred in commercial and backyard poultry flocks in Slovenia. In all submissions, infection with ILT virus (ILTV) was confirmed by PCR, which is the first report of ILT in Slovenia. Circulating ILT strains were characterized by the sequence and phylogenetic analysis of two fragments of the ICP4 gene. Four strains—three detected in non-vaccinated flocks and one in a flock vaccinated against ILT—were identical or very similar to the chicken embryo–origin live virus vaccines, and the other three were closely related to Russian, Chinese, Australian, and American field strains and to tissue culture origin vaccine strains. As in other diseases, coinfections with other respiratory pathogens in confirmed ILT cases may cause a more severe condition and prolong the course of the disease. In our study, coinfections with Mycoplasma synoviae (7/7 tested flocks), infectious bronchitis virus (5/5 tested flocks), Mycoplasma gallisepticum (4/7 tested flocks), Ornithobacterium rhinotracheale (3/4 tested flocks), and avian pox virus (1/2 tested flocks) were confirmed, indicating the importance of these pathogens in the occurrence of ILT infections.

The causes of embryonic mortality in Hermann’s tortoises (Testudo hermanni) during artificial incubation were determined. Total egg failure at the end of the hatching period was investigated. The hatching artefacts represented 19.2% (N = 3557) of all eggs (N = 18,520). The viability rate of incubated eggs was 80.8%. The eggs, i.e., embryos, were sorted according to the cause of unsuccessful hatching and subsequently analyzed. Some of the eggs were divided into two or more groups. Unfertilized eggs were confirmed in 61.0%, infected eggs in 52.5%, and eggs in various stages of desiccation in 19.1%. This group also included mummified embryos. Pseudomonas aeruginosa, Bacillus sp., Purpureocillium lilacinum, and Escherichia coli were frequently confirmed in infected eggs. Embryos were divided into three groups: embryos up to 1.0 cm—group 1 (2.2%), embryos from 1.0 cm to 1.5 cm—group 2 (5.4%) and embryos longer than 1.5 cm—group 3 (7.3%) of all unhatched eggs. Inability of embryos to peck the shell was found in 1.3%. These tortoises died shortly before hatching. Embryos still alive from the group 2 and group 3 were confirmed in 0.7% of cases. Dead and alive deformed embryos and twins were detected in the group 3 in 0.5% and 0.1% of cases, respectively. For successful artificial hatching, it is important to establish fumigation with disinfectants prior to incubation and elimination of eggs with different shapes, eggs with broken shells, and eggs weighted under 10 g. Eggs should be candled before and periodically during artificial incubation, and all unfertilized and dead embryos must be removed. Heartbeat monitor is recommended. Proper temperature and humidity, incubation of “clean” eggs on sterile substrate and control for the presence of mites is essential. Monitoring of the parent tortoises is also necessary.

Istrian shorthaired hound is an old indigenous Croatian dog breed with historical traces of its origin, which date back to the 14th century. Due to its intelligence and great hunting abilities, it is considered an excellent hunting dog. Despite its ancient origin, there is no data on genetic diversity, population structure, and degree of inbreeding that could be used for advanced management and conservation of this breed. Our study aimed to provide a high-resolution population structure of the Istrian shorthaired hound using a 220K HD SNP array, to compare the obtained data with the genealogical records and to place the breed in a broader context of world dog populations. Relatively high population size and low inbreeding coefficient estimated from genealogical data indicate a preserved genetic diversity in this breed. The principle component analysis, the NeighborNet network, and TreeMix were used to determine the genetic relationship between the Istrian shorthaired hound and other breeds. The Istrian shorthaired hound was found to be genetically related to Italian hunting dogs sharing the same branch with the Segugio Italiano a Pelo Raso and Segugio Italiano a Pelo Forte. The ADMIXTURE analysis indicated that the Istrian shorthaired hound could be involved in the development of some other hunting dog breeds. The estimated effective population size (Ne) based on SNP data was similar to Ne calculated from genealogical data indicating the absence of bottlenecks and well-balanced use of breeding animals. The low genomic inbreeding coefficient, together with the higher number of short runs of homozygosity, observed in the Istrian shorthaired hound, confirms the ancient origin of the breed based on historical documents. The analysis of selective sweeps identified genomic regions with the strongest selection signals in the vicinity of the genes associated with cognitive performance and behavior. Genome analysis proved to be a useful tool for estimating population parameters and can be implemented in the conservation plan for this indigenous breed.

