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Neorickettsia helminthoeca (NH), the agent of salmon poisoning disease or canine neorickettiosis (CN), is a bacterial endosymbiont of the nematode Nanophyetus salmincola, and infections are spreading among specific fish-eating mammalians. This article describes the pathologic and immunohistochemical findings associated with spontaneous NH-induced infections in dogs from Southern Brazil. The principal pathologic findings were hypertrophy of Peyer patches and lymphadenopathy with lymphocytic proliferation, chronic interstitial pneumonia, and chronic enteritis associated with positive intralesional immunoreactivity to antigens of NH within macrophages and histiocytes. Positive immunoreactivity against canine parvovirus-2 (CPV-2) or/and canine distemper virus was not detected in the evaluated intestinal segments or in the samples from the cerebellum and lungs, respectively, from the dogs evaluated. These findings demonstrated that NH was involved in the enteric, pulmonary, and lymphoid lesions herein described, and provide additional information to confirm the occurrence of this bacterial endosymbiont within this geographical location. It is proposed that chronic pneumonia should be considered as a pathologic manifestation of NH-induced infections. Additionally, our results show that the occurrences of CN seem to be underdiagnosed in Southern Brazil due to the confusion with the incidence of CPV-2.

An analysis of daily water samples collected from an index site on Big Soos Creek, Washington indicated intra-annual differences in the concentrations of waterborne Nanophyetus salmincola. Waterborne concentrations, quantified as gene copies/L, peaked during the fall (October-November 2016), decreased to very low concentrations over the winter (January-March 2017), and then increased in the spring and throughout the summer. High waterborne concentrations of N. salmincola DNA (2 × 106 gene copies/L) corresponded with live N. salmincola cercariae (mean = 3 cercariae/L) that were detected in companion water samples. Spikes in waterborne N. salmincola concentrations in October and November typically coincided with increases in streamflow; this combination resulted in elevated infection pressures during high water events in the fall. The peak in waterborne N. salmincola concentrations corresponded with an accompanying peak in tissue parasite density (metacercariae/posterior kidney) in Coho Salmon Oncorhynchus kisutch that were reared in the untreated water.

Out-migrating steelhead Oncorhynchus mykiss from four Puget Sound rivers and associated marine basins of Puget Sound in Washington State were examined for the parasite, Nanophyetus salmincola in 2014 to determine whether recent trends in reduced marine survival are associated with the presence of this pathogen. A subset of steelhead from three of these river-marine basin combinations was analyzed for the presence of persistent organic pollutants (POPs) to assess whether exposure to these contaminants is a contributing factor to their reduced marine survival. The prevalence and parasite load of N. salmincola were significantly higher in fish from central and southern Puget Sound than in fish from river systems in northern Puget Sound. The proportion of steelhead samples with concentrations of POPs higher than adverse effects thresholds (AETs) or concentrations known to cause adverse effects was also greater in fish from the central and southern regions of Puget Sound than in those from the northern region. Polybrominated diphenyl ether concentrations associated with increased disease susceptibility were observed in 10% and 40% of the steelhead sampled from central and southern Puget Sound regions, respectively, but in none of the fish sampled from the northern region. The AET for polychlorinated biphenyls was exceeded in steelhead collected from marine habitats: 25% of the samples from the marine basins in the central and southern regions of Puget Sound and 17% of samples from northern Puget Sound region. Both N. salmincola and POP levels suggest there are adverse health effects on out-migrating steelhead from one southern and one central Puget Sound river that have lower early marine survival than those from a river system in northern Puget Sound.

Intestinal flukes of fish (mainly salmonids) belonging to the genus Nanophyetus (Trematoda: Troglotrematidae) are the causative agents of nanophyetiasis, a zoonotic disease of animals and humans, which is widespread in countries in the northern Pacific. Two geographical forms, one from North America and the other from the eastern Eurasia were described within this genus; however, their taxonomic status was debatable. A multilocus analysis of nuclear rDNA sequences applied in this study has shown that these forms are independent nominal species: Nanophyetus salmincola and Nanophyetus schikhobalowi. This study, based on sequencing the mtDNA nad1 gene, has evaluated the genetic variability of N. schikhobalowi from eastern Eurasia (Russia) and compared our data with the results obtained for N. salmincola from North America (United States). The genetic differentiation within the Eurasian sample was 1.4%, that for the North American sample was 0.8%, and differentiation between the samples was 15.5%. High values of genetic divergence and completed sorting of mitochondrial haplotypes confirmed the species independence of the compared geographic forms of Nanophyetus.

A parasitological survey of 651 northern fur seals Callorhinus ursinus L. from five subpopulations was conducted on St. Paul Island, Alaska, during July–August 2012–2014. Digenean trematodes were found in 210 of 651 fur seals with a total prevalence of 32.3%. Intensity of infection varied from 1 to 1540 parasites with mean intensity 18.4 ± 111.1 SD and median intensity of 2 specimens per host. Significant differences in prevalence and intensity of infection in northern fur seals between separate rookeries was not observed (Mann–Whitney test; p > 0.05). Four species of digeneans belonging to the families Heterophyidae (Apophallus zalophi Price, 1932, Phocitrema fusiforme Goto and Ozaki, 1930, and Galactosomum ubelakeri (Dailey, 1969)) and Troglotrematidae (Nanophyetus salmincola (Chapin, 1926)) were found. Nanophyetus salmincola is reported from C. ursinus for the first time. We obtained partial 28S rDNA sequences for all digenean species and conducted molecular phylogenetic analysis to demonstrate their phylogenetic relationships.

