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U priručniku je dan prikaz imunološkog mehanizma u lubina i komarče, najvažnijih bakterijskih bolesti te postupaka cijepljenja u mrijestilištu i docijepljivanja na uzgajlištima u jadranskim uzgajalištima riba

APPLICATION OF EPIDEMIOLOGICAL MODELLING OF Sparicotyle chrysophrii TRANSMISSION IN Sparus aurata FARMS Elisa Stella1, *, Roberto Pastres1 , Damiano Pasetto1 , Matko Kolega2 , Danijel Mejdandžić2 , Slavica Čolak2 , Lorenzo Mari3 , Enrico Bertuzzo1 1 Department of Environmental Sciences, Informatics and Statistics, Ca’ Foscari University of Venice Scientific Campus, Via Torino, 155, 30172, Mestre- Venice, Italy 2 Cromaris d.d., Zadar, Croatia 3 Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano Via Ponzio 34/5, 20133, Milan, Italy E-mail: elisa.stella@unive.it Introduction Mediterranean aquaculture production is known to have impacts on the environmental and marine habitat, impairing water and bottom sediment quality. Furthermore, rapid development of cage fish farming has been associated with propagation of infectious diseases, pathogens and parasites. Sparicotyle chrysophrii is a common parasite of cultured Gilthead sea bream (Sparus aurata), an important species for Mediterranean aquaculture production. The parasite attaches on fish gills and can cause lethal epizootics in sea cages. Infections depend on environmental factors, in particular on water temperature (Antonelli et al., 2010). Epidemiological modeling represents a valid tool to help fish farmers understand parasite transmission and evaluate possible control measures. To this purpose, in the following we analyze a novel epidemiological model of S. chrysophrii transmission. Methods We developed a novel stratified compartmental model where each compartment Xja represents the abundance of fish infected with j juvenile parasites and a adults. The model further accounts for environmental abundance of eggs and miracidia, where the latter determines the force of infection, i.e. the rate at which parasite larvae attach to fish. The dynamics further accounts for egg production and hatching, parasite development, baseline mortality for parasites and fish, and parasite-induced fish mortality. Critical parameters controlling the progression of the disease are assumed to be temperature dependent. We applied the model to data collected in six cages of a sea bream farm managed by Cromaris (Bisage, Croatia). A controlled experiment was run between February and November 2021 in which 30 fish were collected each month and, for each fish, all eight arc gills were examined to count the number of attached parasites. We estimated model parameters in a Bayesyan framework, sampling the posterior distribution with a Markov Chain Monte Carlo algorithm. Specifically, the model estimates the temporal dynamics of the probability that a sampled fish hosts a certain number of adult parasites. The likelihood of a monthly sample is calculated assuming a multinomial distribution, where the event probabilities are estimated through the deterministic model simulation given a certain parameter set. We further included the total number of fish alive at any time in the determination of the likelihood. Results Results show that the model is able to reproduce the distribution of the number of parasites hosted by the sampled fish (Figure 1), as well as the temporal progression of the prevalence of infection (i.e., the number of fish hosting at least one parasite). Data shows that prevalence has a peak in August, after the period with the warmest water temperature, and then decreases. Discussion and conclusions We applied a novel epidemiological model to a dataset of S. chrysophrii infections collected in a sea bream farm. The model succeeds in simulating the effects of environmental factors (e.g., water temperature) on the infection dynamics. Posterior distribution of parameters shed insights on crucial process rates that control disease transmission. Future developments could involve coupling the epidemiological model with a fish growth model to simulate the effect of parasite burden on fish metabolism. The knowledge gained through this combined experimental and modeling exercise could be used in the future for designing and implementing strategies to control the spreading of the infection and, accordingly, to improve both fish health and aquaculture production. (Continued on next page) 1250 Acknowledgements The research leading to these results has received funding from the European Union’s HORIZON 2020 Framework Programme under Grant Agreement no. 862658. References Antonelli, L., Quilichini, Y., & Marchand, B (2010). Sparicotyle chrysophrii (Van Beneden and Hesse, 1863) (Monogenea: Polyopisthocotylea) parasite of cultured gilthead sea bream Sparus aurata (Linnaeus 1758) (Pisces: Teleostei) from Corsica: Ecological and morphological study. Parasitology Research, 107:389–398.