An amendment to this paper has been published and can be accessed via the original article.

Background: Cryptorchidism is one of the most frequent congenital birth defects in male children and is present in 2–4% of full-term male births. It has several possible health effects including reduced fertility, increased risk for testicular neoplasia, testicular torsion, and psychological consequences. Cryptorchidism is often diagnosed as comorbid; copresent with other diseases. It is also present in clinical picture of several syndromes. However, this field has not been systematically studied. The aim of the present study was to catalog published cases of syndromes which include cryptorchidism in the clinical picture and associated genomic information.Methods: The literature was extracted from Public/Publisher MEDLINE and Web of Science databases, using the keywords including: syndrome, cryptorchidism, undescended testes, loci, and gene. The obtained data was organized in a table according to the previously proposed standardized data format. The results of the study were visually represented using Gephi and karyotype view.Results: Fifty publications had sufficient data for analysis. Literature analysis resulted in 60 genomic loci, associated with 44 syndromes that have cryptorchidism in clinical picture. Genomic loci included 38 protein-coding genes and 22 structural variations containing microdeletions and microduplications. Loci, associated with syndromic cryptorchidism are located on 16 chromosomes. Visualization of retrieved data is presented in a gene-disease network.Conclusions: The study is ongoing and further studies will be needed to develop a complete catalog with the data from upcoming publications. Additional studies will also be needed for revealing of molecular mechanisms associated with syndromic cryptorchidism and revealing complete diseasome network.

Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope “hot-spots” of modified and optimized norovirus–host interactions.

The document presents recommendations for the comprehensive treatment of patients with haemophilia in Slovenia. It enables health workers at all three levels of health care to become well-acquainted with all the possible aspects of treatment based on the best practices of major centres worldwide, and on the studies and experience of health professionals at the National Haemophilia Centre, University Medical Center Ljubljana. The document contains definitions, treatment algorithms and lists of medications with their characteristics and appropriate dosages. It specifically defines indications for the exclusive competence of the tertiary level in Ljubljana due to the actual availability of teams and laboratory options. It also contains an extensive list of the literature on haemophilia.

The rationale, the concept and key challenges of the H2020 project TREASURE dealing with local pig breeds is presented and discussed. The action addresses the phenotypic and genetic characterization, performance of local pig breeds in diverse production systems and their environmental impact, specific quality of their products and market potential. The goal is to build up the capacities to develop sustainable pork chains based on local pig breeds. A special emphasis is given to describe the workplan for Black Slavonian and Turopolje local pig breeds from Croatia and Slovenian Krškopolje pig.

Genetic diversity and phylogenetic relationship of three Croatian autochthonous cattle breeds was analyzed using a sequence of the mtDNA D-loop region. Among Busha, Istrian, and Slavonian Syrmian Podolian cattle 146 unrelated animals were tested. The sequencing of 780 base pairs of the mtDNA D-loop region revealed 39 polymorphic sites representing 28 different haplotypes. The highest numbers of haplotypes were observed in the Busha population and the lowest in the population of Slavonian Syrmian Podolian cattle, while the highest level of sequence diversity within a population was observed in the Istrian cattle. Our results indicate a high level of mtDNA diversity in the populations of Busha and Istrian cattle and a low level of preserved diversity in the population of Slavonian Syrmian Podolian cattle. The sequence analysis showed substantial subdivision between the breeds (FST = 0.1434), and a large fraction of variation within the breeds. Although the dominant haplotypes are classified as the T3 haplogroup, some of the haplotypes are classified as the rarer T2 and T5 haplogroups. Genetic information based on mtDNA typing has a great importance for the future conservation management and preservation of genetic diversity in autochthonous cattle breeds.