SUMMARYHuman and animal nanophyetiasis is caused by intestinal flukes belonging to the genus Nanophyetus distributed on both North American and Eurasian coasts of Northern Pacific. In spite of the wide geographical distribution and medical and veterinary importance of these flukes, the intra-generic taxonomy of Nanophyetus spp. remains unresolved. The two most widely distributed nominal species, Nanophyetus salmincola and Nanophyetus schikhobalowi, both parasitizing humans and carnivorous mammals, were described from North America and eastern Eurasia, respectively. However, due to their high morphological similarity their interrelationships remained unclear and taxonomic status unstable. In this study, we explored genetic diversity of Nanophyetus spp. from the Southern Russian Far East in comparison with that of samples from North America based on the sequence variation of the nuclear ribosomal gene family (18S, internal transcribed spacers, ITS1-5·8S-ITS2 and 28S). High levels of genetic divergence in each rDNA region (nucleotide substitutions, indels, alterations in the secondary structures of the ITS1 and ITS2 transcripts) as well as results of phylogenetic analysis provided strong support for the status of N. salmincola and N. schikhobalowi as independent species.

Miscellaneous intestinal fluke species causing zoonotic human infections include 8 species (7 genera); Brachylaima cribbi (Brachylaimidae), Cotylurus japonicus (Strigeidae), Gynaecotyla squatarolae (Microphallidae), Isoparorchis hypselobagri (Isoparorchidae), Microphallus brevicaeca (Microphallidae), Nanophyetus salmincola and Nanophyetus schikhobalowi (Troglotrematidae), and Prohemistomum vivax (Cyathocotylidae). The number of human infections with each of these species is only 1, to a few, to numerous; only 1 case (C. japonicus, G. squatarolae, and P. vivax), 3 cases (I. hypselobagri), 12 cases (M. brevicaeca), about 20 cases (B. cribbi and N. salmincola), or numerous cases (N. schikhobalowi). The source of human infection is variable, and the clinical significance is also variable depending on the species involved.

The ability of formalin, PEROX-AID (hydrogen peroxide), and seawater to kill waterborne Nanophyetus salmincola cercariae was evaluated in vitro. Newly emerged cercariae survived for extended periods in freshwater, with 53-73% survival occurring in negative control groups after 24 h. Exposure to dilutions of formalin reduced this survival time, with 0% of cercariae surviving after 30 min in 450 μL/L, 40 min in 225 μL/L, and 300 min in 113 μL/L. Exposure to PEROX-AID (hydrogen peroxide) for 1 h resulted in reduced cercarial survival (16.4%) only at the highest concentration (100 μL/L), compared with 100% survival in the untreated controls and all lesser concentrations. Exposure to dilutions of seawater resulted in reduced cercarial survival only at high salinities (15.2-30.3‰), where 10-min exposures resulted in 0-20% survival. These results provide insights into options for prophylactic water treatment at salmonid enhancement facilities that experience high mortalities due to infections with Nanophyetus salmincola. Further, the intolerance of live cercariae to high salinities indicates that exposure to fish occurs primarily in the freshwater portions of watersheds.

The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon Oncorhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmincola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations.

We report the development and validation of two quantitative PCR (qPCR) assays to detect Nanophyetus salmincola DNA in water samples and in fish and snail tissues. Analytical and diagnostic validation demonstrated good sensitivity, specificity, and repeatability of both qPCR assays. The N. salmincola DNA copy number in kidney tissue was significantly correlated with metacercaria counts based on microscopy. Extraction methods were optimized for the sensitive qPCR detection of N. salmincola DNA in settled water samples. Artificially spiked samples suggested that the 1-cercaria/L threshold corresponded to an estimated log10 copies per liter ≥ 6.0. Significant correlation of DNA copy number per liter and microscopic counts indicated that the estimated qPCR copy number was a good predictor of the number of waterborne cercariae. However, the detection of real-world samples below the estimated 1-cercaria/L threshold suggests that the assays may also detect other N. salmincola life stages, nonintact cerca...

We examined 1454 juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), captured in nearshore waters off the coasts of Washington and Oregon (USA) from 1999 to 2004 for infection by Renibacterium salmoninarum, Nanophyetus salmincola Chapin and skin metacercariae. The prevalence and intensities for each of these infections were established for both yearling and subyearling Chinook salmon. Two metrics of salmon growth, weight residuals and plasma levels of insulin-like growth factor-1, were determined for salmon infected with these pathogens/parasites, both individually and in combination, with uninfected fish used for comparison. Yearling Chinook salmon infected with R. salmoninarum had significantly reduced weight residuals. Chinook salmon infected with skin metacercariae alone did not have significantly reduced growth metrics. Dual infections were not associated with significantly more severe effects on the growth metrics than single infections; the number of triple infections was very low and precluded statistical comparison. Overall, these data suggest that infections by these organisms can be associated with reduced juvenile Chinook salmon growth. Because growth in the first year at sea has been linked to survival for some stocks of Chinook salmon, the infections may therefore play a role in regulating these populations in the Northeast Pacific Ocean.

