A phylogeny based on cytochrome-c oxidase gene sequences identifies sympatric Ichthyophonus genotypes in the NE Pacific Ocean.

In recent decades, evidence has accumulated to suggest that the widespread and highly variable parasite Ichthyophonus hoferi is actually a species complex. Highly plastic morphology and a general lack of defining structures has contributed to the likely underestimate of biodiversity within this group. Molecular methods are a logical next step in the description of these parasites, but markers used to date have been too conserved to resolve species boundaries. Here we use mitochondrial encoded cytochrome-c oxidase (MTCO1) gene sequences and phylogenic analysis to compare Ichthyophonus spp. isolates from several marine and anadromous fish hosts. The resulting phylogeny displays lineage separation among isolates and possible host/niche segregation not previously described. The parasite type that infects Pacific herring Clupea pallasii, Atlantic herring C. harengus, Atlantic salmon Salmo salar, and Pacific staghorn sculpin Oligocottus maculosus (Clade A) is different from that which infects Chinook salmon Oncorhynchus tshawytscha, walleye pollock Gadus chalcogrammus, Greenland halibut Reinhardtius hippoglossoides, and Pacific halibut Hippoglossus stenolepsis (Clade B). MTCO1 sequences confirmed the presence of a more divergent Ichthyophonus sp. isolated from American shad Alosa sapidissima in rivers of eastern North America (Clade C), while American shad introduced to the Pacific Ocean are infected with the same parasite that infects Pacific herring (Clade A). Currently there are no consensus criteria for delimiting species within Ichthyophonidae, but MTCO1 sequences hold promise as a potential species identifying marker and useful epizootiological tool.

A case report and statewide surveillance of "weak meat" condition of Alaska weathervane scallops, Patinopecten caurinus, linked to a recently identified pathogenic parasite, Merocystis kathae (Apicomplexa: Aggregatidae).

Weathervane scallop, Patinopecten caurinus, the largest scallop species in the world, is distributed from northern California, U.S.A., to the Bering Sea, and is only commercially harvested in Alaska. The fishery is considered well managed by the State of Alaska (U.S.A) Department of Fish and Game (ADF&G) and federal government, with many precautionary measures in place to avoid overharvest. There have been episodic declines in some management areas due to unknown causes. Fishermen also encounter scallops with abnormal adductor muscles, a condition colloquially termed "weak meat", characterized by the retention of muscle when shucked, an obvious darkened discoloration, and/or an abnormal texture making the product unacceptable for marketing. A similar syndrome in Atlantic sea scallops, Placopecten magellanicus, described as "gray meat", occurs in the eastern U.S. and Canada, and proposed causes include senescence, loss of bioenergetics due to chronic infestations, or a synergism of these factors. Recently a severe apicomplexan infection was found to cause a gray meat condition in Iceland scallops, Chlamys islandica, and the collapse of that stock. This parasite was subsequently detected in Atlantic sea scallops with the gray meat condition off the U.S. East Coast. Studies that followed identified the parasite as Merocystis kathae, previously described from the common whelk, Buccinum undatum, more than 100 years ago. In 2015 Bering Sea fishermen reported weak meat in their catch, so samples were submitted to ADF&G for diagnosis. Adductor muscles from all affected scallops had many large foci of an apicomplexan associated with necrosis, fibrosis, and muscular atrophy. Given the reduced quality, marketability, and possibly fitness of affected scallops, we performed a survey to estimate prevalence, intensity, and geographic distribution of this apicomplexan in Alaskan weathervane scallops. We sampled 180 scallops, from individual beds within each of the three major geographically broad scallop areas in Alaska. Overall prevalence was about 82%, ranging from 69 to 100% by district. Overall mean infection intensity, based on the number of parasite foci/section, was about 9 (range of 5-29, by location), with scallops from the Bering Sea and Southwest Kodiak being most severely infected. Molecular analyses confirmed that the Alaskan parasite is M. kathae, i.e., the same apicomplexan that caused the collapse of Icelandic scallops and a suspected cause for gray meat and mass mortality of Atlantic sea scallops in northeast North America.