In this study, Hermann’s tortoise (Testudo hermanni boettgeri) eggs were studied. The aim was to evaluate the basic composition, amino and fatty acid profiles, as well as the presence of certain trace elements in the eggs. The average size of THB eggs was 29.9 × 39.5 mm and weight 20.7 g. The shell accounted for 12.5%, albumen 46.9% and yolk 40.6% of the entire THB egg. The refractive index was 1.3341 in the albumen and 1.5120 in the yolk. Albumen contained 98.2% water, 0.9% proteins, 0.7% ash and traces of fat. Yolk contained 60.6% water, 21.0% protein, 14.2% fat and 4.0 % ash. The pH of albumen was 8.8. The fatty acid composition was measured in egg yolk; oleic acid, palmitic acid, palmitoleic acid and vaccenic acid were the most abundant. The amino acid composition was measured in egg albumen, and 18 amino acids were detected. The major amino acids present in albumen were glutamic acid, aspartic acid, leucine, phenylalanine, lysine, glycine, serine, threonine and alanine. Concentrations of the trace elements, in descending order were in the shell: iron (Fe), nickel (Ni), copper (Cu), manganese (Mn), cobalt (Co), zinc (Zn), chromium (Cr), arsenic (As) and selenium (Se); in the albumen: Fe, Cu, Cr, Se; and in the yolk: Fe, Zn, Cu, Mn, Se, Cr, Ni, Pb, Co. Key words: Testudo hermanni; eggs; chemical composition; fatty acids; amino acids; trace elements DOLOČANJE RAZLIČNIH KEMIČNIH SESTAVIN V JAJCIH GRŠKE KORNJAČE (Testudo hermanni) Namen raziskave je bil proučiti sestavo jajc grške kornjače (Testudo hermanni boettgeri), vsebnost aminokislin in maščobnih kisli, kot tudi vsebnost nekaterih elementov v sledovih. Povprečna velikost jajc THB je bila 29,9 × 39,5 mm, masa pa 20,7 g. Celotno jajce je bilo sestavljeno iz 12,5 % lupine, 46,9 % beljaka in 40,6 % rumenjaka. Indeks refrakcije beljaka je znašal 1,3341 in rumenjaka 1,5120. Beljak je vseboval 98,2 % vode, 0,9 % beljakovin, 0,7 % pepela in sledove maščobe. Rumenjak je vseboval 60,6 % vode, 21,0 % beljakovin, 14,2 % maščob in 4,0 % pepela. Vrednost pH beljaka je bila 8,8. Najvišji delež maščobnih kislin v rumenjaku so predstavljale oleinska kislina, palmitinska kislina, palmitooleinska kislina in vakcenska kislina. V beljaku smo potrdili 18 različnih aminokislin. Najvišji delež le-teh so predstavljale glutaminska kislina, asparginska kislina, levcin, fenilalanin, lizin, glicin, serin, treonin in alanin. Koncentracije elementov v sledovih v lupini so bile v naslednjem padajočem zaporedju: železo (Fe), nikelj (Ni), baker (Cu), mangan (Mn), kobalt (Co), cink (Zn), krom (Cr), arzen (As), selen (Se); v beljaku: Fe, Cu, Cr, Se, v rumenjaku pa: Fe, Zn, Cu, Mn, Se, Cr, Ni, Pb, Co. Ključne besede: Testudo hermanni; jajca; kemična sestava; maščobne kisline; aminokisline; elementi v sledovih

Mitochondrial transcription factor A (TFAM) is a nuclear-encoded mitochondrial protein that plays an important role in energy metabolism and is a candidate gene for fat deposition in cattle and human. In the present study, we characterized the methylation status of bovine TFAM promoter flanking the single nucleotide polymorphism (SNP) rs42159487C>T, previously reported to affect fat deposition. Our results showed that the cytosine at this SNP position is methylated and therefore results in gain/loss of the functional CpG locus (5mC>T). Promoter constructs developed based on three TFAM SNPs showed that the C/C/C haplotype associated with fat deposition in beef cattle had lower promoter function/activity than A/T/T haplotype. Our results imply that genetic variability underlying gain/loss of the CpG sites in the nuclear-encoded mitochondrial proteins might be good candidate loci for fat deposition phenotypes.

Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing.

The opinions and arguments, claiming that Anton Janša stayed in Carinthia from 1765 until his departure for Vienna in the year 1766, that he was a member of the Carinthian agricultural society and similar, are a novelty in the knowledge of the life and work of the first c. k. beekeeping teacher in Vienna. This statement was for the first time published in the book of Ehrenfels in 1829 and for the second time in the book of Heinrich in 1832. This notice was later reproduced in other works. The message of these publications is: Anton Janša was a Carinthian, he brought the Carinthian way of beekeeping to Vienna, he originates from the Carinthian agricultural society, which also recommended him in Vienna, so he was invited by the Empress Maria Theresia to Vienna and so on. Among Slovenian authors Perc (1925a,b) and Stabej (1955) drew attention to these records. Janša describes in his books and stresses his Carniolan origin, Carniolan way of beekeeping and Carniolan wooden hive and does not refer to his life and work in Carinthia. To confirm these records and publications on Anton Janša in Carinthia, it is necessary to find archival documents in provincial archive in Klagenfurt to confirm or to reject this hypothesis.