Histology is often underutilized in aquatic animal disease screening and diagnostics. The agreement between histological classifications of infection and results using diagnostic testing from the American Fisheries Society’s Blue Book was conducted with 4 common salmon pathogens: Aeromonas salmonicida, Renibacterium salmoninarum, Ceratomyxa shasta, and Nanophyetus salmincola. Adult Chinook salmon ( Oncorhynchus tshawytscha) in Oregon were evaluated, and agreement between tests was calculated. Live and dead (both pre- and postspawning) salmon were collected from the Willamette River, Oregon, its tributaries, the Willamette Hatchery, and after holding in cool, pathogen-free water during maturation at Oregon State University. Sensitivity and specificity of histology compared to Blue Book methods for all fish, live fish only, and dead (pre- and postspawned combined) fish only were, respectively, as follows: A. salmonicida ( n = 105): specificity 87.5%, 87.5%, 87.5% and sensitivity 38.6%, 14.8%, 60.0%; R. salmoninarum ( n = 111): specificity 91.9%, 85.7%, 97.7% and sensitivity 16.0%, 7.1%, 27.2%; C. shasta ( n = 136): specificity 56.0%, 63.3%, 28.6% and sensitivity 83.3%, 86.2%, 71.4%; N. salmincola ( n = 228): specificity 68.2%, 66.7%, not possible to calculate for dead fish and sensitivity 83.5%, 80.5%, 87.3%. The specificity was good for bacterial pathogens. This was not the case for C. shasta, likely due to detection of presporogenic forms only by histology. Sensitivity of histology for bacterial pathogens was low with the exception of dead fish with A. salmonicida. Kappa analysis for agreement between Blue Book and histology methods was poor to moderate. However, histological observations revealed the presence of other pathogens that would not be detected by other methods.

The trematode Nanophyetus salmincola is known as the carrier of Neorickettsia helminthoeca, an obligate intracellular endosymbiotic bacterium that causes salmon poisoning disease (SPD), a fatal disease of dogs. The bacteria are maintained through the complex life cycle of N. salmincola that involves snails Juga plicifera as the first intermediate host, salmonid fishes as the second intermediate host and fish-eating mammals as definitive hosts. N. salmincola was also found to harbor a second species of Neorickettsia that causes the Elokomin fluke fever disease (EFF) which has clinical signs similar to SPD in bears, but only low grade illness in dogs. The EFF agent has not been sequenced. In this study we identified N. salmincola as the vector of yet additional species of Neorickettsia known as Stellanchasmus falcatu (SF) agent using DNA sequencing.

We are studying the impacts of parasites on threatened stocks of Oregon coastal coho salmon ( Oncorhynchus kisutch ). In our previous studies, we have found high infections of digeneans and myxozoans in coho salmon parr from the lower main stem of West Fork Smith River (WFSR), Oregon. In contrast parr from tributaries of this river, and outmigrating smolts, harbor considerably less parasites. Thus, we have hypothesized that heavy parasite burdens in parr from this river are associated with poor overwintering survival. The objective of the current study was to ascertain the possible effects these parasites have on smolt fitness. We captured parr from the lower main stem and tributaries of WFSR and held them in the laboratory to evaluate performance endpoints of smolts with varying degrees of infection by three digeneans ( Nanophyetus salmincola , Apophallus sp., and neascus) and one myxozoan ( Myxobolus insidiosus ). The parameters we assessed were weight, fork length, growth, swimming stamina, and gill Na + ,K + -ATPase activity. We repeated our study on the subsequent year class and with hatchery reared coho salmon experimentally infected with N. salmincola . The most significant associations between parasites and these performance or fitness endpoints were observed in the heavily infected groups from both years. We found that all parasite species, except neascus, were negatively associated with fish fitness. This was corroborated for N. salmincola causing reduced growth with our experimental infection study. Parasites were most negatively associated with growth and size, and these parameters likely influenced the secondary findings with swimming stamina and ATPase activity levels.

We are conducting studies on the impacts of parasites on Oregon coastal coho salmon (Oncorhynchus kistuch). An essential first step is documenting the geographic distribution of infections, which may be accomplished by using different methods for parasite detection. Thus, the objectives of the current study were to (1) identify parasite species infecting these stocks of coho salmon and document their prevalence, density, and geographic distribution; (2) assess the pathology of these infections; and (3) for the first time, determine the sensitivity and specificity of histology for detecting parasites compared with examining wet preparations for muscle and gill infections. We examined 576 fry, parr, and smolt coho salmon in total by histology. The muscle and gills of 219 of these fish also were examined by wet preparation. Fish were collected from 10 different locations in 2006-2007. We identified 21 different species of parasites in these fish. Some parasites, such as Nanophyetus salmincola and Myxobolus insidiosus, were common across all fish life stages from most basins. Other parasites, such as Apophallus sp., were more common in underyearling fish than smolts and had a more restricted geographic distribution. Additional parasites commonly observed were as follows: Sanguinicola sp., Trichodina truttae , Epistylis sp., Capriniana piscium, and unidentified metacercariae in gills; Myxobolus sp. in brain; Myxidium salvelini and Chloromyxum majori in kidney; Pseudocapillaria salvelini and adult digenean spp. in the intestine. Only a few parasites, such as the unidentified gill metacercariae, elicted overt pathologic changes. Histology had generally poor sensitivity for detecting parasites; however, it had relatively good specificity. We recommend using both methods for studies or monitoring programs requiring a comprehensive assessment of parasite identification, enumeration, and parasite-related pathology.

