Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Residual tissue magnesium concentration in jellyfish (Aurelia aurita and Cassiopea andromeda) following magnesium chloride euthanasia.

Magnesium chloride in high concentration is used for euthanasia of jellyfish to limit overpopulation and for predatory species consumption, but its use could lead to magnesium bioaccumulation and subsequent negative effects in consumers. Two species of scyphozoan jellyfish (Cassiopea andromeda and Aurelia aurita) were subjected to freezing (control), or magnesium chloride baths (144 g/L), with subsequent 30 min baths (one or two) in fresh artificial saltwater and submitted for inductively coupled plasma analysis to determine tissue concentration. Frozen jellyfish consistently yielded the lowest magnesium concentrations, while magnesium chloride euthanized individuals contained the highest concentrations in both species. C. andromeda displayed a significantly higher (p < .05) magnesium absorption capacity than A. aurita in both trials. Single and double baths significantly decreased magnesium concentrations (p < .05) in both species, however, magnesium remained consistently elevated compared to frozen specimens. This study demonstrated species-specific magnesium accumulation in jellyfish posteuthanasia and that rinsing was an effective method to limit excessive magnesium that could be deleterious to animals in public display aquaria. Magnesium concentrations of tissue and receiving water should be tested if magnesium chloride euthanasia is utilized for dietary supplementation in small bodies of water.

Magnesium concentration influences size and pulse rate in the upside-down jellyfish, Cassiopea andromeda.

Magnesium is involved in a variety of physiological processes in marine animals and is known to be deleterious in both excess and deficiency. The effects of magnesium concentration ranging from 700 mg/L (low), 1344 mg/L (control), and 2000 mg/L (high) on size and pulse rate in upside-down jellyfish (Cassiopea andromeda) medusae were examined in two separate 28-day trials. Exposure to low magnesium resulted in significantly (p < .05) higher pulse rates and decreased bell diameter and also produced oral arm degradation. Exposure to high magnesium resulted in significantly (p < .05) lower pulse rates and decreased bell diameter as well as oral arm cupping. In both low and high magnesium, almost all specimens changed color from pale blue on Day 1, to brown by Day 28, suggesting a loss of zooxanthellae. The decrease in bell diameter and color change was more pronounced and occurred more rapidly in low magnesium. The results of both trials demonstrate the deleterious effects of high and low magnesium on C. andromeda and emphasize the importance of monitoring magnesium concentration to maintain healthy display animals in public aquaria.

Rare neural crest tumor in teleost species: olfactory neuroblastoma in a spotted pike-characin Boulengerella maculata.

One spotted pike-characin Boulengerella maculata, managed in a public display aquarium for 7 yr, developed a small unilateral pink growth associated with the right nare. The growth eventually extended through both nares after 5 mo. B. maculata is a species of piscivorous fish that relies on visual acuity when hunting prey. The rapidly-growing bilateral tumor would have impeded vision if left untreated. The fish was managed for over 10 mo using only surgical debulking, topical liquid nitrogen, and occasional nonsteroidal anti-inflammatory medications as needed to reduce tumor size and restore sight. Diagnosis proved challenging due to superimposed inflammation, neoplasm cellular inconsistencies, and a lack of proven immunohistochemical stains or other diagnostic stains. Using tumor location and appearance, histopathology, advanced imaging, electron microscopy, special staining, and immunohistochemistry, the ultimate diagnosis confirmed olfactory neuroblastoma, or esthesioneuroblastoma. Although long-term prognosis is poor, hunting strategy of the affected species may dictate quality of life, as appetite, body condition, and behavior remained largely unchanged in this animal until time of euthanasia.

Assessment of Probiotic Viability during Cheddar Cheese Manufacture and Ripening Using Propidium Monoazide-PCR Quantification.