The genetic variability for a sample of 107 animals from three autochthonous Croatian horse breeds was estimated using 20 microsatellites. The average number of alleles per locus (6.3) and proportion of heterozygosity (0.732) indicated a moderate variability. The expected heterozygosity was similar among all breeds and ranged between 0.724 in the Posavina horse, and 0.737 in the Croatian Coldblood and Murinsulaner horse. The inbreeding coefficient FIS was low and non-significant over the three populations. The genetic differentiation among the three populations was low (FST=0.026), suggesting that only 2.6% of the total genetic variability was due to differences between the breeds, and 97% to individual differences. The results of pairwise genetic differentiation suggest that the Posavina horse and the Croatian Coldblood were the most closely related populations (FST=0.016). These results are confirmed by Nei’s genetic distances with the highest value observed between the Posavina horse and the Murinsulaner (0.082) and the lowest between the Posavina horse and the Croatian Coldblood (0.044). An assignment test correctly assigned 82% of individuals to the correct breed. Strategies for preserving the original native genes in the Croatian native horse breeds should be considered in order to prevent these breeds from becoming extinct and include them in the future breeding programmes.

Genetic variation in three Croatian coldblood horse populations was analysed using a sequence analysis of the proximal part (nt 15 498-15 821) of the D-loop region of mtDNA. Twenty unrelated horses were chosen from the Posavina horse and the Croatian Coldblood breeds and fifteen horses from the Murinsulaner horse population. Sequencing of the proximal part of the mtDNA D-loop region revealed 26 polymorphic sites representing thirty haplotypes which were clustered into eight haplogroups. A wide variety of mitochondrial haplotypes of the analysed horse breeds were clustered into eight different haplogroups. Two haplogroups (D and F) were specific to Posavina horse, five haplogroups were shared among the Croatian Coldblood and Murinsulaner horse, the fact that can be explained by selection strategy at the beginning of the 20th century and possible gene flow between the two populations. These results indicate the presence of many ancient maternal lineages with high diversity in mtDNA. The genetic information based on mtDNA typing has a great importance for the future breed conservation strategy, especially for the critically endangered breed such as Murinsulaner horse.

Abstract Microarrays allow researchers to measure the expression of thousands of genes in a single experiment. Before statistical comparisons can be made, the data must be assessed for quality and normalisation procedures must be applied, of which many have been proposed. Methods of comparing the normalised data are also abundant, and no clear consensus has yet been reached. The purpose of this paper was to compare those methods used by the EADGENE network on a very noisy simulated data set. With the a priori knowledge of which genes are differentially expressed, it is possible to compare the success of each approach quantitatively. Use of an intensity-dependent normalisation procedure was common, as was correction for multiple testing. Most variety in performance resulted from differing approaches to data quality and the use of different statistical tests. Very few of the methods used any kind of background correction. A number of approaches achieved a success rate of 95% or above, with relatively small numbers of false positives and negatives. Applying stringent spot selection criteria and elimination of data did not improve the false positive rate and greatly increased the false negative rate. However, most approaches performed well, and it is encouraging that widely available techniques can achieve such good results on a very noisy data set.

Abstract A large variety of methods has been proposed in the literature for microarray data analysis. The aim of this paper was to present techniques used by the EADGENE (European Animal Disease Genomics Network of Excellence) WP1.4 participants for data quality control, normalisation and statistical methods for the detection of differentially expressed genes in order to provide some more general data analysis guidelines. All the workshop participants were given a real data set obtained in an EADGENE funded microarray study looking at the gene expression changes following artificial infection with two different mastitis causing bacteria: Escherichia coli and Staphylococcus aureus. It was reassuring to see that most of the teams found the same main biological results. In fact, most of the differentially expressed genes were found for infection by E. coli between uninfected and 24 h challenged udder quarters. Very little transcriptional variation was observed for the bacteria S. aureus. Lists of differentially expressed genes found by the different research teams were, however, quite dependent on the method used, especially concerning the data quality control step. These analyses also emphasised a biological problem of cross-talk between infected and uninfected quarters which will have to be dealt with for further microarray studies.