Multiple analytical techniques were used to evaluate the impact of multiple parasite species on the mortality of threatened juvenile coho salmon (Oncorhynchus kisutch) from the West Fork Smith River, Oregon, USA. We also proposed a novel parsimonious mathematical representation of macroparasite distribution, congestion rate, which (i) is easier to use than traditional models, and (ii) is based on Malthusian parameters rather than probability theory. Heavy infections of Myxobolus insidiosus (Myxozoa) and metacercariae of Nanophyetus salmincola and Apophallus sp. occurred in parr (subyearlings) from the lower mainstem of this river collected in 2007 and 2008. Smolts (yearlings) collected in 2007–2010 always harboured fewer Apophallus sp. with host mortality recognised as a function of intensity for this parasite. Mean intensity of Apophallus sp. in lower mainstem parr was 753 per fish in 2007 and 856 per fish in 2008, while parr from the tributaries had a mean of only 37 or 13 parasites per fish, respectively. Mean intensity of this parasite in smolts ranged between 47 and 251 parasites per fish. Over-dispersion (variance to mean ratios) of Apophallus sp. was always lower in smolts compared with all parr combined or lower mainstem parr. Retrospective analysis based on smolt data using both the traditional negative binomial truncation technique and our proposed congestion rate model showed identical results. The estimated threshold level for mortality involving Apophallus sp. was at 400–500 parasites per fish using both analytical methods. Unique to this study, we documented the actual existence of these heavy infections prior to the predicted mortality. Most of the lower mainstem parr (approximately 75%) had infections above this level. Heavy infections of Apophallus sp. metacercariae may be an important contributing factor to the high over-wintering mortality previously reported for these fish that grow and develop in this section of the river. Analyses using the same methods for M. insidiosus and N. salmincola generally pointed to minimal parasite-associated mortality.

The potential effect of the freshwater trematode Nanophyetus salmincola on early marine survival of Pacific salmon was assessed by monitoring the prevalence and intensity of metac- ercarial infection in yearling coho salmon Oncorhynchus kisutch, and yearling and subyearling Chi- nook salmon O. tshawytscha caught off Oregon and Washington during May, June, and September of 1999 to 2002. Annual prevalences of N. salmincola infection in yearling coho salmon were 62 to 78% and were significantly greater each year than in both yearling and subyearling Chinook salmon (19.3 to 53.8% and 40.5 to 53.5%, respectively). Yearling coho salmon also had significantly higher intensities of infection (from approximately 2-fold to 12-fold) than yearling and subyearling Chinook salmon. Prevalences and intensities in coho salmon caught in September were significantly lower (by approximately 21%) than in coho salmon caught in May or June in 3 of the 4 years. Variance to mean ratios of parasite abundance in coho salmon were also lowest in September, suggesting parasite- associated host mortality during early ocean residence. There was no evidence for a seasonal decline in infection in yearling or subyearling Chinook salmon. Infection intensities, but not prevalences, were significantly greater in naturally produced (wild) coho salmon than in hatchery produced coho salmon and could be due to differences in exposure to the trematode. Highly infected naturally pro- duced coho salmon were not caught in September. This study suggests that coho salmon with high intensities of N. salmincola may not survive early marine residence, and that disease processes need to be considered as a factor affecting marine survival of juvenile salmon.

Although the adverse impact of pathogens on salmon populations in the Pacific Northwest is often discussed and recognized, little is currently known regarding the incidence and corresponding significance of delayed disease-induced mortalities. In the study reported herein, we surveyed the presence and prevalence of selected micro- and macroparasites in out-migrant juvenile coho salmon Oncorhynchus kisutch and Chinook salmon O. tshawytscha from 12 coastal estuaries in the Pacific Northwest over a 6-year period (1996–2001). The major finding of this study was the widespread occurrence of pathogens in wild salmon from Pacific Northwest estuaries. The six most prevalent pathogens infecting both juvenile Chinook and coho salmon were Renibacterium salmoninarum, Nanophyetus salmincola, an erythrocytic cytoplasmic virus (erythrocytic inclusion body syndrome or erythrocytic necrosis virus), and three gram-negative bacteria (Listonella anguillarum, Yersinia ruckeri, and Aeromonas salmonicida). The most pre...

Previous studies have shown that juvenile chinook salmon Oncorhynchus tshawytscha exposed in the field or the laboratory to polychlorinated biphenyls (PCBs), an anthropogenic stressor, are immunosuppressed. It is not known whether simultaneous exposure to natural stressors can increase this immunosuppression. To examine the effects of natural and anthropogenic stressors on immune function, we infected juvenile chinook salmon with metacercariae of the trematode Nanophyetus salmincola by exposing the fish to infected freshwater snails Juga plicifera. Infected (>300 metacercariae per fish) and noninfected salmon were then injected with either the commercial PCB mixture Aroclor 1254 or an acetone–emulphor carrier. B cell function was examined by in vitro hemolytic plaque-forming cell (PFC) assay. Nanophyetus salmincola infection resulted in significantly lower anterior kidney primary PFCs and lower splenic secondary PFCs. The combination of N. salmincola infection and Aroclor 1254 exposure caused a l...

