RESUMEN
Myxobolus cerebralis (Mc) is a myxozoan parasite causing whirling disease in hatchery- and natural-origin salmonids. To minimize spread of this parasite and the incidence of its associated disease, fish health professionals routinely screen fish for Mc before stocking or moving the fish to Mc-free waters. Sample collection for Mc traditionally entails lethal sampling of cranial tissue followed by pepsin-trypsin digest (PTD) and screening of the sample for mature myxobolid myxospores (PTD method); however, nonlethal sampling methods would be advantageous in some circumstances, such as when dealing with rare or otherwise valuable fish. Accordingly, we compared Mc detections in cranial cartilage by using the PTD method with PCR assays of fin biopsies collected from juvenile Chinook Salmon Oncorhynchus tshawytscha and adult steelhead O. mykiss. Cranial samples were also analyzed using PCR methods for comparative purposes. Results indicated that Mc could be detected by PCR in fin clips, but the results generated by this approach differed significantly from those associated with PTD- and/or PCR-based analysis of cranial cartilage samples. Polymerase chain reaction-based analysis-of individual head samples and head digest pools in both species as well as fins in steelhead-yielded more positive detections than PTD analysis alone. The PCR-based analysis of head and fin tissues yielded different Mc detection rates in both species, but the nature of the detection disparity varied depending on the species and/or life stage of the fish. We conclude that for lethal cranial samples, neither PTD nor PCR should be used alone, but using these techniques in concert may provide the most complete and accurate estimation of Mc presence in a group of salmonids. If imperiled or highly valuable fish are in question, nonlethal fin samples may be used to generate some information regarding Mc status, with the understanding that parasite DNA detections do not necessarily signify mature infections or disease.
Asunto(s)
Enfermedades de los Peces/parasitología , Myxobolus/genética , Oncorhynchus mykiss , Salmón , Aletas de Animales/parasitología , Animales , ADN Protozoario/análisis , Enfermedades de los Peces/diagnóstico , Myxobolus/aislamiento & purificación , Enfermedades Parasitarias en Animales/diagnóstico , Enfermedades Parasitarias en Animales/genética , Pepsina A/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Esporas Protozoarias , Tripsina/metabolismoRESUMEN
Nonlethal sampling techniques have previously been evaluated for detection of a variety of viral salmonid pathogens. However, many of these studies have used molecular assays in lieu of widely accepted cell culture techniques to evaluate the sampled tissues. Samples were collected from female steelhead Oncorhynchus mykiss broodstock using three potential nonlethal sampling methods (mucus/skin scrape, pectoral fin clip, and gill tissue biopsy) and evaluated for the presence of infectious hematopoietic necrosis virus (IHNV) via cell culture techniques. The results were compared with those from samples collected using a standard lethal sampling method (pooled anterior kidney and spleen tissues) applied to the same fish. Of the three nonlethal sampling techniques that were evaluated, fin clipping was the easiest and least invasive method. Furthermore, fin tissue was as sensitive as or more sensitive than kidney/spleen tissue for detecting IHNV in this population of fish. However, with the exception of gill tissue, the nonlethal samples did not appear to be appropriate surrogates for lethally collected tissues with regard to identifying an active infection in a particular fish. Nevertheless, nonlethal sampling coupled with cell culture appears to be suitable for helping to define the IHNV status of a steelhead population. Received July 27, 2016; accepted December 11, 2016.
Asunto(s)
Enfermedades de los Peces/diagnóstico , Virus de la Necrosis Hematopoyética Infecciosa/aislamiento & purificación , Oncorhynchus mykiss , Infecciones por Rhabdoviridae/veterinaria , Animales , Células Cultivadas/virología , Femenino , Branquias , Infecciones por Rhabdoviridae/diagnósticoRESUMEN
In the 1990s, the Tubifex tubifex aquatic oligochaete species complex was parsed into 6 separate lineages differing in susceptibility to Myxobolus cerebralis, the myxozoan parasite that can cause whirling disease (WD). Lineage III T. tubifex oligochaetes are highly susceptible to M. cerebralis infection. Lineage I, IV, V and VI oligochaetes are highly resistant or refractory to infection and may function as biological filters by deactivating M. cerebralis myxospores. We designed a 2-phased laboratory experiment using triactinomyxon (TAM) production as the response variable to test that hypothesis. A separate study conducted concurrently demonstrated that M. cerebralis myxospores held in sand and water at temperatures ≤15°C degrade rapidly, becoming almost completely non-viable after 180 d. Those results provided the baseline to assess deactivation of M. cerebralis myxospores by replicates of mixed lineage (I, III, V and VI) and refractory lineage (V and VI) oligochaetes. TAM production was zero among 7 of 8 Lineage V and Lineage VI T. tubifex oligochaete groups exposed to 12500 M. cerebralis myxospores for 15, 45, 90 and 135 d. Among 4 mixed lineage exposure groups, TAM production averaged 14641 compared with 2202495 among 12 groups of Lineage III oligochaetes. Among the 6 unexposed Lineage III experimental groups seeded into original Phase 1 substrates for the 45, 90 and 135 d treatments during the Phase 2 portion of the study, TAM production was reduced by 98.9, 99.9 and 99.9%, respectively, compared with the average for the 15 d exposure groups. These results are congruent with the hypothesis that Lineage V and Lineage VI T. tubifex oligochaetes can deactivate and destroy M. cerebralis myxospores.
Asunto(s)
Myxobolus , Oligoquetos/fisiología , Esporas , Animales , Interacciones Huésped-Parásitos , Factores de TiempoRESUMEN
While whirling disease was first observed in Rainbow Trout Oncorhynchus mykiss in 1893, the complete life cycle of Myxobolus cerebralis (Mc), the causative agent of the disease, was not understood until 1984, when it was shown to involve two obligate hosts, a salmonid fish and the aquatic oligochaete Tubifex tubifex (Tt). The viability of the triactinomyxon (TAM) actinospores produced by Tt has been well studied, and is known to be temperature dependent and measured in days and weeks. Assertions that Mc myxospores produced by infected fish remain viable for years or even decades were made during the mid-20th century, decades before the Mc life cycle was described. Moreover, the duration of myxospore viability has not been well studied since the life cycle was elucidated. In a series of time-delay treatments, we assessed the long-term viability of Mc myxospores by exposure to Mc-susceptible Tt oligochaetes and quantified TAM production. As the time delay between inoculation and incubation of Mc myxospores in sand and water and exposure to Tt oligochaetes increased, TAM production decreased exponentially. Production among the 15-d time-delay replicates was reduced 74.7% compared with the 0-d treatment. Likewise, total TAM production was reduced 94.5, 99.4, and 99.9%, respectively, in the 90-, 120-, and 180-d time-delay treatments. Linear regression analysis of our data and the absence of TAM production among replicates of Mc myxospores held at 5°C for 365 d prior to exposure to Mc-susceptible Tt oligochaetes indicate that the long-term viability of Mc myxospores is less than 1 year under the conditions of this study.