Survey on Myxobolus infection of the bleak (Alburnus alburnus L.) in the river Danube and in Lake Balaton

In a three-year survey of myxosporean infections of the bleak (Alburnus alburnus), involving the examination of 205 fish specimens from the River Danube and 50 from Lake Balaton, four Myxobolus species (two gill parasites, one fin parasite and a species parasitising the skeletal muscles) were detected. Two of the species could be identified as M. alburni and M. obesus. Of the other two species, the gill parasite proved to be a hitherto undescribed species which is described here as a new species by the name of M. margitae. One of the two gill-parasitic species, M. obesus, formed plasmodia in the respiratory lamellae of the gill filaments, while the plasmodia of M. margitae n. sp. were formed in the afferent artery of the primary gill filaments. The plasmodia containing spores morphologically identifiable with the species M. alburni were located in the connective tissue between the fin rays. The less frequently found muscle-parasitic Myxobolus species has not been identified precisely. The plasmodia of M. obesus were found in the fish in May and June, while those of M. alburni and M. margitae n. sp. in July and August. The prevalence of infection in fish examined in these periods was 15.5% for M. obesus, 11.5% for M. margitae and 14.0% for M. alburni.     

Remarks on the seasonal occurrence and identification of young plasmodial stages of Myxobolus spp. infecting cyprinid fishes in Hungary

During a survey on the Myxobolus infection of two cyprinid fishes, the ide (Leuciscus idus) and the roach (Rutilus rutilus), myxosporean developmental stages were found around the arteries of the gill filaments and in the gill lamellae. An analysis of the 18S rDNA sequences of these stages revealed that plasmodia developing in the ide belonged to Myxobolus elegans, those developing in the gill lamellae of the roach corresponded to M. intimus, while plasmodia developing in close contact with the cartilaginous gill rays proved to be developmental stages of M. feisti. A strict seasonal cycle with a very long intrapiscine development was recorded for M. elegans and M. intimus. Developing plasmodia of the latter Myxobolus spp. occurred from early summer to next spring, and spore formation took place only in April. No seasonality associated with M. feisti infections was found. Developing plasmodia and mature spores of this species occurred simultaneously in different seasons of the year. Myxobolus feisti spore formation always occurred in close contact with the cartilaginous tissue of the gill filaments but spores were rarely encapsulated in the cartilaginous gill rays.     

Myxobolus infections of common carp (Cyprinus carpio) in Syrian fish farms

During a survey on Myxobolus infection of pond-cultured common carp in Syria three Myxobolus spp. were found. Myxobolus dispar infected the gill arteries, forming large elongated plasmodia in the gill filaments. The plasmodia of M basilamellaris were located in the gill arches at the base of the filaments. Elongated filiform plasmodia of M. encephalicus were found in the blood vessels of the brain. Despite the common occurrence of the above parasites, no disease symptoms were observed in the infected fish specimens. This is the first report on myxosporean infection of fish from Syrian waters.     

Molecular biology can differentiate morphologically indistinguishable Myxosporean species: Myxobolus elegans and M. hungaricus

Two, morphologically indistinguishable myxosporean species, Myxobolus elegans Kashkovsky, 1966 and M. hungaricus Jaczó, 1940 were differentiated using molecular biological methods. Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify an approximately 1600 base pairs (bp) long fragment of the 18S ribosomal RNA gene. In restriction fragment length polymorphism (RFLP) study with HinfI, MspI and TaqI enzymes, the two parasite species were easily distinguishable. The genetic distinctness was also confirmed by the DNA sequence of their PCR products. Although M. elegans and M. hungaricus are morphologically very similar, based on the results of the PCR-RFLP and the DNA sequences, we concluded that they are valid species.     

Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp. parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 ± 1.8 μm in total length, 12.9 ± 0.8 μm in body length, 3.4 ± 0.3 μm in width, 3.1 ± 0.1 μm in thickness and 24.6 ± 2.2 μm in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 ± 0.5 μm in length and 0.7 ± 0.1 μm in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size.     

Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions.

