Keith A. Johnson

Discriminating between a Neurotropic Myxobolus sp. and M. cerebralis, the Causative Agent of Salmonid Whirling Disease

Abstract While screening salmonids for Myxobolus cerebralis, the causative agent of whirling disease, we detected a neurotropic Myxobolus sp. that is morphologically similar to the M. cerebralis spore in brain and other nervous tissue. We developed a polymerase chain reaction (PCR)–based diagnostic technique to differentiate the two species of Myxobolus. Primers were designed for a 277-base-pair (bp) sequence within the 18S ribosomal DNA (rDNA) gene, and the restriction enzyme Mse-I was chosen to cut the amplified product into diagnostic fragments. When electrophoresed in an acrylamide gel, M. cerebralis yielded a two-band pattern while the neurotropic Myxobolus sp. yielded a three-band pattern. This diagnostic method has resolved cases in which the pepsin–trypsin digest screening test indicated Myxobolus spores but histological examination or PCR was negative for M. cerebralis. We were also able to eliminate M. kisutchi as the neurotropic Myxobolus sp. using spores from infected coho salmon Oncorhynchus ...

Management of Bacterial Kidney Disease in Chinook Salmon Hatcheries Based on Broodstock Testing by Enzyme-Linked Immunosorbent Assay: A Multiyear Study

Abstract From the mid-1980s through the early 1990s, outbreaks of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum continued in Chinook salmon Oncorhynchus tshawytscha in Idaho Department of Fish and Game (IDFG) hatcheries despite the use of three control methods: (1) injection of returning adult fish with erythromycin to reduce prespawning BKD mortality and limit vertical transmission of R. salmoninarum, (2) topical disinfection of green eggs with iodophor, and (3) prophylactic treatments of juvenile fish with erythromycin-medicated feed. In addition, programs to manage BKD through measurement of R. salmoninarum antigen levels in kidney tissues from spawning female Chinook salmon by an enzyme-linked immunosorbent assay (ELISA) were tested over 13–15 brood years at three IDFG hatcheries. The ELISA results were used for either (1) segregated rearing of progeny from females with high ELISA optical density (OD) values (usually ≥0.25), which are indicative of high R. salmoninarum antigen le...

Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum.

In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.

Assessment of Formalin and Hydrogen Peroxide Use during Egg Incubation of North American Burbot

Abstract Control of aquatic fungi in the family Saprolegniacea during egg incubation was investigated as part of a program aimed at developing aquaculture methods for Kootenai River burbot Lota lota maculosa, a species relatively unknown to North American aquaculture. The concentration effectiveness of two antifungal control methods, formalin and hydrogen peroxide, was compared over two consecutive breeding seasons in a newly developed micro-incubation system. The results indicated that daily 15-min treatments of 1,667 mg of formalin/L and 500 mg of hydrogen peroxide/L inhibited fungal growth on eggs and increased egg survival by up to 200% during the incubation period relative to the untreated controls. Lower concentrations of 1,000 mg/L formalin and 250 mg/L hydrogen peroxide also yielded increased survival but were not sufficient to completely inhibit fungal growth on eggs. Concentrations up to 5,000 mg/L formalin and 500 mg/L hydrogen peroxide did not appear to negatively influence egg survival during...

Distribution of Myxobolus cerebralis within a Free-Flowing River System during the Migration Period for Juvenile Anadromous Salmonids in Idaho

Abstract To gain a better understanding of the pathogen-associated risk to resident and anadromous salmonid populations, the distribution, prevalence, and intensity of Myxobolus cerebralis infections were assessed during the smolt migration period in the Salmon, Snake, and Clearwater rivers of Idaho. Sentinel exposures of rainbow trout Oncorhynchus mykiss fry (0.6 g) occurred at 10 locations beginning at the headwaters of the Salmon River and extending downstream to the Snake and Clearwater rivers from April 18–28 and May 15–25, 2001. Following exposure, fish were examined for the prevalence and severity of infection by means of histological, pepsin−trypsin digest, and nested polymerase chain reaction analyses. Infections did not occur in the portions of the Snake or Clearwater rivers included in this study but were found in the Salmon River at five locations during April and seven locations during May. The results during April demonstrated moderate to high prevalence of infection (65% and 75% at 900 and ...

