Mining the red deer genome (CerEla1.0) to develop X-and Y-chromosome-linked STR markers.

Microsatellites are widely applied in population and forensic genetics, wildlife studies and parentage testing in animal breeding, among others, and recently, high-throughput sequencing technologies have greatly facilitated the identification of microsatellite markers. In this study the genomic data of Cervus elaphus (CerEla1.0) was exploited, in order to identify microsatellite loci along the red deer genome and for designing the cognate primers. The bioinformatics pipeline identified 982,433 microsatellite motifs genome-wide, assorted along the chromosomes, from which 45,711 loci mapped to the X- and 1096 to the Y-chromosome. Primers were successfully designed for 170,873 loci, and validated with an independently developed autosomal tetranucleotide STR set. Ten X- and five Y-chromosome-linked microsatellites were selected and tested by two multiplex PCR setups on genomic DNA samples of 123 red deer stags. The average number of alleles per locus was 3.3, and the average gene diversity value of the markers was 0.270. The overall observed and expected heterozygosities were 0.755 and 0.832, respectively. Polymorphic Information Content (PIC) ranged between 0.469 and 0.909 per locus with a mean value of 0.813. Using the X- and Y-chromosome linked markers 19 different Y-chromosome and 72 X-chromosome lines were identified. Both the X- and the Y-haplotypes split to two distinct clades each. The Y-chromosome clades correlated strongly with the geographic origin of the haplotypes of the samples. Segregation and admixture of subpopulations were demonstrated by the use of the combination of nine autosomal and 16 sex chromosomal STRs concerning southwestern and northeastern Hungary. In conclusion, the approach demonstrated here is a very efficient method for developing microsatellite markers for species with available genomic sequence data, as well as for their use in individual identifications and in population genetics studies.

DNA of Theileria orientalis, T. equi and T. capreoli in stable flies (Stomoxys calcitrans).

BACKGROUND: From a veterinary-medical point of view, the stable fly, Stomoxys calcitrans, is perhaps the economically most important blood-sucking muscoid fly species (Diptera: Muscidae), owing to its worldwide occurrence, frequently high local abundance, direct harm caused to livestock, pet animals and humans, as well as its vector role. Considering the latter in the context of protozoan parasites, the stable fly is a mechanical vector of trypanosomes and Besnoitia besnoiti. However, its role as a vector of piroplasms appears to be seldom studied, despite old data suggesting mechanical transmission of babesiae by dipteran flies. METHODS: In this study 395 stable flies (and one Haematobia stimulans) were collected at a cattle farm with known history of bovine theileriosis, and at further nine, randomly chosen locations in Hungary. These flies were separated according to sex (30 of them also cut into two parts: the head with mouthparts and the thorax-abdomen), followed by individual DNA extraction, then screening for piroplasms by PCR and sequencing. RESULTS: In stable flies, Theileria orientalis and T. capreoli were identified at the cattle farm and T. equi was identified in three other locations. At the cattle farm, significantly more male stable flies carried piroplasm DNA than females. There was no significant difference between the ratio of PCR-positive flies between the stable (void of cattle for at least two hours) and the pen on the pasture with cattle at the time of sampling. Among dissected flies (29 S. calcitrans and 1 H. stimulans), exclusively the thoracic-abdominal parts were PCR-positive, whereas the head and mouthparts remained negative. CONCLUSIONS: Theileria DNA is detectable in stable flies, in the case of T. orientalis at least for two hours after blood-feeding, and in the case of T. capreoli also in the absence of infected hosts (i.e. roe deer). Male flies rather than females, and thoracic-abdominal (most likely crop) contents rather than mouthparts may pose a risk of mechanical transmission. These data suggest that it is worth to study further the vector role of stable flies in the epidemiology of theilerioses, in which not the immediate, but rather the delayed type transmission seems possible.

Sarcocystis rileyi emerging in Hungary: is rice breast disease underreported in the region?

Reports of Sarcocystis rileyi-like protozoa ('rice breast disease') from anseriform birds had been rare in Europe until the last two decades, when S. rileyi was identified in northern Europe and the UK. However, despite the economic losses resulting from S. rileyi infection, no recent accounts are available on its presence (which can be suspected) in most parts of central, western, southern and eastern Europe. Between 2014 and 2019, twelve mallards (Anas platyrhynchos) were observed to have rice breast disease in Hungary, and the last one of these 12 cases allowed molecular identification of S. rileyi, as reported here. In addition, S. rileyi was molecularly identified in the faeces of one red fox (Vulpes vulpes). The hunting season for mallards in Hungary lasts from mid-August to January, which in Europe coincides with the wintering migration of anseriform birds towards the south. Based on this, as well as bird ringing data, it is reasonable to suppose that the first S. rileyi-infected mallards arrived in Hungary from the north. on the other hand, red foxes (Vulpes vulpes), which are final hosts of S. rileyi, are ubiquitous in Hungary, and our molecular finding confirms an already established autochthonous life cycle of S. rileyi in the region. Taken together, this is the first evidence for the occurrence of S. rileyi in Hungary and its region.