From early April into mid-June 1977, sequential groups of juvenile rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were each exposed for 10 days to the parasite Myxobolus cerebralis by immersion in a stream inhabited by infected wild trout. Following incubation in a M. cerebralis-free facility, trout were subsequently killed, and heads and gill arches were examined by routine histologic methods. A grading scale to quantify lesion severity was developed and applied. Percentage infected, lesion severity scores, effects of water temperature and flow rates on percentage infected and lesion severity scores, and resulting pathology were determined for each species at each exposure period. The percentage of rainbow trout infected with M. cerebralis was significantly higher than the percentage of brown trout infected for each exposure period. The percentages of rainbow trout infected in exposure periods later in the calendar year were significantly higher than those in earlier periods. The percentages of brown trout infected were not significantly different among exposure periods. Overall average lesion severity scores were significantly higher in rainbow than in brown trout. Lesion severity scores in rainbow trout increased over time (a positive correlation with exposure period). Lesion severity scores were not significantly different for brown trout among exposure periods. A significant correlation existed between water temperature and percentage of rainbow trout infected; a significant correlation also existed between water temperature and lesion severity scores in rainbow trout. Similar correlations did not exist for percentage of brown trout infected or accompanying lesion severity scores. In rainbow trout, ventral calvarium was the most common site of M. cerebralis replication, followed by gill arches. In brown trout, lesions were virtually confined to gill arches. Early lesions consisted of foci of cartilage necrosis with small numbers of M. cerebralis developmental stages. More advanced lesions consisted of multifocal areas of cartilage necrosis with numerous M. cerebralis developmental stages and/or mature myxospores bordered and/or infiltrated by mono- and multinuclear leukocytes. Lesions in brown trout were smaller and had fewer associated leukocytes and M. cerebralis developmental stages and/or mature myxospores. Higher infection rates, lesion severity scores, and differences in lesion location in rainbow versus brown trout explain in part why numbers of rainbow but not brown trout have fallen in western rivers inhabited with M. cerebralis-infected trout.     

Occurrence and pathology of Sinergasilus lieni (Copepoda: Ergasilidae), a parasite of the silver carp and bighead, in Hungarian ponds

Sinergasilus lieni Yin, 1949, a well-known and pathogenic parasitic copepod in China and Russia, has been detected in Hungarian carp farms for the first time. The parasite infected the third-year generation of silver carp (Hypophthalmichthys molitrix) and bighead (Aristichthys nobilis). The gills of the infected fish specimens showed severe pathological changes. At the attachment sites of female copepods clubbing and fusing of the gill filaments were observed and in some parts of the pale or whitish hemibranchia deep indentations were recorded in places where the tips of the damaged filaments had broken off. Silver carp and bighead were infected at a similar rate, having 8 to 27 copepods attached to the end of the clubbed filaments or the proliferated epithelium of 2 to 10 fused filaments. In histological sections the head part of the parasite was found in a deep cavity of the proliferated epithelium, piercing its antennae deep into the tissues. Only the end of the filaments showed changes. In this part the proliferated epithelium was infiltrated by eosinophilic granular cells. In the central and basal parts of the hemibranchia the original structure of the filaments was preserved with intact secondary lamellae.     

Myxobolus cyprini Doflein, 1898, in Peamouth: First Report of This Myxosporean in the Western Hemisphere

Abstract

A survey of 3–8-year-old peamouths Mylocheilus caurinus collected from the lower Fraser River, British Columbia, revealed 100%, infection by a myxosporean identified as Myxobolus cyprini based on spore morphology, site of infection, and associated pathological changes. Myxobolus cyprini is a common myxosporean parasite of several species of cyprinid fishes (family Cyprinidae) in Europe, the former USSR, and China, but this is the first report of this myxosporean in the Western Hemisphere. The source of the infection in peamouths—whether the parasite is endemic to North America or has been imported with exotic cyprinids from Europe or Asia—is unknown. It is also possible that the myxosporean from peamouths is very closely related to, but a distinct species from, M. cyprini of Eurasia. As reported from European cyprinid fishes, the myxosporean in peamouths developed in the skeletal muscle, and disseminated spores were found associated with chronic inflammation in the visceral organs.     

