A Multi-Site Assessment of Anesthetic Overdose, Hypothermic Shock, and Electrical Stunning as Methods of Euthanasia for Zebrafish (Danio rerio) Embryos and Larvae.

Euthanasia in zebrafish (Danio rerio) younger than 5 days post fertilization (dpf) is poorly described in the literature, and standardized protocols are lacking, most likely because larvae not capable of independent feeding are often not protected under national legislations. We assessed the euthanasia efficacy in laboratories in different countries of a one hour anesthetic overdose immersion with buffered lidocaine hydrochloride (1 g/L, with or without 50 mL/L of ethanol), buffered tricaine (1 g/L), clove oil (0.1%), benzocaine (1 g/L), or 2-phenoxyethanol (3 mL/L), as well as the efficacy of hypothermic shock (one hour immersion) and electrical stunning (for one minute), on zebrafish at <12 h post fertilization (hpf), 24 hpf, and 4 dpf. Based on the survival/recovery rates 24 h after treatment, the most effective methods were clove oil, lidocaine with ethanol, and electrical stunning. For 4 dpf larvae, signs of aversion during treatment demonstrated that all anesthetics, except lidocaine, induced aversive behavior. Therefore, the most suited euthanasic treatment was lidocaine hydrochloride 1 g/L, buffered with 2 g/L of sodium bicarbonate and mixed with 50 mL/L of ethanol, which euthanized both embryos and larvae in an efficient and stress-free manner. Electrical stunning also euthanized embryos and larvae efficiently and without signs of aversion; this method needs further assessment in other laboratories to draw firm conclusions.

Occurrence of intersex in wild freshwater fish in Slovenian rivers: a histological evaluation.

The aim of this preliminary research was to establish if there are intersex occurrences in wild freshwater fish in Slovenian rivers and streams. In the first study we evaluated all fish species of both sexes obtained from the river Ljubljanica from its source to mouth. In the second study we focused on the rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) males from 30 rivers and streams in different parts of Slovenia. The male gonads were histologically assessed for the presence of oocytes to determine the frequency and degree of intersex. Oocytes were found in the testicular tissue of a single grayling (Thymallus thymallus) and in the adipose tissue adjacent to the testis of a single common barbel (Barbus barbus), both from the Ljubljanica. Several cyst-like structures that resemble degenerated presumptive oocytes were also present in several trout testes. This preliminary report is the first of its kind in Slovenia. To gain a better insight into the intersex issue in Slovenia, we plan to regularly biomonitor freshwater pollution by histologically examining fish gonads and, if possible, by determining vitellogenin plasma levels in fish.

Mycobacterium angelicum sp. nov., a non-chromogenic, slow-growing species isolated from fish and related to Mycobacterium szulgai.

The name 'Mycobacterium angelicum' dates back to 2003 when it was suggested for a slowly growing mycobacterium isolated from freshwater angelfish. This name is revived here and the novel species is proposed on the basis of the polyphasic characterization of four strains including the original one. The four strains presented 100 % 16S rRNA gene sequence similarity with Mycobacterium szulgai but clearly differed from M. szulgai for the milky white aspect of the colonies. The sequence similarity with the type strain of M. szulgai ranged, in eight additionally investigated genetic targets, from 78.9 to 94.3 %, an evident contrast with the close relatedness that emerged at the level of 16S rRNA gene. The average nucleotide identity between the genomes of M. szulgai DSM 44166T and strain 126/5/03T (type strain of the novel species) was 92.92 %, and supported the status of independent species. The confirmation of the name Mycobacterium angelicum sp. nov. is proposed, with strain 126/5/03T ( = CIP 109313T = DSM 45057T) as the type strain.

Aphanomyces astaci in wild crayfish populations in Slovenia: first report of persistent infection in a stone crayfish Austropotamobius torrentium population.

All 5 crayfish species inhabiting Slovenian freshwaters, of which 3 are indigenous crayfish species (ICS: Astacus astacus, Austropotamobius pallipes, and A. torrentium) and 2 are non-indigenous (NICS: Pacifastacus leniusculus and Cherax quadricarinatus), were inspected for the presence of Aphanomyces astaci, the causative agent of crayfish plague. Wild crayfish populations showing no clinical signs of infection were inspected using A. astaci-specific real-time PCR. In addition, a conventional PCR assay was employed and confirmative sequencing was performed. Out of 88 analyzed crayfish, 15/27 (55.6%) specimens of A. torrentium from Borovniscˇ%%KERN_ERR%%ica Brook and 4/35 (11.4%) of P. leniusculus from the Mura River tested positive, showing low to moderate levels of infection (agent levels A1-A4 and A1-A3, respectively). Results revealed the presence of A. astaci not only in the resistant NICS but also in ICS, since the infected population of A. torrentium presumably had no contact with the NICS carrier and appeared to sustain A. astaci infection in the 2 sampling years. Although the A. astaci genotype has not yet been identified, a connection between the latent infection in ICS and a Group A strain of A. astaci, co-evolving with A. torrentium since its first introduction to Slovenia, is suggested as the most plausible conclusion. This is the first reported population of the genus Austropotamobius with persistent infection, in addition to the already known populations of the genus Astacus. Findings of the presumed co-evolution of A. astaci and ICS hosts open new perspectives, necessitating additional studies on the presence of A. astaci genotypes in the persistently infected ICS populations.

First isolation and genotyping of viruses from recent outbreaks of viral haemorrhagic septicaemia (VHS) in Slovenia.

In November and December 2007, the virus causing viral haemorrhagic septicaemia (VHS) was detected in rainbow trout Oncorhynchus mykiss from 2 fish farms in Slovenia. During 2008 and 2009 the infection spread only among rainbow trout farms and 4 new outbreaks were confirmed. High mortality and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides [nt]) and 9 partial (600 nt) glycoprotein gene nucleotide sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Genotype I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis and epidemiological investigations confirmed that the VHS virus had been (re)introduced with imported live fish, and that subsequent outbreaks were linked to the initial infection. Our study shows that direct nucleotide sequencing of RT-PCR products, amplified from the tissue of VHSV-infected fish, represents a reliable tool for fast routine genotyping in diagnostic laboratories. This is the first report of a natural epidemic associated with VHSV infection in Slovenia since the eradication of the disease in 1977.

Abamectin effects on rainbow trout (Oncorhynchus mykiss).

The effect of abamectin (ABM) on rainbow trout (Oncorhynchus mykiss) was studied. The acute toxicity of ABM on rainbow trout was established, following the target 58-h water bath exposure of ABM concentrations from 0.6 to 4.5 microg/l, on the basis of which LD75 (4.0 microg/l) was calculated. The histological changes in organs showed a direct toxicity of ABM for rainbow trout since degenerative changes in brain and kidney and--to a minor extent--in liver were established. The values of the ABM residues in fish muscle tissue with skin were proportional to the exposed concentrations of ABM.

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods.

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.