Tomislav Gomerčić

Records and genetic diversity of striped dolphins (Stenella coeruleoalba) from the Croatian coast of the Adriatic Sea

The striped dolphin (Stenella coeruleoalba) is a small, pelagic and cosmopolitan cetacean. Though it is the most common dolphin species in the Mediterranean Sea, it is not considered resident in the Croatian part of the Adriatic Sea. Fifteen striped dolphins were found dead in the Croatian part of the Adriatic Sea in the last eight years (1999– 2007). More specimens were found in southern than in the northern part of the Adriatic. Analysis of twelve microsatellite loci and sequencing of a 882 base pair (bp) fragment of the mitochondrial DNA (mtDNA) control region were performed for genetic characterization. The mean allelic diversity (7 ± 0.78) and mean expected heterozygosity haplotypes with 22 polymorphic sites in ten individuals. The haplotype diversity (0.911 ± 0.077) was high, while nucleotide diversity was strikingly low (0.006 ± 0.003). Results presented here support the notion of the striped dolphin not being resident species in Croatian part of the Adriatic Sea.

Extensive polymorphism and evidence of selection pressure on major histocompatibility complex DLA-DRB1, DQA1 and DQB1 class II genes in Croatian grey wolves

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric-Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning-sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (d(N)/d(S)) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved.

Three Anisakis spp. isolated from toothed whales stranded along the eastern Adriatic Sea coast.

Knowledge concerning cetacean ecology in the Mediterranean is limited but important for sustainable planning and enforcement of appropriate conservation measures. Any information that might help to elucidate their ecology is essential. We explored the population and genetic structures of Anisakis spp. nematodes isolated from four toothed whale species - bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), Rissos dolphins (Grampus griseus) and Cuviers beaked whales (Ziphius cavirostris) - stranded along the eastern Adriatic Sea coast (1990-2012) to reveal more information on host ecological patterns. Lower parasite prevalence was observed in resident dolphin species compared with occasionally occurring species, as well as in young compared with adult dolphins, indicating different feeding habits related to age. No unequivocal relationship between the biological traits of a host (age, body length, body mass and blubber depth) and Anisakis population parameters was observed. Phylogenetic analysis revealed a new geographical record of Anisakis simplex sensu stricto (1.96%) and Anisakis physeteris (1.31%) in the Adriatic Sea in addition to resident Anisakis pegreffii (96.73%). In an assessment of the Adriatic Sea and oceans worldwide, the genetic structure of Anisakis revealed that A. pegreffii populations do not differ among various final host species but do differ with respect to geographical location in contrast to previously accepted Anisakis panmixia.

Stomach contents of two Cuvier’s beaked whales (Ziphius cavirostris) stranded in the Adriatic Sea

The stomach contents of two Cuvier’s beaked whales (Ziphius cavirostris) stranded along the eastern Adriatic coast were examined. One was a male stranded in 2004 and the other was a newborn stranded in 2008. Milk was found in the newborn’s stomach. The prey of the male was only cephalopods belonging mainly to the Teuthoidea. Five families and seven species were identified. Octopoteuthis sicula represented about 50% of estimated prey weight and Chiroteuthis veranyi was the most frequent species (estimated from the quantity of lower beaks). Three species are not listed in the Adriatic fauna, while the other species are found in middle and lower slope cephalopod assemblages in the southern Adriatic Sea.

Presence of the Mediterranean monk meal (Monachus monachus) in the Croatian part of the Adriatic sea

All findings and observations included in this note suggest that monk seals can easily reach all parts on the Croatian side of the Adriatic Sea.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health.

Histological structure of the adrenal gland of the bottlenose dolphin (Tursiops truncatus) and the striped dolphin (Stenella coeruleoalba) from the Adriatic Sea.

The structure of the adrenal gland was studied in 11 bottlenose dolphins (Tursiops truncatus), and five striped dolphins (Stenella coeruleoalba). These species are legally protected in Croatia. All examined animals died of natural causes and were found stranded along eastern Adriatic coast. In both species the adrenal gland consists of a cortex and a medulla; the cortex is divided into three zones. Whereas in the bottlenose dolphin, there is a zona arcuata which contains columnar cells arranged in the form of arches; in the striped dolphin this zone is replaced by zona glomerulosa containing rounded clusters of polygonal cells. In both species, the zona fasciculata consists of radially oriented cords of polygonal cells, whereas in zona reticularis cells are arranged in branching and anastomosing cords. The adrenal medulla in both species contains dark, epinephrine‐secreting cells and light norepinephrine‐secreting cells. Epinephrine‐secreting cells are localized in the outer part of the medulla, whereas norepinephrine‐secreting cells are found in the inner part, arranged in clusters and surrounded by septa of thin connective tissue. The gland is surrounded by a thick connective‐tissue capsule, from where thick trabeculae extend towards the interior. In the bottlenose dolphin, group of cells resembling both medullar and cortical cells can be seen within the capsule; whereas only groups of cells resembling cortical cells are found within the capsule of the striped dolphin. In the bottlenose dolphin invagination of the adrenal cortex into the medulla is obvious as well as medullary protrusions extending through cortex to the connective tissue capsule.