Three Myxosporeans Found in the Cranial and Branchial Tissues of Rainbow Trout in California

Abstract

Three myxosporeans were encountered in the cranial tissues of a California population of rainbow trout Oncorhynchus mykiss examined for the presence of Myxobolus cerebralis, the causative agent of whirling disease. Typical spores of M. cerebralis and a previously undescribed species of Myxobolus were found in the cranial tissues prepared by the pepsin HCl-trypsin digestion method. Henneguya zschokkei was also detected in digest preparations of cranial tissues, but was more numerous when branchial cartilage was included in the preparations. Microscopic examinations of tissues of individual rainbow trout showed occasional infections with both myxobolid species. Myxobolus cerebralis trophozoites and spores were found in the cranial and gill cartilage, and Myxobolus sp. was found in the brain and spinal cord. Henneguya zschokkei was also found within granulomas in the connective tissues below the gill arch. Both M. cerebralis and H. zschokkei were associated with a chronic inflammatory response in their respective tissues. In contrast, the Myxobolus sp. spores were found in pockets within the nervous tissues with no detectable host response. The spore measurements, calculated from fresh digests of infected tissues for the three myxosporeans (N = 20), for length × width × thickness in micrometers (SD) were 11.7 (0.6) without tails and 42.6 (5.2) with tails × 7.7 (0.8) × 7.0 (0.1) for H. zschokkei, 9.9 (0.4) × 8.4 (0.1) × 6.5 (0.3) for M. cerebralis, and 12.7 (0.7) × 10.5 (1.0) × 9.5 (0.8) for Myxobolus sp. Examined under scanning electron microscopy, the latter two species were morphologically similar although distinctive in size.     

Light and scanning electron microscopy of Henneguya arapaima n. sp. (Myxozoa: Myxobolidae) and histology of infected sites in pirarucu (Arapaima gigas: Pisces: Arapaimidae) from the Araguaia River, Brazil

In this report, we describe Henneguya arapaima n. sp., a parasite of the gill arch and gall bladder of Arapaima gigas (pirarucu) collected in the Araguaia River, in the municipality of Nova Crixás, Goiás State, central Brazil. The plasmodia were white, round or ellipsoidal and measured 200-600mum. Parasite development was asynchronous and the mature spores were fusiform and had smooth wall. The spores measurements were (range, with means+/-S.D. in parentheses): total length-48.4-53.1mum (51.6+/-3.4mum), body length-13.5-15.2mum (14.2+/-0.8mum), body width-5.1-6.1mum (5.7+/-0.5mum), body thickness-4.7-5.3mum (4.9+/-0.2mum) and caudal process length-38.0-41.2mum (38.3+/-2.9mum). The polar capsules were elongated and of unequal size, with lengths of 6.3-6.8mum (6.5+/-0.2) and 6.2-6.6mum (6.3+/-0.1) for the longest and shortest axes, respectively. Capsule width was 1.4-1.6mum (1.5+/-0.1). Histological analysis showed that the plasmodia occurred in the tunica adventitia of the gall bladder and were delimited by a thin capsule of connective tissue. In the gill arch, the plasmodia were also surrounded by connective tissue similar to the endomesium of striated skeletal muscle cells. Sixty-five juvenile specimens of A. gigas weighing 1.0-25.0kg were examined, 17 (26.1%) of which were infected. Of these, 14 (82.3%) had cysts in the gall bladder, two (11.7%) had cysts in the gill arch and only one (5.9%) had cysts in both organs. When the fish were grouped by weight, the prevalence of infection in fish weighing up to 10.0kg (20.7%) was significantly lower than in fish weighing 10.1-25.0kg (50%) (G=3.93; d.f.=1; p<0.05).     