Introduction Monogenean outbreaks threaten the health of farmed fish stocks and severely affect aquaculture production. The meagre Argyrosomus regius is a promising species for diversification in Mediterranean fish farming: in the wild, this species is known to host a variety of ectoparasites, however reports of infection and pathology in farmed fish are rather infrequent. The present investigation reports the occurrence of an outbreak of monogenean infection in A. regius broodstock, with identification of the parasite species involved and associated histopathological findings. Methodology During a mortality outbreak registered in a meagre broodstock batch, parasites were isolated from gills and skin and preserved in 70% ethanol and 10% buffered formalin. Gill samples were also fixed in 10% buffered formalin for histology. The parasites were subjected to morphological analysis in light and scanning electron microscopy, and to molecular analysis amplifying and sequencing the complete ITS and partial 28S rDNA. Results and Conclusions Two monogenean species were identified, based on their morphology: the calceostomatid Ktariella polyorchis, reported for the first time in farmed A. regius, and the diplectanid Diplectanum sciaenae, already reported from A. regius broodstock in Spain. K. polyorchis were mainly found on the skin, mainly along the dorsal part of the body and tail, and in the oral cavity, while D. sciaenae were recovered from the gills. At histological level, the presence of D. scienae was associated to severe gill damage with diffuse epithelial hyperplasia and sloughing off in the lamellae, focal necrosis, inflammatory infiltration, hyperemia, telangiectasias and hemorrhages. Histological lesions were similar to those observed in previous reports and confirmed D. scienae as health-threatening parasite of meagre. Our findings suggest a role of both K. polyorchis and D. sciaenae as pathogenic parasites of A. regius farmed in the Mediterranean area, although at the moment they have been reported only from broodstock. In addition, we provide the first sequence data for these two monogenean species, as a further diagnostic tool for their correct taxonomical identification in possible future outbreaks.

INTRODUCTION. Monogeneans of the Family Calceostomatidae Parona & Perugia, 1890 parasitize different species of Sciaenids worldwide: among these, the calceostomatid Ktariella polyorchis Vala & Euzet, 1977 has been described infecting the gills of wild meagre Argyrosomus regius (Asso, 1801) caught off Tunisia and Egypt. The present investigation reports for the first time the occurrence of K. polyorchis in broodstock of A. regius from a Croatian farm. MATERIALS AND METHODS. At the farm, gills samples and skin scrapings were collected from heavily infected fish and preserved in 70% ethanol and 10% buffered formalin. Ethanol-fixed parasites were subjected to morphological analysis following clarification in Amman’s lactophenol. Before clearing the parasites, a section of the body without diagnostic characters was excised with a sterile scalpel and processed for molecular analysis, amplifying and sequencing the complete ITS and partial 28S rDNA. For observation in Scanning Electron Microscopy (SEM), formalin-fixed specimens were dehydrated in a graded ethanol series, critical point dried and sputter coated with gold-palladium. RESULTS AND CONCLUSIONS. Although being reported mainly from the gills, in our samples K. polyorchis were mainly found on the fins and skin, while the gills were infected with another monogenean species, the diplectanid Diplectanum sciaenae Van Beneden & Hesse, 1863; this finding may indicate a possible displacement of K. polyorchis in concurrent heavy infections with D. sciaenae. Originally assigned to the genus Calceostoma Van Beneden, 1958 based on the morphology of the anterior region and of the armature in the haptor, K. polyorchis was subsequently assigned to a separate genus based on the morphology of the male reproductive system. Our study adds further morphological information, providing the first detailed description of the parasite surface by SEM analysis, and the first molecular data, useful in future taxonomical and phylogenetic studies.

Bacterial adhesion to fish surface and survival in gastrointestinal tract are essential characteristics in order to select probiotic candidate for aquaculture products. In our previous research we isolated Lactobacillus plantarum O1 from the gut of sea bream (Sparus aurata) and characterized it as plantaricin producer and bioprotective culture. This strain was successfully used for biopreservation of fresh sea bass (Dichentrarchus labrax), mussels (Mytilus galloprovincialis) and oysters (Ostrea edulis) with extension of shelf life to ten days and thirteen days, respectively. In order to characterize it as potential fish probiotic, L. plantarum O1 was examined for in vitro adhesion to sea bass mucus, survival in 10% sea bass bile and gastric contents as well as survival in sea water and hemolytic activity. Results showed good adhesion capacity to fish surface mucus as well as high survival rate in 10% bile and stomach content. L. plantarum O1 also withstood sea water environment and didn’t lyse red blood cells. These results suggest that L. plantarum O1 has potential application as fish probiotic and further studies are required to evaluate these properties in aquatic animals.