Most pathogen detection tests are imperfect, with a sensitivity < 100%, thereby resulting in the potential for a false negative, where a pathogen is present but not detected. False negatives in a sample inflate the number of non-detections, negatively biasing estimates of pathogen prevalence. Histological examination of tissues as a diagnostic test can be advantageous as multiple pathogens can be examined and providing important information on associated pathological changes to the host. However, it is usually less sensitive than molecular or microbiological tests for specific pathogens. Our study objectives were to 1) develop a hierarchical occupancy model to examine pathogen prevalence in spring Chinook salmon Oncorhynchus tshawytscha and their distribution among host tissues 2) use the model to estimate pathogen-specific test sensitivities and infection rates, and 3) illustrate the effect of using replicate within host sampling on sample sizes required to detect a pathogen. We examined histological sections of replicate tissue samples from spring Chinook salmon O. tshawytscha collected after spawning for common pathogens seen in this population: Apophallus/echinostome metacercariae, Parvicapsula minibicornis, Nanophyetus salmincola/ metacercariae, and Renibacterium salmoninarum. A hierarchical occupancy model was developed to estimate pathogen and tissue-specific test sensitivities and unbiased estimation of host- and organ-level infection rates. Model estimated sensitivities and host- and organ-level infections rates varied among pathogens and model estimated infection rate was higher than prevalence unadjusted for test sensitivity, confirming that prevalence unadjusted for test sensitivity was negatively biased. The modeling approach provided an analytical approach for using hierarchically structured pathogen detection data from lower sensitivity diagnostic tests, such as histology, to obtain unbiased pathogen prevalence estimates with associated uncertainties. Accounting for test sensitivity using within host replicate samples also required fewer individual fish to be sampled. This approach is useful for evaluating pathogen or microbe community dynamics when test sensitivity is

Case Description—2 captive sun bears (Helarctos malayanus) were evaluated because of acute onset of vomiting, mucoid diarrhea, lethargy, and anorexia 1 week after eating live trout from a northern California reservoir. Clinical Findings—In 1 of the bears, a CBC and serum biochemical analyses revealed mild anemia, mild eosinophilia, moderate lymphopenia, moderate hypoalbuminemia, and high serum G-glutamyltransferase activity. Ultrasonographic examination of the same bear revealed ascites and mesenteric lymphadenopathy. Histologic examination of gastrointestinal tract biopsy specimens revealed moderate to severe lymphoplasmacytic and eosinophilic gastritis, enteritis, and colitis. Ova of Nanophyetus salmincola, the trematode vector of Neorickettsia helminthoeca (a rickettsial organism that causes salmon poisoning disease), were detected in fecal samples from both bears. Treatment and Outcome—The bears were treated with oxytetracycline, doxycycline, praziquantel, and famotidine. Within 1 week after initiation of treatment, the appetite and fecal consistency of each bear were considered normal. Fecal ova shedding began 4 days after onset of clinical signs and ceased 9 days later. Clinical Relevance—Salmon poisoning disease can be rapidly fatal in untreated animals, but if diagnosed early and treated appropriately, full recovery can be achieved. Domestic dogs and captive exotic bears are highly susceptible to clinical disease after ingestion of trematode-infected fish. Salmon poisoning disease may develop outside the geographic range in which the causative organism is endemic as a result of the transplantation of infected fish for sport fishing; veterinarians practicing in areas where infected fish may be transplanted should be aware of appropriate diagnostic and treatment protocols.

Trematodes transmitted through fish are reviewed and species naturally infecting humans are listed. Their medical importance, geographical distribution, and animal hosts are summarized. The most important group is liver flukes: Amphimerus sp., Clonorchis sinensis, Metorchis spp., and Opisthorchis spp. The others are clinostomes, echinostomes, heterophyids, and nanophyetids. The human infection Amphimerus sp. can be found in Ecuador; and Ascocotyle longa is a heterophyid that can be found in Latin American countries. Apophallus donicus, Metorchis conjunctus, and Nanophyetus salmincola can be found in North America; Opisthorchis felineus, Metorchis bilis, and Nanophyetus s. schikhobalowi exist in Europe and Northeast Asia. The species that can be found in Asia are Clonorchis sinensis, Metorchis orientalis, Opisthorchis noverca, O. viverrini, clinostomes, echinostomes, and most heterophyids.

Metacercariae of an unidentified species of Apophallus Luhe, 1909 are associated with overwinter mortality in coho salmon, Oncorhynchus kisutch (Walbaum, 1792), in the West Fork Smith River, Oregon. We infected chicks with these metacercariae in order to identify the species. The average size of adult worms was 197 × 57 μm, which was 2 to 11 times smaller than other described Apophallus species. Eggs were also smaller, but larger in proportion to body size, than in other species of Apophallus. Based on these morphological differences, we describe Apophallus microsoma n. sp. In addition, sequences from the cytochrome c oxidase 1 gene from Apophallus sp. cercariae collected in the study area, which are likely conspecific with experimentally cultivated A. microsoma, differ by >12% from those we obtained from Apophallus donicus (Skrjabin and Lindtrop, 1919) and from Apophallus brevis Ransom, 1920. The taxonomy and pathology of Apophallus species is reviewed.