Detection of Myxobolus cerebralis in Rainbow Trout and Oligochaete Tissues by Using a Nonradioactive in Situ Hybridization (ISH) Protocol

Abstract

A nonradioactive in situ hybridization (ISH) protocol was developed to detect Myxobolus cerebralis, the causative organism of whirling disease, in its primary host, rainbow trout Oncorhynchus mykiss, and in its alternate oligochaete host, Tubifex tubifex. A cocktail of three oligonucleotide primers (derived from the small subunit ribosomal DNA sequence) directed at target sequences of the parasite DNA was tailed at the 3′ end with digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP). Labeled probes were hybridized to parasite DNA present in deparaffinized tissue sections from infected trout and oligochaetes. The bound probes were visualized after modifications of existing ISH protocols. By using the new ISH procedure, the parasite was found in target tissues of subclinically and clinically infected fish and tubificid oligochaetes after exposures of these hosts to triactinomyxons and mature spores, respectively. The probe did not bind with salmonid tissues infected with two other myxosporean parasites, Ceratomyxa shasta or the PKX organism, or to a Myxobolus sp. infecting the cartilage of plain sculpin Myoxocephalus jaok. These initial results indicate that ISH is an effective and specific test for detecting Myxobolus cerebralis in its fish and oligochaete hosts.     

Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.     

Aspects of the pathology of Lamproglena clariae (Copepoda: Lernaeidae) on gills of Clarias gariepinus from the Vaal River system,South Africa

Lamproglena clariae attaches to gill filaments of Clarias gariepinus, penetrates the gill tissue and consumes blood. Hence, the purpose of this study was to investigate the mechanical damage caused by this parasite on gill filaments of the host. Infestation intensity and haematocrit values of fish blood were determined and correlated. Fish were killed, gills dissected out and observed with a dissection microscope. Gill filaments with adult females in situ were fixed in a solution of alcohol, formaldehyde and acetic acid and preserved in 70% ethanol. Fixed specimens were studied by histological examination following staining with Heidenhein’s azan solution. Gross morphology revealed varying degrees of swelling of gill filaments near the attached specimens of L. clariae. Epithelial hyperplasia, resulting in fusion of gill lamellae and necrosis of the host tissue in the vicinity of the parasite were revealed through histological examination. No correlation was evident between the infestation intensity and fish haematocrit values. Although results showed that L. clariae causes localized infestation, high intensity of infestation may be detrimental to the fish. Hyperplasia of epithelial tissue resulting in fusion of lamellae would result in restriction of air passages and thus hinder the process of respiration of the host.     

Experimental Induction of Proliferative Gill Disease in Specific-Pathogen-Free Channel Catfish

Abstract

Specific-pathogen-free channel catfish Ictalurus punctatus were exposed to sediment and mud from a pond containing channel catfish with proliferative gill disease. In one experiment, fish were to exposed to mud and sediment for 2 months in water maintained at 19°C. Fish were necropsied weekly, and certain tissues were examined histologically and ultrastructurally. Four trials were conducted with sediment samples from different epizootics of proliferative gill disease. In a second experiment, fish were exposed to sediment for 7 d in water maintained at 16, 19, or 26°C; the fish were then moved to clean water held at 16, 19, or 26°C. Fish were necropsied before transfer to clean water and weekly thereafter for 2 months. Channel catfish held at 19°C developed proliferative gill disease within 2 d of exposure to sediment. Primary cells of a uninucleate myxosporean parasite were present in the gills at the base of lamellae. These developed into plasmodia with numerous secondary cells, and some primary cells disintegrated, releasing their internal secondary cells. Similar development was observed in internal organs 1 week after appearance of the parasite in gills. Complete sporogony did not occur over the 2 months of this study. Plasmodia became necrotic and were not detected after 60 d. In fish exposed to sediment for 7 d at 16, 19, and 21°C, similar organisms were detected, but clinical disease occurred only at 19 and 26°C. Proliferative gill disease may be attributed to extrasporogonic stages of a myxosporean resembling Sphaerospora spp.     

Prevalence and Pathogenicity of a Heterophyid Trematode Infecting the Gills of an Endangered Fish,the Fountain Darter,in Two Central Texas Spring-Fed Rivers

Abstract

Gills of 194 fountain darters Etheostoma fonticola collected from the Comal River in Texas from May 1997 through May 1998 were found to be parasitized with 8–1,524 metacercarial cysts of a heterophyid trematode tentatively identified as Centrocestus formosanus. The intensity of infection varied among three sites on the Comal River. In contrast, of 130 darters from the nearby San Marcos River that were examined, only 4 (3%) were infected, and these had 1–2 cysts per fish. Of 2,279 Melanoides tuberculata snails from the Comal River that were examined, 139 (6.1%) were infected with the trematode. Only 1 snail in 2,241 from the San Marcos River that were examined was infected. The presence of metacercariae in darters was associated with flared opercula, shortened or thickened gill filaments, epithelial hyperplasia, and engorged lamellae. The normal cartilage support of the filaments was distorted and displaced, leading to severe deformities of filament structure. Gill damage was severe and possibly life threatening for the darters with more than 800 cysts per fish (9% of examined fish). We suspect that fountain darter deaths were caused by the parasite in the Comal River during this study.     