The cymothoid isopod (Ceratothoa oestroides, Risso 1916) is protandric hermaphroditic parasite. It is parasitizing in the buccal cavity and can cause significant production losses in cage reared sea bass (Dicentrarchus labrax). Biology of this parasite is often studied in laboratory conditions, while the literature data on the biology of this parasite in farming conditions are scarce. Therefore, the aim of this study is to collect data on the biology C. oestroides in the sea bass cage farming conditions in the Adriatic Sea. The experiment was carried out at farm within 14 commercial net cages of 16 m diameter stocked with 200.000 fingerlings. Visual control of the entire population of fish in each cage was carried out consecutively between 2300 and 4700 degreedays (DD) of production. Total of 100 infected individuals were excluded at each sampling. Afterwards, biometric analyses were performed on infected fishes and on C. oestroides males and females. Sexual maturity and fecundity on C. oestroides females was determined. In addition, sea temperature were recorded daily. Fish infected with C. oestroides were found in all cages. Overall, the percentage of infection ranged from 0.19% to 2.53%. Also, mature females were found in all cages, in range from 2% to 18%. Pearson correlation coefficient showed that there was a statistically significant and moderate positive correlation between DD and length of female parasites (r = 0.512, p

Introduction: The cymothoid isopod Ceratothoa oestroides (Risso, 1816) is a protandric hermaphrodite fish parasite. It is a ubiquitous parasite with significant impact on fish health, usually found in Sparidae, Carangidae, Clupeidae, Maenidae, Scorpaenidea and Mugilidae families (Charfi-Cheikhrouha et al., 2000). Its common effects on fish in aquaculture can vary from growth retardation and decreased immunocompetency to mass mortalities of juvenile fishes, causing significant economic consequences in aquaculture (Mladineo, 2003). Most studies on this parasite in the Mediterranean aquaculture have described its influence on sea bass and sea bream (Horton and Okamura, 2001 ; Bouboulis et al., 2004). Last few years meagre (Argirosomus regius) has been introduced in fish farming in Croatia (Kružić et al., 2016). In this study, we examined C. oestroides infestation in the cultured meagre to provide new insights into importance and incidence of this parasite in this geographic area, and in order to improve existing fish farm management strategies. Methodology: Research was carried out at two commercial fish farms in the Central Adriatic, Croatia. First sampling and biometry of meagre was performed at the age of seven months (January 2016) and afterwards in February, March, April, May and July. A total of 2.028 fish were sampled. The incidence of parasites and weight and growth of meagre were monitored. Results: Parasites were found at both commercial fish farms. A total of 2.028 fish were examined and 84 (4.14%) of them were infested with parasites. The percentage of infestation was highest in February (7.0%) and lowest in May (2.4%). Biometric measurements revealed that meagre infested with the parasite had a significantly (P

U ovom diplomskom radu obradili smo uzročnike tuberkuloze i drugih mikobakterioza koji su u istraživane divljači (divlja svinja) izolirani u razdoblju od 1998. do 2006. godine. Također smo obradili zoologiju divlje svinje i njezin način života. Poglavlja o mikobakterijama sadržavaju podatke o klasifikaciji i rasprostranjenost mikobakterija u okolišu, te podatke od građe i tenaciteta do dijagnostike i profilakse mikobakterioza. U radu smo prikazali i tuberkulozu kao bolest koja se javlja u domaćih i divljih životinja ali i ljudi (zoonoza). Mikrobiološkom pretragom i molekularnim postupcima izdvojeni sojevi u divlje svinje su identificirani kao: M. avium subsp. avium (jedan izolat), M. avium hominissuis (deset izolata), M. fortuitum (dva izolata), M. caprae (jedan izolat) i M. peregrinum (jedan izolat). Velika nazočnost M. avium kompleksa je očekivana jer je sustavnim provođenjem mjera gotovo suzbijena vrsta M. bovis. Uz to mikobakterije iz M. avium kompleksa su vrlo proširene vrste u okolišu, pogotovo u vlažnim staništima u kojima obitava i divlja svinja. Dobiveni rezultati su odraz epizootiološke situacije s obzirom na tuberkulozu i mikobakterioze u domaćih i divljih životinja. Zbog daljnjeg nastojanja iskorjenjivanja tuberkuloze i mikobakterioza, te otkrivanja uloge divljih životinja u održavanju i širenju uzročnika bolesti, potrebno je još istraživati kronologiju prijenosa među vrstama. Zbog očite prisutnosti mikobakterija u divljih svinja logično je da one predstavljaju prirodne rezervoare tih uzročnika, a time i opasnost za druge divlje i domaće životinje te čovjeka.