Establishment of Fascioloides magna in a new region of Hungary: case report.

During the monitoring of red deer (N = 124) and fallow deer (N = 13) populations in four neighbouring areas, the presence of Fascioloides magna was confirmed in southwestern Hungary. The prevalence and the mean intensity of the infection within the host populations ranged between 0 and 100% and 0-36.3, respectively. The determined prevalences are similar to that observed earlier in other European natural foci. The authors hypothesise that the appearance of F. magna in this region should have been a partly natural- and partly human-influenced process.

Tick- and fly-borne bacteria in ungulates: the prevalence of Anaplasma phagocytophilum, haemoplasmas and rickettsiae in water buffalo and deer species in Central Europe, Hungary.

BACKGROUND: Hunting constitutes an important industry in Europe. However, data on the prevalence of vector-borne bacteria in large game animal species are lacking from several countries. Blood or spleen samples (239 and 270, respectively) were taken from red, fallow and roe deer, as well as from water buffaloes, mouflons and wild boars in Hungary, followed by DNA extraction and molecular analyses for Anaplasma phagocytophilum, haemoplasmas and rickettsiae. RESULTS: Based on blood samples, the prevalence rate of A. phagocytophilum infection was significantly higher in red deer (97.9%) than in fallow deer (72.7%) and roe deer (60%), and in all these compared to mouflons (6.3%). In addition, 39.2% of the spleen samples from wild boars were PCR positive for A. phagocytophilum, but none of the buffalos. Based on blood samples, the prevalence rates of both Mycoplasma wenyonii (Mw) and 'Candidatus M. haemobos' (CMh) infections were significantly higher in buffaloes (Mw: 91.2%; CMh: 73.3%) than in red deer (Mw: 64.6%; CMh: 45.8%), and in both of them compared to fallow deer (Mw: 30.3%; CMh: 9.1%) and roe deer (Mw: 20%; CMh: 1.5%). The prevalence of Mw and CMh infection significantly correlated with the body sizes of these hosts. Furthermore, Mw was significantly more prevalent than CMh in buffaloes, red and roe deer. Mycoplasma ovis was detected in mouflons, M. suis in wild boars, R. helvetica in one fallow deer and one mouflon, and an unidentified Rickettsia sp. in a fallow deer. CONCLUSIONS: Forest-dwelling game animal species were found to be important carriers of A. phagocytophilum. In contrast, animals grazing grassland (i.e. buffaloes) were less likely to get infected with this Ixodes ricinus-borne pathogen. Water buffaloes, deer species, mouflons and wild boars harbored haemoplasmas that may affect domestic ungulates. Evaluated animals with larger body size had significantly higher prevalence of infection with haemoplasmas compared to smaller deer species. The above host species rarely carried rickettsiae.

The red deer Cervus elaphus genome CerEla1.0: sequencing, annotating, genes, and chromosomes.

We present here the de novo genome assembly CerEla1.0 for the red deer, Cervus elaphus, an emblematic member of the natural megafauna of the Northern Hemisphere. Humans spread the species in the South. Today, the red deer is also a farm-bred animal and is becoming a model animal in biomedical and population studies. Stag DNA was sequenced at 74× coverage by Illumina technology. The ALLPATHS-LG assembly of the reads resulted in 34.7 × 103 scaffolds, 26.1 × 103 of which were utilized in Cer.Ela1.0. The assembly spans 3.4 Gbp. For building the red deer pseudochromosomes, a pre-established genetic map was used for main anchor points. A nearly complete co-linearity was found between the mapmarker sequences of the deer genetic map and the order and orientation of the orthologous sequences in the syntenic bovine regions. Syntenies were also conserved at the in-scaffold level. The cM distances corresponded to 1.34 Mbp uniformly along the deer genome. Chromosomal rearrangements between deer and cattle were demonstrated. 2.8 × 106 SNPs, 365 × 103 indels and 19368 protein-coding genes were identified in CerEla1.0, along with positions for centromerons. CerEla1.0 demonstrates the utilization of dual references, i.e., when a target genome (here C. elaphus) already has a pre-established genetic map, and is combined with the well-established whole genome sequence of a closely related species (here Bos taurus). Genome-wide association studies (GWAS) that CerEla1.0 (NCBI, MKHE00000000) could serve for are discussed.

Evidence for host specificity of Theileria capreoli genotypes in cervids.