We evaluated the ability of 5 muscle- or skin-dwelling parasites to persist in naturally infected coho salmon, Oncorhynchus kisutch, from the West Fork Smith River, Oregon, by holding them in captivity from late summer to early spring (parr stage to the typical time of smoltification). These parasites included metacercariae of 3 digeneans, Nanophyetus salmincola, Apophallus sp., and neascus sp., and 2 myxozoans, Myxobolus insidiosus and Myxobolus fryeri. Two groups of wild-caught fish were evaluated in the laboratory, i.e., heavily infected fish from the lower main stem and less severely infected fish collected from tributaries of this river. All parasites survived in these fish for the 7-month experiment. Only 2 parasites had a statistically significant lower median abundance between host life stages. The mean abundance of N. salmincola declined 45% in the tributary fish and Apophallus sp. declined 43% in the lower main stem fish. However, more than 50% of each species persisted until the end of the study, with smolts still harboring relatively high infections.

Neorickettsia helminthoeca is an obligate intra-cytoplasmic bacterium that causes salmon poisoning disease (SPD), an acute, febrile, fatal disease of dogs. The complex life-cycle of this pathogen involves stages in an intestinal fluke (Nanophyetus salmincola), a river snail (Oxytrema silicula), in fish, and in fish-eating mammals. This complexity has created confusion with respect to the various bacterial and parasitic infections associated with the disease and its significance in dogs in specific geographical locations has likely to have previously been under-estimated. This paper addresses the history, taxonomy, microbiology of N. helminthoeca and summarises the pathogenesis, clinical signs and pathological features associated with infection. Furthermore, the biological cycles, treatment, control, and both public and veterinary health impacts associated with this pathogen and the intestinal fluke N. salmincola are discussed.

The first US case of human nanophyetiasis that does not involve ingestion of raw or undercooked salmonid fishes is reported. The patient worked with highly infected fish. Hand contamination with the infectious metacercariae of the digenetic trematode Nanophyetus salmincola (family Troglotrematidae) occurred during the handling of fresh-killed, juvenile coho salmon, Oncorhynchus kisutch. Diagnosis of nanophyetiasis was based on the clinical findings of chronic diarrhea, nausea, abdominal discomfort, and a peripheral blood eosinophilia of 43% and was confirmed on finding characteristic bipolar eggs in a stool specimen. The patient rarely ate seafood products and never ate raw or cold smoked fish. He responded favorably to praziquantel and was asymptomatic after therapy.

Salmon poisoning disease (SPD) was experimentally induced in juvenile coyotes (Canis latrans). The disease was lethal in 11 of 12 coyotes within 15 days after inoculation with 1,000 or 4,000 metacercariae of Nanophyetus salmincola. Clinical manifestations of the disease included lymph node enlargement, anorexia, pyrexia, diarrhea and death. Coccoid bodies indistinguishable from rickettsiae were observed in macrophages of spleen, liver, lymph nodes, and duodenum. Percentage recovery of adult trematodes from metacercariae administered was 23% from 12 inoculated coyotes, compared to 13% in one inoculated dog. Juvenile coyotes appear to be highly susceptible to experimental SPD.

Praziquantel at dosages of 10, 20 or 100 mg/kg of body weight was evaluated against metacercariae of Nanophyetus salmincola in chinook salmon (Oncorhynchus tshawytscha). Ten salmon were used in each of four treated groups and 10 salmon were nontreated controls. Three wk after treatment, viability of metacercariae was determined by histologic evaluation, and by feeding the salmon to coyotes and subsequently determining the numbers of trematode eggs/g of feces and numbers of N. salmincola recovered at necropsy. Results of the experiment indicated that praziquantel at the dosages and routes of administration used was not effective against metacercariae in chinook salmon.

Human nanophyetiasis is a zoonotic disease recently recognized in the coastal US Pacific Northwest and is caused by the digenetic trematode Nanophyetus salmincola. Ten additional stool-proven cases from the same geographic area have been identified; five presented with histories of gastrointestinal complaints and five had eosinophilia. Nine of 10 patients had histories of fish ingestion (incompletely cooked or home-smoked salmon or steelhead trout) and were treated with praziquantel. Several individuals noted prompt resolution of symptoms following therapy. In all treated cases subsequent stool examinations were negative for eggs of Nanophyetus. Nanophyetiasis may be the most commonly encountered trematodiasis endemic to North America, and the use of praziquantel in its treatment appears to be efficacious.

Forty-two seals and sea lions found dead along the Oregon Coast were examined for parasites and associated pathology. Nematode infections of the lung and/or gastrointestinal tract were the primary cause of death in 5 of 42 animals examined. New distribution records were established for Pricetrema zalophi and Zalophotrema hepaticum. New host records include Z. hepaticum and Diphyllobothrium cordatum in the Steller's sea lion (Eumetopias jubatus); Nanophyetus salmincola in the California sea lion (Zalophus californianus); P. zalophi in the harbor seal (Phoca vitulina); and P. zalophi, Trigonocotyle sp. and Otostrongylus circumlitus in the northern elephant seal (Mirounga angustirostris).