Isolation of Clostridium tertium from a Striped Dolphin (Stenella coeruleoalba) in the Adriatic Sea

Bacteriologic examination of an abscess found between blubber and musculature of a striped dolphin (Stenella coeruleoalba) found dead on 30 March 2002 in the Adriatic Sea, Croatia, was performed and an aerotoler-ant, nontoxogenic Clostridium tertium was isolated in pure culture. National Collections of Industrial Food and Marine Bacteria (NCIMB Ltd., Aberdeen, Scotland, UK) confirmed the results. Sequencing results showed it to be C. tertium with 100% similarity. The strain was named Clostridium tertium strain Zagreb, acceded to the culture collection and assigned the accession number NCIMB 13970. This is the first record of C. tertium in marine mammals.

PCR-based Y chromosome marker for discriminating between golden jackal (Canis aureus) and domestic dog (Canis lupus familiaris) paternal ancestry

AbstractNumerous reports of hybridization between wild Canis species and domestic dog suggest that hybridization between golden jackal and dog is likely. Here we present an assay of a Y chromosome marker for discriminating between golden jackal (Canis aureus) and dog (Canis lupus familiaris) paternal ancestry. Taking advantage of an insertion found in a dog Zfy intron haplotype but not in a golden jackal haplotype, we developed a three-primer PCR system in which species are differentiated based on the number of amplicons. Two amplicons are produced from domestic dog DNA template, while one amplicon is produced from golden jackal DNA template. Both amplicons can be analyzed in a single agarose gel electrophoresis run, while the longer amplicon also serves as an internal control. The method was validated using 27 golden jackal and 25 dog samples; all samples showed the expected amplicons. This simple and cost-effective method should prove useful for determining the direction of golden jackal–dog hybridization.

HEMATOLOGICAL AND SERUM BIOCHEMICAL REFERENCE VALUES FOR THE POSAVINA AND CROATIAN COLDBLOOD HORSE BREEDS

Abstract In this study the hematological and biochemical parameters in the blood of indigenous Croatian working horse breeds were investigated. The Posavina and Croatian Coldblood horses are adapted to harsh environmental conditions and their blood parameters might differ from other horse breeds. The study was carried out on 100 mares and 12 stallions of ages from 2 to 19 years. Fifteen hematological and 19 biochemical parameters were analysed. Values of 22 parameters showed considerable overlapping with values obtained for other horse breeds, and substantial resemblance is evident with values reported for Pakistani working horses. Several reference values showed statistically significant effect of sex (Hb, ALB, CREA, TBIL and Ca where significantly higher in stallions; EOS, CK, ALP, UREA and Mg were significantly higher in mares). None of the parameters studied showed any differences associated with age. The adaptation of Posavina and Croatian Coldblood horses to the harsh environment of flooded pastures and the way of breeding might be reflected in their specific reference values. The obtained results enabled the establishment of hematological and biochemical reference values in the blood of Posavina and Croatian Coldblood horses that will in turn support clinical diagnosis and further research into horse physiology. Abstract U studiji je obavljeno ispitivanje hematoloških i biohemijskih parametara krvi kod radnih hrvatskih autohtonih rasa konja. Posavski i hrvatski hladnokrvnjak su dve rase konja koje su adaptirane na oštre uslove spoljašnje sredine pa se pretpostavlja da njihovi parametri krvi mogu da budu različiti u poređenju sa drugim rasama. Ispitivanje je obavljeno na 100 kobila i 12 pastuva starosti od 2 do 19 godina. Obavljena je analiza u odnosu na pedeset hematoloških i 19 biohemijskih parametara. Vrednosti koje su dobijene u odnosu na 22 parametra pokazale su značajna preklapanja u poređenju sa drugim rasama konja, pri čemu je ustanovljena značajna podudarnost sa vrednostima koje su karakteristične za pakistanskog radnog konja. Nekoliko referentnih vrednosti je pokazalo statistički značajnan utacaj pola (Hb, ALB, CREA, TBIL i Ca vrednosti su bile značajno veće kod pastuva; EOS, CK, ALP, UREA i Mg vrednosti su bile značajno veće kod kobila). Nijedan parametar koji je ispitivan nije pokazao bilo kakvu varijaciju u odnosu na starost životinja. Prilagođenost posavskog i hrvatskog hladnokrvnog konja na oštre i teške uslove spoljašnje sredine koji podrazumevaju ispašu na poplavljenim pašnjacima, kao i način uzgoja, može da ima posledice na specifične referentne vrednosti ispitivanih parametara. Dobijeni podaci omogućavaju definisanje hematoloških i biohemijskih referentnih vrednosti krvi posavske i hrvatske rase hladnokrvnih konja, a te vrednosti bi omogućavale kliničku dijagnostiku i buduća ispitivanja fiziologije konja.