Abstract Microarray analyses have become an important tool in animal genomics. While their use is becoming widespread, there is still a lot of ongoing research regarding the analysis of microarray data. In the context of a European Network of Excellence, 31 researchers representing 14 research groups from 10 countries performed and discussed the statistical analyses of real and simulated 2-colour microarray data that were distributed among participants. The real data consisted of 48 microarrays from a disease challenge experiment in dairy cattle, while the simulated data consisted of 10 microarrays from a direct comparison of two treatments (dye-balanced). While there was broader agreement with regards to methods of microarray normalisation and significance testing, there were major differences with regards to quality control. The quality control approaches varied from none, through using statistical weights, to omitting a large number of spots or omitting entire slides. Surprisingly, these very different approaches gave quite similar results when applied to the simulated data, although not all participating groups analysed both real and simulated data. The workshop was very successful in facilitating interaction between scientists with a diverse background but a common interest in microarray analyses.

Alternative intron splicing is a process where introns are excised from pre-mRNA following different patterns. The consequence of it is a presence of several different forms of mature mRNAs differing in length and in content of the whole exons or their parts. Studies in the past decade lead to the view that a complex and extensively coupled network has evolved to coordinate the activities of gene expression pathway, which includes transcription, several pre-mRNA processing steps and the export of mature mRNA to the cytoplasm. Coupling of transcription to splicing was revealed in studies showing that transcription of pre-mRNA by different promoters can generate different alternatively spliced mRNAs. The main goal of this study was to determine the impact of different milk gene promoters on alternative splicing under in vitro conditions. Besides, we also investigated how different cell types influence the splice site selection. With this experiment, we confirmed our predictions that promoter structure and cell-type have an impact on alternative splicing. Differences were observed in quantity of transcripts, as well as in splice-site selection.

Breeding mice positive for Myocoptes musculinus and Myobia musculi were treated with ivermectin in doses of 200 μg/kg of body weight (6μl of active substance) as a spot application on the back of the neck. Application was repeated three times in seven-day intervals. Different development forms (adults, nymphs, larvae, and eggs) were observed in groups of adult and young mice before the treatment as well as during and after the treatment. Before treatment, the fur pluck technique and sticky paper technique (sampling from two different places: from back and abdomen) were evaluated. The fur pluck technique was used as the gold standard. The sticky paper technique has 91.5 to 93.2 % sensitivity for Myocoptes musculinus and 10.8 to 13.5 % for Myobia musculi. We observed that Myobia musculi place eggs next to the skin, on the bases of two or more hairs together; consequently, the sampling is not always satisfactory. The effectiveness of therapy with ivermectin for Myocoptes musculinus and Myobia musculi was shown after the second treatment. Before the treatment, as well as during and after the treatment, a significantly higher percentage of positive mice was observed among old than in young ones. After the third treatment, adults and developmental stages (eggs, larvae, and nymphs) were still found only in Myocoptes musculinus. Almost all eggs of Myocoptes musculinus and Myobia musculi were drained and damaged after the third treatment.

Abstract Sequencing of the mtDNA control region (385 or 695 bp) of 212 Lipizzans from eight studs revealed 37 haplotypes. Distribution of haplotypes among studs was biased, including many private haplotypes but only one haplotype was present in all the studs. According to historical data, numerous Lipizzan maternal lines originating from founder mares of different breeds have been established during the breed's history, so the broad genetic base of the Lipizzan maternal lines was expected. A comparison of Lipizzan sequences with 136 sequences of domestic- and wild-horses from GenBank showed a clustering of Lipizzan haplotypes in the majority of haplotype subgroups present in other domestic horses. We assume that haplotypes identical to haplotypes of early domesticated horses can be found in several Lipizzan maternal lines as well as in other breeds. Therefore, domestic horses could arise either from a single large population or from several populations provided there were strong migrations during the early phase after domestication. A comparison of Lipizzan haplotypes with 56 maternal lines (according to the pedigrees) showed a disagreement of biological parentage with pedigree data for at least 11% of the Lipizzans. A distribution of haplotype-frequencies was unequal (0.2%–26%), mainly due to pedigree errors and haplotype sharing among founder mares.

Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample.