1. 1. Qualitative and quantitative analysis of the amino acid content in the digestive gland of the unparasitized and parasitized Oxytrema silicula and the rediae of Nanophyetus salmincola resulted in the detection of seventeen amino acids plus ammonia. 2. 2. Ammonia, lysine, aspartic acid, threonine, serine, proline, glycine and alanine are found in relatively high concentrations. 3. 3. Histidine, glutamic acid, valine, methionine, leucine, isoleucine and arginine are present in moderate amounts; while tyrosine, phenylalanine and cystine exist in low and trace amounts. 4. 4. In parasitized snails considerable depletion of amino acids is encountered in those present in high concentrations, while increases of amino acids are observed in most of those present in low and moderate concentrations.

1. 1. Quantitative analysis of the fatty acid content in the hepatopancreas of the unparasitized and parasitized Oxytrema silicula and the rediae of Nanophyetus salmincola resulted in the detection of seven free fatty acids. 2. 2. Oleic acid was found in the highest concentration. 3. 3. An increase of 15 per cent in free fatty acid content of the infected hepatopancreas was observed with no appreciable change in the percentage composition with respect to individual fatty acid.

The agent of so-called “salmon poisoning disease” of dogs on the Pacific Coast of the USA, first reported by Cordy and Gorham (1950) , is an intracytoplasmic, rickettsia-like, sometimes pleomorphic microorganism found particularly in the reticulo-endothelial cells of lymphoid tissues of infected Canidae. It is transmitted in nature to members of the Canidae by ingestion of fish containing encysted cercariae of the intestinal fluke, Nanophyetus salmincola. The natural, developmental cycle of this trematode includes snails, Goniobasis pliciera silicula, and fish of the family Salmonidae. The infection is transmissible in series by blood and by fresh or frozen node tissues of infected to susceptible dogs as well as by suspension of adult flukes. There is an incubation period of 8 to 10 days followed by several days of an acute febrile episode. Mortality is high. When there is occasional recovery, the dogs are solidly immune. Reasons are presented for assigning this pathogen to the family Rickettsiaceae by modifying the vector character of the family from “arthropod-borne” to “invertebrate-borne” agents, and for having given this the name of Neorickeltsia helmintheca Philip, Hadlow, and Hughes (1953) .

Between 1974 and 1985, 10 patients were studied who presented with either gastrointestinal complaints or unexplained peripheral blood eosinophilia, and had eggs typical for N. salmincola recovered from their stools. Clinical findings in 8 of the 10 included increased frequency of bowel movements or diarrhea (6), peripheral blood eosinophilia (6), abdominal discomfort (5), nausea and vomiting (3), weight loss (2), and fatigue (2). Two were asymptomatic. Eight recalled eating fish prior to the onset of symptoms. Anthelminthic treatment consisting of three 2-g doses of niclosamide (2 patients) or two 50 mg/kg doses of bithionol (1 patient) proved effective. In the remaining individuals symptoms resolved slowly over several months.

Clinical salmon poisoning disease (SPD), and survival of Neorickettsia helminthoeca and metacercariae of Nanophyetus salmincola in fish were evaluated experimentally in 12-wk-old coyotes (Canis latrans) to determine the potential of SPD for biological control of coyotes. Coyotes readily ate fish that contained metacercariae and rickettsiae. They developed diarrhea, anorexia and lethargy within 7 days after feeding. Infected coyotes lost 58% of their body weight when compared to uninfected controls. They died or became moribund and were euthanatized within 17 days after feeding. Rickettsiae were present in the macrophages of lymph nodes of all affected coyotes. Clinical disease occurred in coyotes fed fresh fish, but not in coyotes fed fish stored at 4 C for greater than or equal to 30 days or at -20 C for 14 days. Metacercariae in fish were viable after 60 days at 4 C. These trematodes developed in coyotes, but clinical SPD did not occur. This indicated survival of metacercariae, but not rickettsiae. Metacercariae were not viable after 14 days at -20 C.

Publisher Summary The digenetic trematode Nanophyetus salmincola (Chapin) is the vector for Neorickettsia helminthoeca, a rickettsia-like organism that causes “salmon poisoning” disease (SPD). The disease is usually fatal for dogs, foxes, and coyotes. Nanophyetus s. salmincola ( N.s. salmincola ) requires multiple hosts for completion of its life cycle. The first intermediate host is the pleurocerid stream snail, Oxytrema silicula. The second intermediate hosts are salmonid and some non-salmonid fishes and the Pacific giant salamander in which the cercariae encyst. The definitive hosts are fish-eating birds and mammals. The chapter reviews several aspects of SPD. Some of them are (1) geographical distribution, (2) etiology, (3) transmission, (4) symptoms, and (5) pathogenesis and pathology. The treatment of SPD is also reviewed. Various sulfanilamides given orally or parenterally have been successful in the treatment. Dosage at therapeutic blood levels should be maintained for at least 3 days. Effective results have also been obtained with penicillin, chlortetracycline, chloramphenicol, and oxytetracycline. The best results follow administration of large divided doses. If the animal is dehydrated, intravenous fluid therapy is essential to avoid nephrotoxic effects. Treatment in the late stages of the disease may not be successful.