The use of a suitable food carrier such as cheese could significantly enhance probiotic viability during storage. The main goal of this study was to assess viability of commercial probiotic strains during Cheddar cheesemaking and ripening (4-6 months) by comparing the efficiency of microbiological and molecular approaches. Molecular methods such as quantitative PCR (qPCR) allow bacterial quantification, and DNA-blocking molecules such as propidium monoazide (PMA) select only the living cells' DNA. Cheese samples were manufactured with a lactococci starter and with one of three probiotic strains (Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus RO011, or Lactobacillus helveticus RO052) or a mixed culture containing B. animalis subsp. lactis BB-12 and L. helveticus RO052 (MC1), both lactobacilli strains (MC2), or all three strains (MC3). DNA extractions were then carried out on PMA-treated and non-treated cell pellets in order to assess PMA treatment efficiency, followed by quantification using the 16S rRNA gene, the elongation factor Tu gene (tuf) or the transaldolase gene (tal). Results with intact/dead ratios of bacteria showed that PMA-treated cheese samples had a significantly lower bacterial count than non-treated DNA samples (P < 0.005), confirming that PMA did eliminate dead bacteria from PCR quantification. For both quantification methods, the addition of probiotic strains seemed to accelerate the loss of lactococci viability in comparison to control cheese samples, especially when L. helveticus RO052 was added. Viability of all three probiotic strains was also significantly reduced in mixed culture cheese samples (P < 0.0001), B. animalis subsp. lactis BB-12 being the most sensitive to the presence of other strains. However, all probiotic strains did retain their viability (log 9 cfu/g of cheese) throughout ripening. This study was successful in monitoring living probiotic species in Cheddar cheese samples through PMA-qPCR.

Neoplasia of captive yellow sea horses (Hippocampus kuda) and weedy sea dragons (Phyllopteryx taeniolatus).

Syngnathidae is the family of fish that includes sea horses, pipefish, and sea dragons. To date, only a single publication has described neoplasia in syngnathids, a fibrosarcoma of the brood pouch in an aquarium-reared lined sea horse (Hippocampus erectus). From 1998 until 2010, the Toronto Zoo submitted 172 syngnathids for postmortem; species included the spotted or yellow sea horse (Hippocampus kuda), the pot-bellied sea horse (Hippocampus abdominalis) and the weedy sea dragon (Phyllopteryx taeniolatus). Seven neoplasms and two neoplastic-like lesions were identified from these cases. Under light microscopy, the neoplasms had morphological characteristics of a cardiac rhabdomyosarcoma, renal adenocarcinoma, renal adenoma, renal round cell tumors, which were likely lymphomas, exocrine pancreatic carcinoma, and intestinal carcinoma. Of these neoplasms, four had clear evidence of metastasis: the pancreatic and intestinal carcinomas and both round cell tumors. As syngnathids are highly fastidious animals, they can be difficult to maintain in captivity. In order to improve their husbandry, preventative and palliative care, as well as treatment, it is important to investigate and document the types of diseases affecting syngnathids.

Mass mortality associated with koi herpesvirus in wild common carp in Canada.

Koi herpesvirus (KHV) was identified as being associated with more than one mortality event affecting common carp in Canada. The first was an extensive mortality event that occurred in 2007 in the Kawartha Lakes region, Ontario, affecting Lakes Scugog and Pigeon. Fish had branchial necrosis and hepatic vasculitis with an equivocal interstitial nephritis. Several fish also had branchial columnaris. Subsequent mortality events occurred in 2008 in additional bodies of water in south-central Ontario, such as Lake Katchewanooka and outside of Ontario in Lake Manitoba, Manitoba. Koi herpesvirus was detected in fish submitted for examination from all of these lakes by polymerase chain reaction (PCR), and sequence of the PCR product revealed 100% homology to KHV strains U and I. Real-time PCR analysis of KHV-infected wild carp revealed viral loads ranging from 6.02×10(1) to 2.4×10(6) copies µg(-1) host DNA. This is the first report of KHV in Canada.

Repeated low-level exposure of the round goby (Neogobius melanostomas) to Clostridium botulinum type E neurotoxin.

In a 4-mo study (June 2004-September 2004), round gobies (Neogobius melanostomas) were dosed orally every 72 hr for up to 21 days with Clostridium botulinum neurotoxin type E (BoNT/E) at one of four doses: 0, 50, 250, and 500 mouse lethal doses (MLD). Fish were observed for changes in pigmentation and behavior for the duration of the experiment. Mortality was observed with all treatments, with the exception of the 0 MLD control. Clinical signs observed were consistent with prior research and appeared to occur in a threshold manner. The mean times to death and percent mortalities were dose dependent. Hazard ratios were determined to have a significant positive (parameter estimate = 0.03) linear relationship with dose. The hazard ratio showed that per one unit dose increase, the instantaneous probability of a fish dying increased 1.02%. Postmortem analysis of experimental fish demonstrated that 11% (3/27) of fish contained detectable BoNT/E in their visceral fraction. The other 89% tested negative for BoNT/E, despite the fact that all fish died as a result of BoNT/E exposure. Therefore, botulism should not necessarily be ruled out as the cause of a fish kill, even if the fish test negative for BoNT/E.