The aim of this work was to determine the MC1R genotype of Black Slavonian pigs and to find an efficient and simple PCR-RFLP method, based on differences in MC1R genotype, to distinguish between purebred Black Slavonian pigs and their crossings with commercial pig breeds and Wild Boars. Sequencing of the MC1R exon was performed to determine the genotype of MC1R in Black Slavonian pig breed, which was shown to be MC1R*2. Digestion reactions of both PCR products representing the majority of MC1R exon revealed presence of the BspHI restriction site at position 121 and absence of the AccII and CrfI restriction site at position 240, which is characteristic for the MC1R*2 genotype. A simple PCR-RFLP method, based on different coat colour MC1R gene genotypes was determined by which it is possible to detect potential crossings of autochthonous Black Slavonian pig with commercial pig breeds and also with Wild Boars.

The frequency of mammary neoplasia in different species varies tremendously. Among women, breast cancer features as one of the most frequent types of cancer globally. Breast cancer is the cause of almost half a million deaths worldwide each year. Of these deaths, more than 58 % are occurring in developing countries. Mammary tumors are rare in cows, mares, goats, ewes, and sows as evident by few cases reported in literature. In dairy cattle and milking goats the udders are inspected daily by palpation when milking and the tumors would be detected soon. The morphological features of bovine mammary gland are more similar to those in human than in rodents, the latter being commonly used for cancerogenic studies. There are several factors that might affect the high rate of tumor development in women and attenuate the development of tumors in ruminants. Besides diet, life style, reproductive history, stress load, the general physiology and metabolism might have an influence. Translational research using different mammary gland cell populations from several species may lead to comparative studies and may help reduce the risk and contribute to discovery of new therapeutic targets to treat breast cancer. In present work the zero occurrence of mammary gland tumors in ruminants will be reviewed focusing on literature records and mechanisms underlying the resistance.

The importance of pluripotent cells, which can differentiate in to different cell lineages and form an entire organism, is fundamental for understanding developmental biology including emerging diseases and offers potential for numerous applications in medicine and biotechnology. However, molecular mechanisms behind differentiation and de-differentiation (reprogramming) remain largely unknown. Until recently it was possible to obtain stem cells only from embryos in the early stages of development (embryonic stem cells – ESCs) or by using very inefficient and technically difficult method of somatic cell nuclear transfer (SCNT) that requires use of egg cells (oocytes). Both methods raised ethical concerns, especially when using human biological material. On the other hand, induced pluripotent stem cells (iPSCs) can be generated by direct reprogramming of differentiated adult somatic cells. iPSCs show similarities to ESCs and represent ethically acceptable and almost unlimited source of individuum-specific pluripotent cells. iPSCs are particularly important for development of regenerative medicine, disease modelling, drug development and testing, basic research, generation of transgenic animals, and for conservation of endangered species. However, before it is possible to exploit their potential in full, reprogramming processes should be investigated and understood in details and safe methods developed – that will enable production of genetically and epigenetically stable cells without tumorigenic potential. This article provides an overview of the field of iPSCs and addresses some of the latest achievements and applications of pluripotent cells in human and farm animals.

Haemolytic disease of the newborn (HDN), also known as haemolytic disease of the fetus and newborn (HDFN), develops in the fetus when erythrocyte alloantibodies produced by the mother pass through the placenta. Severe form of HDFN is mainly caused by anti-D antibodies, but it can also be caused by many other types of antibodies, most often by anti-Kell (anti-K), anti-c, anti-E in anti-C. In order to prevent sensitisation to erythrocyte antigens, every pregnant woman is tested for blood group AB0, D, K and for irregular antibodies (IAT–indirect antiglobulin test). For D-negative women IAT is repeated in the 28th week of pregnancy, followed by an injection of anti-D immnunoglobulin (Ig anti-D) when indicated. At the Blood Transfusion Centre of Slovenia, we have recently started to determine the fetal D status from a maternal blood sample in order to avoid the unnecessary application of Ig anti-D to women bearing Dnegative fetuses.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated reaction occurring in 3 – 5 % of patients receiving heparin therapy for more than 5 days, which can lead to arterial and venous thrombosis. It is caused by IgG antibodies directed against platelet factor 4/heparin complexes, leading to platelet activation, platelet depletion and thrombocytopenia. Prolonged therapy with heparin can result in serious adverse consequences, so prompt diagnosis is of key importance for the introduction of supplemental therapy. The diagnosis of HIT is based on clinical criteria as well as on serological and functional laboratory assays. Serological assays that identify the presence of platelet factor 4/heparin antibodies in the serum are not sufficient for confirmation of diagnosis because all detected antibodies are not capable of causing thrombocytopenia and thrombosis. Since the usual enzyme-immune assays detect the presence of antibodies but not also their pathogeneity it is recommended to confirm HIT with a functional assay thatconfirms the capacity of HIT antibodies to activate or aggregate platelets. One of newer functional methods is the flow cytometric analysis of P-selectin (CD62P) expression on the standard platelets after exposure to the patient’s serum. In our experience, this test, in parallele with standard serological tests, proved its reliability for the confirmation of clinical HIT diagnosis.