Data on the prevalence of piroplasms in buffaloes and large game animal species are lacking from several central European countries. Therefore, to investigate the presence of Babesia/Theileria DNA in these hosts, 239 blood and 270 spleen samples were taken from cervids (red, fallow, and roe deer), as well as from water buffaloes, mouflons, and wild boars in southwestern Hungary, followed by DNA extraction and molecular analysis for piroplasms. All samples from buffaloes and wild boars were PCR negative. Based on spleen samples, the prevalence of piroplasms was significantly higher in red deer (41.7%) than in fallow deer (23.5%). Two genotypes of Theileria capreoli were identified, which showed significant association with their host species (i.e. genotype "capreoli-CE1" was exclusively found in roe deer, whereas red and fallow deer harbored only genotype "elaphi-CE1"). Genotype "elaphi-CE1" of T. capreoli was also detected in one mouflon. No Babesia spp. were identified. In conclusion, in the evaluated region, genotypes of T. capreoli show host-associations among cervids, and at least one of these genotypes may infect mouflons.

Benzimidazole resistance in Haemonchus contortus recovered from farmed red deer.

Thirty Haemonchus contortus male worms were collected from farmed red deer yearlings in order to determine whether routine administration of albendazole for a long-term period (17 years) could select anthelmintic resistance. PCR-RFLP method based on single-nucleotide polymorphism of codon 200 in isotype 1 ß-tubulin gene (Phe200Tyr) was applied. The results showed a significant frequency of either the resistant allele (85 %) or the homozygous resistant genotype (70 %). By chi-square test, Hardy-Weinberg equilibrium of the population was accepted (p = 0.334, power of test 0.01). True prevalence of the resistant genotype (RR) was estimated to be 46.5-87.2 % (confidence interval 95 %) calculated by Sterne's exact method. These results confirmed that long-term use of benzimidazoles could change the relative allele frequency of genes associated with drug resistance and may cause a large-scale spread of the resistant allele. To our knowledge, this study supported benzimidazole resistance in Haemonchus contortus in red deer for the first time.

Genetic diversity and population genetics of large lungworms (Dictyocaulus, Nematoda) in wild deer in Hungary.

Dictyocaulus nematode worms live as parasites in the lower airways of ungulates and can cause significant disease in both wild and farmed hosts. This study represents the first population genetic analysis of large lungworms in wildlife. Specifically, we quantify genetic variation in Dictyocaulus lungworms from wild deer (red deer, fallow deer and roe deer) in Hungary, based on mitochondrial cytochrome c oxidase subunit 1 (cox1) sequence data, using population genetic and phylogenetic analyses. The studied Dictyocaulus taxa display considerable genetic diversity. At least one cryptic species and a new parasite-host relationship are revealed by our molecular study. Population genetic analyses for Dictyocaulus eckerti revealed high gene flow amongst weakly structured spatial populations that utilise the three host deer species considered here. Our results suggest that D. eckerti is a widespread generalist parasite in ungulates, with a diverse genetic backround and high evolutionary potential. In contrast, evidence of cryptic genetic structure at regional geographic scales was observed for Dictyocaulus capreolus, which infects just one host species, suggesting it is a specialist within the studied area. D. capreolus displayed lower genetic diversity overall, with only moderate gene flow compared to the closely related D. eckerti. We suggest that the differing vagility and dispersal behaviour of hosts are important contributing factors to the population structure of lungworms, and possibly other nematode parasites with single-host life cycles. Our findings are of relevance for the management of lungworms in deer farms and wild deer populations.

Occurrence of Coxiella burnetii and Chlamydiales species in abortions of domestic ruminants and in wild ruminants in Hungary, Central Europe.

Coxiella burnetii and certain members of the Chlamydiales order are zoonotic, intracellular, Gram-negative bacteria, with abortigenic potential in ruminants. These pathogens have a broad host range and worldwide geographical distribution. The current study aimed to reveal the importance of C. burnetii and Chlamydiales spp. in abortions in domestic ruminants and their occurrence in wild ruminants with real-time polymerase chain reaction (qPCR) assays, histology, and immunohistochemical staining (IHC). From the 111 abortion cases of domestic ruminants examined, C. burnetii was detected in 33 placenta samples (cattle, n = 22; sheep, n = 10; goat, n = 1), and members of the Chlamydiales order were detected in 32 placenta samples (cattle, n = 14; sheep, n = 16; goat, n = 2) using qPCR. Coinfection with both C. burnetii and Chlamydiales spp. were identified in 12 cases (cattle, n = 3; sheep, n = 8; goat, n = 1) out of the qPCR-positive samples. The presence of the relevant antigen was confirmed by IHC in 20 cases (C. burnetii, n = 2, in sheep; Chlamydiaceae, n = 17, in sheep [n = 15] and goat [n = 2]; and both pathogens in 1 sheep). Coxiella burnetii was identified in 2.2% (2/91) of the wild ruminants, but the samples were negative by IHC. Uncultured Chlamydiales spp. were detected in 4.4% (4/91) of the placenta samples by qPCR. In conclusion, Q fever is widespread among domestic ruminants in Hungary, and, in several cases, C. burnetii was implicated as the primary cause of abortions. Waddlia chondrophila, Parachlamydia spp., and uncultured Chlamydiales spp. were present only sporadically in samples from cattle and wild ruminants.