Redescription and molecular characterization of Myxobolus kisutchi Yasutake & Wood, 1957.

The genus Myxobolus Butschli, 1882 (Myxozoa, Myxosporea, Myxobolidae) includes over 700 described species primarily from fish hosts (Eiras, Molnar & Lu 2005). Spore morphology, in conjunction with host and tissue specificity, has been the accepted means of species identification (Lom & Dykova 1992). Although fresh material is preferable (Lom & Arthur 1989), species have been described from histological material that has been formalin or alcohol fixed (Schuberg & Schröder 1905; Wyatt 1979; Yasutake & Wood 1957). Spore size comparisons are sometimes difficult as fixatives and stains tend to cause spore shrinkage (Parker & Warner 1970). In the light of current DNA analysis technologies, species descriptions solely based on morphological characteristics of spores may be inadequate. In identifying a neurotropic Myxobolus species found in Idaho (Hogge, Campbell & Johnson 2008), the Eagle Fish Health Laboratory (EFHL) of the Idaho Department of Fish and Game wanted to compare this neurotropic species with other Myxobolus species characterized in the cranial tissues of salmonids. One of the species we wanted to investigate was Myxobolus kisutchi, described by Yasutake & Wood (1957) in the spinal cord of coho salmon, Oncorhynchus kisutch (Walbaum), collected at Minter Creek, Washington. The original description of M. kisutchi, made from histological sections, was insufficient for comparisons with other Myxobolus spp. whose characterizations were from fresh material. Therefore, the objectives of this study were to augment the original data with morphological and morphometrical data using fresh spores and to provide the 18S rDNA sequence. To maximize the likelihood of obtaining the same species, samples were collected from the same tissue, host and location of the original material. Juvenile coho salmon were obtained from Minter Creek in northern Washington State (47.372062N 122.702494W) in 2003, 2006 and 2007. In 2003, five salmon heads were split in half and fixed in 10% neutral buffered formalin for histological examination. These were processed using standard techniques, sectioned and then stained with May–Grunwald Giemsa and haematoxylin and eosin. Slides were examined using a compound microscope for the presence of parasite developmental stages and myxospores in brain and spinal cord. In 2006, three salmon heads were disinfected with 70% isopropanol. Using a fresh scalpel blade for each head, the dorsal cranium was removed to reveal the brain and spinal cord, which were placed into three individual tubes for DNA extraction. Genomic DNA was isolated using a QIAGEN DNeasy tissue kit (Qiagen, Valencia, CA, USA) according to the manufacturer s protocol. To sequence the 18S rDNA of the three isolates, myxozoan generic primers 18E (CTGGTTGATCCTGCCAGT) (Hillis & Dixon 1991) and 18R Journal of Fish Diseases 2008, 31, 707–712 doi:10.1111/j.1365-2761.2008.00936.x

A Simple Isolation Incubator for Specialized Rearing of Salmonid Eggs and First-Feeding Fry

Abstract The construction of an isolation incubator for small-scale fish culture and research is described. The simple, inexpensive (US

Detection of Renibacterium salmoninarum in chinook salmon, Oncorhynchus tshawytscha (Walbaum), using quantitative PCR

5) isolation incubator is routinely used to incubate as many as 100 green eggs of sockeye salmon Oncorhynchus nerka and Chinook salmon O. tshawytscha through the first-feeding fry stage of development at the Idaho Department of Fish and Games Eagle Fish Hatchery. The incubator is small (2.5-L volume) and portable, requires a small amount of water per individual unit (500–1,200 mL/min), and provides a way to incubate multiple rearing groups in a quarantine environment through the early stages of fish development. Although these incubators have been used exclusively for incubation of salmonid eggs and fry, we expect this design can be used to successfully incubate eggs of other fish species.