Large-Scale Analysis of Flavobacterium psychrophilum Multilocus Sequence Typing Genotypes Recovered from North American Salmonids Indicates that both Newly Identified and Recurrent Clonal Complexes Are Associated with Disease.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species.IMPORTANCEFlavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.

Comparison of fixatives and fixation time for PCR detection of Mycobacterium in zebrafish Danio rerio .

Mycobacteriosis is a common disease of laboratory zebrafish Danio rerio. Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more widespread and is not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture followed by molecular or biochemical identification is the traditional approach, but DNA of diagnostic value can also be retrieved from paraffin blocks. Here we investigated the type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection as parameters to determine whether the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for 3, 7, 21, and 45 d. Subsequently, fish were evaluated by hematoxylin and eosin and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective and obtained PCR product from 75 and 88% of the M. chelonae- and M. marinum-infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test.

Paramecium caudatum enhances transmission and infectivity of Mycobacterium marinum and M. chelonae in zebrafish Danio rerio.

Mycobacterial infections in laboratory zebrafish Danio rerio are common and widespread in research colonies. Mycobacteria within free-living amoebae have been shown to be transmission vectors for mycobacteriosis. Paramecium caudatum are commonly used as a first food for zebrafish, and we investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 d post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 d post hatch) showed 31% incidence (14/45). Adult fish fed a gelatin food matrix containing mycobacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure as follows: M. marinum OSU 214 47% (21/45), M. marinum CH 47% (9/19), and M. chelonae 38% (5/13). In contrast, fish feed mycobacteria alone in this diet did not become infected, except for 2 fish (5%) in the M. marinum OSU 214 low-dose group. These results demonstrate that P. caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures.

Proliferative kidney disease in Alaskan salmonids with evidence that pathogenic myxozoans may be emerging north.

Proliferative kidney disease (PKD) of salmonids, a chronic immunopathology caused by the myxozoan parasite Tetracapsuloides bryosalmonae, is exacerbated by increased water temperatures. PKD causes economic concerns to trout farmers and contributes to the decline of wild salmonid populations in North America and Europe. The parasite occurs as far north as Norway and Iceland in Europe and was confirmed from California to southern British Columbia in the American continent. In mid-September 2011 adult chum salmon (Oncorhynchus keta) were sampled from Kantishna River, a tributary to Yukon River in Alaska. Clinical PKD was diagnosed based on the macroscopic appearance of mottled kidneys that were uniformly swollen and by the detection of tumultuous histozoic extrasporogonic and coelozoic sporogonic stages of T. bryosalmonae in renal tissue by histopathology. Archived samples provided the molecular confirmation and local strain identification, representing the first confirmed case of PKD in wild adult chum salmon, also co-infected with Parvicapsula minibicornis that represents another novel myxozoan detection in Alaska. Our investigation was extended to another case from August/September 1997, with mortality following furunculosis and ectoparasite co-infections, in sockeye salmon (Oncorhynchus nerka) pre-smolts net-pen reared in English Bay Lakes, Alaska. Immunohistochemistry on archived histological preparations confirmed T. bryosalmonae sporogonic and extrasporogonic stages, indicating a severe to resolving PKD, with concomitant Chloromyxum spp. infection. Those cases provide the first documentation that this parasite is present in Alaska and causes PKD in wild and cultured salmonids in the region. The known geographic range of T. bryosalmonae can be extended to ~267 km south of the Arctic Circle, representing the northernmost detection in America. Given the vast size of Alaska and small resident population, it is likely that T. bryosalmonae remained undetected, but more recently became evident due to the clinical manifestation of PKD, possibly linked to increasing water temperatures reported at the sample locations.

Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species.