In the nineteenth and early twentieth century the Medžimurje horse played an important role as a draft and working horse in the river Mura region. Nowadays, this area is split between Hungary, Austria, Slovenia and Croatia. In the frame of the project "Management of traditional transboundary breeds on example of a nearly forgotten breed the Murinsulaner", we collected data for cold blooded horses similar to the Medžimurje horse. Horses of this type do not exist in Austria anymore, in Croatia they were registered as an authothonous breed »Medjimurje horse«, and in Slovenia and Hungary this horse has a special section in the stud book for cold blooded horses as a Medžimurje type of horse. The Slovenian part of the population of the Medžimurje horse is quite heterogeneous regarding body measures. With respect to the scoring system used in the above mentioned project, the majority of scored animals were classified as »desired« or »satisfactory«. In order to further develop or to preserve this type of horse or to form a consolidated breed we would have to make a breeding program which would prevent mating among related animals and to cooperate with neighbouring countries in the field of breeding and selection. The only hope to preserve this type of horse on the long term in the region is by using it as a tourist attraction for carriage riding and/or as a draft horse in agriculture in marginal regions.

Mycobacterium celatum, a slowly growing potentially pathogenic mycobacterium first described in humans, is regarded as an uncommon cause of human infection, though capable of inducing invasive disease in immunocompromised hosts. According to some reports, a serious disease due to M. celatum may also occur in individuals with no apparent immunodeficiency. In animals, an M. celatum-related disease has been described in three cases only: twice in a domestic ferret (Mustela putorius furo) and once in a white-tailed trogon (Trogon viridis). In this paper, we report the first detection of M. celatum in a domestic pig (Sus scrofa domestica) and roe deer (Capreolus capreolus). A nation-wide overview of human M. celatum infections recorded in Slovenia between 2000 and 2010 is also given. Pulmonary disease due to M. celatum was recognized in one patient with a history of a preexisting lung disease.

Rodovniški podatki kontroliranih tropov so bistvenega pomena za rejce ovc in izvajanje selekcijskih programov. V tej študiji smo preverili, če je možno z osmimi molekularno genetskimi označevalci ter uporabo računalniškega programa ATLAS preveriti skladnost podatkov v več-generacijskih rodovnikih jezersko-solčavske in oplemenjene jezersko-solčavske pasme. Ugotovili smo 90,9 % točnost beleženja rodovniških podatkov, medtem ko smo neskladja ugotovili pri štirih družinah od 44-ih. V dveh primerih je bil potomcu nepravilno pripisan oče ali mati, pri ostalih dveh primerih pa sta bila jagnjetu napačno pripisana oba starša. V drugem delu raziskave smo poskušali z istim nizom mikrosatelitnih označevalcev določiti očetovstva štirim jagnjetom istrske pramenke, kjer v času paritev v trop pripuščajo dvajset ovnov in tako jagnje nima poznanega očeta. Trem jagnjetom smo lahko nedvoumno določili očeta z izločitvijo ostalih devetnajstih. V enem primeru smo ugotovili, da niz osmih označevalcev ni dovolj informativen za določitev očeta potomcu, zato smo analizo razširili in z vključitvijo dodatnih štirih mikrosatelitnih označevalcev uspešno določili očetovstvo tudi v tem primeru. Z izbranim nizom molekularnih označevalcev in obdelave podatkov je možno učinkovito preverjati obstoječe rodovniške podatke in napovedati očetovstvo jagnjetom v tropih, kjer pripuščajo večje število ovnov. Rezultati take analize so lahko v pomoč rejcem in selekcijskim službam za izboljšanje točnosti rodovniških podatkov in učinkovitejše načrtovanje reje in selekcije pri ovcah.