Publisher Summary This chapter discusses the trematode life cycles involving wild mammals of Northwestern North America. It describes five life cycles of trematodes of wild mammals, all of which involve changing hosts in fast-running water. Nanophyetus salmincola, adult in dogs, coyotes, and mink, develops to microcercous creeping cercariae in Oxytrema silicula (Pleroceridae) and emerges into the stream, producing copious masses of thick mucus upon which the cercariae cling. Acanthatrium oregonense and Allassogonoporus marginalis develop in either Oxytrema silicula or Flumenicola virens. Two heterophyid life cycles are similar, but with an interesting alternate reduction in hosts required in one. Euryhelmis squamula in Oregon has been found in Mustela vison. The snail host is the nearly microscopic Bythinella hemphilli found only in quiet seepage areas near fast cold streams.

The agent of so-called “salmon poisoning disease” of dogs on the Pacific Coast of the USA, first reported by Cordy and Gorham (1950), is an intracytoplasmic, rickettsia-like, sometimes pleomorphic microorganism found particularly in the reticulo-endothelial cells of lymphoid tissues of infected Canidae. It is transmitted in nature to members of the Canidae by ingestion of fish containing encysted cercariae of the intestinal fluke, Nanophyetus salmincola. The natural, developmental cycle of this trematode includes snails, Gonidbasis plicifera silicula, and fish of the family Salmonidae. The infection is transmissible in series by blood and by fresh or frozen node tissues of infected to susceptible dogs as well as by suspension of adult flukes. There is an incubation period of 8 to 10 days followed by several days of an acute febrile episode. Mortality is high. When there is occasional recovery, the dogs are solidly immune. Reasons are presented for assigning this pathogen to the family Rickettsiaceae by modifying the vector character of the family from “arthropod-borne” to “invertebrate-borne” agents, and for having given this the name of Neorickettsia helminthēcaPhilip, Hadlow, and Hughes (1953).

Publisher Summary This chapter discusses the transmission of two rickettsia-like disease agents of dogs by endoparasites in Northwestern USA. Theoretically, helminth parasites should make ideal vectors and reservoirs of disease. They are present in profusion throughout the animal kingdom. The natural transmission of Neorickettsia heiminthoeca , the causative agent of salmon poisoning disease of dogs, by Nanophyetus salmincola , an intestinal fluke with a dog-small-fish life cycle, has been repeatedly demonstrated. Elokomin fluke fever is acquired by the dog after ingestion of salmon and trout infected with the metacercariae of Nanophyetus salmincola . The disease causes a marked rise in heterophile antibody. Serological studies using complement fixation and neutralization tests have shown the disease to be distinct from salmon poisoning. Ferrets and bears are susceptible to Elokomin fluke fever and refractory to salmon poisoning disease.

An estimated 650,000 Nanophyetus salmincola were recovered from the small intestine of a wild female cougar kitten (Felis concolor). The trematodes stimulated marked mucosal thickening in the duodenum and jejunum and caused a marked enteritis. Malnutrition was listed as the cause of death due to impaired nutrient absorption resulting from the combined effects of the diarrhea and thickening of the intestine.

Silver salmon (Oncorhynchus kisutch) infected with the metacercariae of the "salmon poisoning" fluke (Nanophyetus salmincola) remain infected after they have migrated to sea. Metacercariae remain viable in such salmon for at least 33(1/2) months. These metacercariae are capable of transmitting salmon disease (Neorickettsiae helminthoeca) to susceptible dogs throughout this period.

The histopathological and histochemical effects on the hepatopancreas of the snail Oxytrema siliqua, caused by the larvae of Nanophyetus salmincola, have been studied. The parasite causes damage to the organ by increased pressure, active ingestion of the hepatic tubules, and by the increased burden placed upon the snail in disposing of the waste of the parasite. Glycogen and lipid uptake from the hepatopancreas by the parasite were tested for and found to occur via two routes: active ingestion of hepatic cells by the parasites, and absorption across the body membranes of the parasite. Acid and alkaline phosphatases were found to be in greater quantities in snails infected with N. salmincola when compared to uninfected snails. Alkaline phosphatase was found to be associated with the parasite and is thought to function in the metabolism of glycogen. ABSTRACT: The histopathological and histochemical effects on the hepatopancreas of the snail Oxytrema siliqua, caused by the larvae of Nanophyetus salmincola, have been studied. The parasite causes damage to the organ by increased pressure, active ingestion of the hepatic tubules, and by the increased burden placed upon the snail in disposing of the waste of the parasite. Glycogen and lipid uptake from the hepatopancreas by the parasite were tested for and found to occur via two routes: active ingestion of hepatic cells by the parasites, and absorption across the body membranes of the parasite. Acid and alkaline phosphatases were found to be in greater quantities in snails infected with N. salmincola when compared to uninfected snails. Alkaline phosphatase was found to be associated with the parasite and is thought to function in the metabolism of glycogen.