While investigating the parasite fauna of wild coho salmon. Oncorhynchus kisutch (Walbaum, 1792), histological examination provided evidence of a new species of Myxobolus (Myxozoa: Myxosporea) infecting nerves of skeletal muscle. Spores were morphologically similar to those of the intramuscular Myxobolus insidiosus Wyatt and Pratt, 1963, both having pyriform spores with clavate polar capsules. However, the former developed exclusively in the nerves of skeletal muscle rather than in myocytes. We examined both species of Myxobolus derived from coho salmon; Chinook salmon, Oncorhynchus tshawytscha (Walbaum, 1792); cutthroat trout, Oncorhynchus clarkii (Richardson, 1836); and rainbow trout Oncorhynchus mykiss (Walbaum, 1792) from freshwater in Oregon. Spore morphology, small subunit ribosomal RNA gene (rDNA) sequences, and site of infection were compared. Myxobolus arcticus Pugachev and Khokhlov, 1979 has pyriform spores, infects the central nervous system of many salmonids, and is found in the Pacific Northwest. It was therefore included in the analyses to rule out conspecificity with the new species. Together, these data show that the Myxobolus sp. from peripheral nerves in the skeletal musculature of coho salmon, rainbow trout, and cutthroat trout is a new species, described herein as Myxobolus fryeri n. sp.

Spores of two fish microsporidia (Pseudoloma neurophilia and Glugea anomala) are highly resistant to chlorine.

Pseudoloma neurophilia (Microsporidia) is the most common pathogen found in zebrafish Danio rerio research facilities. The parasite is associated with marked emaciation. Zebrafish laboratories usually disinfect eggs to prevent transmission of pathogens, typically with chlorine at 25 to 50 ppm for 10 min. The ability of chlorine to kill spores of P. neurophilia and 2 other microsporidia, Glugea anomala and Encephalitozoon cuniculi, was evaluated using 2 viability stains. SYTOX Green was used to visualize dead spores, and live spores were identified by their ability to extrude polar tubes in Fungi-Fluor solution following UV exposure. Results with both stains were similar at various chlorine concentrations for P. neurophilia and G. anomala, but Fungi-Fluor was not useful for E. cuniculi, due to the much smaller spore size. Using the SYTOX stain, we found that 5 ppm chlorine for 10 min causes 100% death in spores of E. cuniculi, which was similar to findings in other studies. In contrast, the spores of P. neurophilia and G. anomala were much more resistant to chlorine, requiring >100 or 1500 ppm chlorine, respectively, to achieve >95% spore death. Repeating chlorine exposures with spores of P. neurophilia using solutions adjusted to pH 7 increased the efficacy of 100 ppm chlorine, achieving >99% spore inactivation. We corroborated our viability staining results with experimental exposures of zebrafish fry, achieving heavy infections in fry at 5 to 7 d post-exposure in fish fed spores treated at 50 ppm (pH 9). Some fish still became infected with spores exposed to 100 ppm chlorine (pH 9.5). This study demonstrates that spores of certain fish microsporidia are highly resistant to chlorine, and indicates that the egg disinfection protocols presently used by most zebrafish research facilities will not prevent transmission of P. neurophilia to progeny.

First report of Cylicospirura felineus in a feral domestic shorthair cat in North America.

CASE SUMMARY: A feral domestic shorthair cat was euthanized owing to acute onset and progression of neurological signs attributed to ethylene glycol toxicity. At post-mortem examination two nodules were identified within the fundus of the stomach. Examination of the gastric nodules revealed an intact mucosal surface, each with multiple red slender nematodes extending through an individual central pore. Histopathological evaluation of the nodules highlighted unique reactive fibroplasia, mimicking feline gastrointestinal eosinophilic sclerosing fibroplasia (FGESF), encasing numerous nematodes with females possessing gravid uteri containing abundant larvated eggs. The latter findings were highly suggestive of the Cylicospirura genus, further supported by an en face evaluation of the buccal cavity, highlighting a distinctive trifid tooth appearance. Together, these findings are consistent with Cylicospirura felineus. PCR for the COX-1 gene was unsuccessful on formalin-fixed specimens, attributed to nucleic acid and protein crosslinking. RELEVANCE AND NOVEL INFORMATION: This represents the first documented case of Cylicospirura species in a feral domestic shorthair cat in North America. This particular cat lived in the highly urban environment of New Orleans, Louisiana. Identification of this case demonstrates the potential for feral cats to serve as reservoir hosts and ultimately support transmission of Cylicospirura species into domesticated cat populations. Gastric cylicospiruriasis may present clinically as a firm abdominal mass, potentially with a history of chronic vomiting. The latter emphasizes the importance of differentiating this condition from a neoplastic process such as alimentary lymphoma and adenocarcinoma. Histologically, the unique thick anastomosing collagenous cords encasing nematodes represent a stereotypical response observed in a broad array of gastrointestinal inflammation in felines, including intralesional bacteria, fungal hyphae, foreign bodies and, in this case, gastric nematodes that closely resemble FGESF. Additionally, these unique histological lesions have previously been misinterpreted as neoplastic conditions, including sclerosing mast cell tumor and extraosseous osteosarcoma.