Wild boar density drives Metastrongylus infection in earthworm.

Larvae of Metastrongylus spp. lungworms infect wild boar (Sus scrofa) definitive hosts through earthworms (Lumbricidae). We compared the abundance and Metastrongylus spp. larval infection measures of earthworms between two areas (both in Zselic, Hungary, 2012) characterized by markedly different wild boar population densities. Estimated wild boar density was 0.03 animal/ha in free range area and 1.03 animal/ha in enclosure. The mean abundance of earthworm populations (mostly Allolobophora, Aporrectodea, and Lumbricus spp.) was assessed by analysing 140-140 soil samples. The assesment of Metastrongylus spp. larval infection measures was based on cca 100-100 earthworms derived from the two areas. The abundance of earthworms and their Metastrongylus spp. larval infection measures (prevalence and mean intensity) were significantly lower in the free range than in the enclosure. Furthermore, using a finer scale within the enclosure, we compared wild boar feeding sites (n = 30) to other sites (n = 75). Earthworm populations were significantly more abundant and carried significantly more prevalent and more abundant larval lungworm infections at the former sites. These results suggest that high wild boar density and forage supplementation in enclosures increase both the abundance and the larval Metastrongylus infections of earthworms.

Phylogenetic analyses of endoparasitic Acanthocephala based on mitochondrial genomes suggest secondary loss of sensory organs.

The metazoan taxon Syndermata (Monogononta, Bdelloidea, Seisonidea, Acanthocephala) comprises species with vastly different lifestyles. The focus of this study is on the phylogeny within the syndermatan subtaxon Acanthocephala (thorny-headed worms, obligate endoparasites). In order to investigate the controversially discussed phylogenetic relationships of acanthocephalan subtaxa we have sequenced the mitochondrial (mt) genomes of Echinorhynchus truttae (Palaeacanthocephala), Paratenuisentis ambiguus (Eoacanthocephala), Macracanthorhynchus hirudinaceus (Archiacanthocephala), and Philodina citrina (Bdelloidea). In doing so, we present the largest molecular phylogenetic dataset so far for this question comprising all major subgroups of Acanthocephala. Alongside with publicly available mt genome data of four additional syndermatans as well as 18 other lophotrochozoan (spiralian) taxa and one outgroup representative, the derived protein-coding sequences were used for Maximum Likelihood as well as Bayesian phylogenetic analyses. We achieved entirely congruent results, whereupon monophyletic Archiacanthocephala represent the sister taxon of a clade comprising Eoacanthocephala and monophyletic Palaeacanthocephala (Echinorhynchida). This topology suggests the secondary loss of lateral sensory organs (sensory pores) within Palaeacanthocephala and is further in line with the emergence of apical sensory organs in the stem lineage of Archiacanthocephala.

Papillomavirus-associated fibropapillomas of red deer ( Cervus elaphus ).

Oval, firm, cutaneous tumours with a rough, hairless, pigmented surface, exhibiting a moderately pronounced papillary structure were detected on the abdominal skin of two young red deer ( Cervus elaphus ). One animal was shot in Lower Austria in 2004, the other at a deer farm in Hungary in 2007. Histological examination of both samples classified the tumours as fibropapillomas, showing marked proliferation of fibroblasts and connective tissue, accompanied by hyperkeratosis, parakeratosis and acanthosis of the overlaying epidermis, and occasional foci of inflammation. The distribution of cytokeratin and vimentin was characterised in the lesion. The presence of papillomavirus (PV) antigen was demonstrated by immunohistochemistry in both cases. Papillomavirus-specific DNA was successfully amplified by PCR from one sample. The obtained partial nucleotide sequence of the L2 ORF exhibited the highest critical identity values with the homologous regions of Delta-papillomaviruses, especially the Roe deer papillomavirus (93%). Phylogenetic analysis of the partial L2 ORF sequence alignment of 10 papillomaviruses by both neighbour-joining and maximum parsimony method confirmed that the Red deer PV is very closely related to the Western roe deer papillomavirus (CcPV1).

Metal levels in tissues of Eurasian otters (Lutra lutra) from Hungary: variation with sex, age, condition and location.

Liver samples of Eurasian otters from various parts of Hungary were analysed for mercury, copper, zinc, lead and cadmium. Only zinc concentration was significantly higher in females. Higher mercury and cadmium concentrations in adults and higher zinc values in immature otters were measured. Accumulation of mercury, copper and zinc in tissues increased with the declining condition of animals. Mercury and copper were detected with higher values in samples from large rivers.