Myxobolus neurotropus infecting rainbow trout in Alaska, a new geographic record.

The Alaska Department of Fish and Game fish pathology laboratory received a rainbow trout Oncorhynchus mykiss from the Alaska Peninsula that was suspected of having whirling disease based on the display of aberrant swimming behavior and a deformed spine. We tested for Myxobolus cerebralis using standard pepsin-trypsin digest and molecular procedures, which yielded negative results. However, many oval shaped myxospores were observed in brain smears and were confirmed to be those of the morphologically similar M. neurotropus based on a diagnostic assay using PCR. The known geographic distribution of this parasite includes Idaho, Washington, Utah, Oregon, California, and now, Alaska. Whether this species is an emerging parasite is not known because it was only described a few years ago. Given the severe infection found in this rainbow trout, perhaps the considerable displacement of neurological tissues and subsequent pressure on peripheral nerves could have contributed to the spinal curvature and accompanied abnormal swimming. Conversely, the M. neurotropus infection may have been incidental and the spinal deformity may have actually been due to one of several nonspecific developmental or congenital causes. Further studies on geographic distribution and impact on host fitness will probably determine the importance of this species to fish health.

Apophallus microsoma N. SP. from chicks infected with metacercariae from coho salmon (Oncorhynchus kisutch) and review of the taxonomy and pathology of the genus Apophallus (Heterophyidae).

Metacercariae of an unidentified species of Apophallus Lühe, 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.

Survey of parasites in threatened stocks of coho salmon (Oncorhynchus kisutch) in Oregon by examination of wet tissues and histology.

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.

Mortality of coho salmon (Oncorhynchus kisutch) associated with burdens of multiple parasite species.

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.

Cylicospirura species (Nematoda: Spirocercidae) and stomach nodules in cougars (Puma concolor) and bobcats (Lynx rufus) in Oregon.

The stomachs and proximal duodena of 160 cougars (Puma concolor) and 17 bobcats (Lynx rufus), obtained throughout Oregon during 7 yr, were examined for Cylicospirura spp. and associated lesions. Prevalence in cougars was 73%, with a range in intensity of 1-562 worms. The mean diameter of nodules was 1.2 cm (SD=0.5), and many extended through the submucosa to the muscularis. About 83% of cougars had nodules; most nodules contained worms, but 14% of the smaller nodules (<0.2 cm) contained porcupine (Erethizon dorsatum) quills. A mean of 12.4 worms/nodule (SD=34.1) was observed, with a maximum of 340 worms/nodule. Prevalence in bobcats was 53%, with an intensity of 1-25 worms. About 65% of bobcats had nodules, which were slightly smaller than those in cougars but appeared to involve similar layers of gastrointestinal tissue. One to 25 Cylicospirura sp. were found in all but two small nodules in bobcats. Cougars killed for livestock damage or safety concerns had a significantly higher median worm intensity than did those that died of other causes. Also, the median worm intensity of older cougars was higher than that of younger lions. There were more males than females killed for livestock damage or safety concerns. The cylicospirurid from cougars was Cylicospirura subaequalis, and that of bobcats was Cylicospirura felineus. These two similar species were separated morphologically by differences in tooth and sex organ morphology. They were also differentiated by DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Worm sequences from cougars differed from those from bobcats by 11%, whereas essentially no difference was found among worms from the same host. Phylogenetic analysis showed that within the order Spirurida, both cylicospirurids were most closely related to Spirocerca lupi, based on this gene sequence.

Persistence of infection by metacercariae of Apophallus sp., Neascus sp., and Nanophyetus salmincola plus two myxozoans (Myxobolus insidiosus and Myxobolus fryeri) in coho salmon Oncorhynchus kisutch.

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.