Gill morphology in two Mediterranean Sea fishes of similar feeding preferences: sea bream (Sparus aurata L) and sea bass (Dicentrarchus labrax)

The aim of the present study was to describe the gill morphology of the gilthead sea bream and sea bass fishes that showed the same feeding habits. Our study focused on the gross morphological, scanning and light microscopical features of the gills, where a many differences in the gill arches and gill rakers surface in both species were explained. Our results marked that; there was an interbranchial median elevation appeared in sea bream. The gill arches length and the gaps between decreased medial wards in both species. The gill rakers had similar arrangement and varied number in both species. The first lateral rakers row was the longest one in sea bass. However the gill filaments appeared in double rows. Gill filaments were long at middle and short at extremities of gill arch in both species. Scanning electron microscopy revealed the surfaces of gill arch covered by a mosaic of pavement cells, varied sized pores of chloride cells and many pointed spines on the rakers ventral border in both species. Two types of taste buds marked on the gill arches and rakers in sea bass. Many lamellae appeared on filaments in both species.     

Massive Lernaea cyprinacea Infestations Damaging the Gills of Channel Catfish Polycultured with Bighead Carp

Abstract

During June and July 1998, at least three Arkansas fish farms polyculturing bighead carp Hypophthalmichthys nobilis with channel catfish Ictalurus punctatus suffered major losses of catfish associated with massive infestations by the crustacean parasite Lernaea cyprinacea. The channel catfish had few adult Lernaea attached to their skin, but there were 8–50 Lernaea copepodids on the surface of each catfish gill filament. The copepodids were found grazing on the gill tissue, and their feeding activity was associated with gill damage including epithelial hyperplasia, telangiectasis, and hemorrhage. Bighead carp in the same ponds were reported to have had numerous adult Lernaea on their skin but did not die during the epizootic. It is possible that the filter-feeding apparatus of the carp captured the copepodids, thus preventing heavy infestation of the gill filaments. Lernaea copepodids have not been implicated previously in fish losses resulting from parasite damage to the gills. The loss of catfish in these cases is likely to be due to their polyculture with the bighead carp, a species that provides an excellent host for adult Lernaea.     

Ultrastructural morphology of the gill of the hybrid striped bass (Morone saxatilis x M. chrysops)

The fine structure of the principal cell types found in the gill filaments and secondary, respiratory lamellae of the hybrid striped bass (Morone saxatilis x M. chrysops) was studied by transmission electron microscopy. Most of the cell types generally resembled those observed at this level of resolution in other euryhyaline, teleostean species, and the mucus cells, epithelial cells, and cartilage cells were non-distinctive. Pillar cells were atypically flattened within the lamellae. The extensive cytoplasmic tubular system of the chloride cells was more densely distributed and was less branched than reported for many other teleosts. The vascular endothelial cells of the marginal, lamellar blood vessels were particularly striking because of their prominent, electron-dense cytoplasmic granules. Although the physiological function and chemical composition of these granules remain obscure, their presence suggests an important role of secretion into the vascular lumen. These gill data from normal specimens of this sport and commercial, hybrid species constitute a useful basis for interpreting gill changes in diseased striped bass.     

Distribution of Myxobolus cerebralis in Salmonid Fishes in Montana

Abstract

Over an approximately 2-year period, 20,974 fish (trout and other salmonid species) from 230 separate waters (creeks, rivers, lakes, reservoirs, ponds, hatcheries, and irrigation ditches) within 21 of the 22 major drainages in Montana were examined for Myxobolus cerebralis. Nine of the major river drainages have waters containing infected fish: Beaverhead, Big Hole, Blackfoot, Clark Fork above the Bitterroot River, Flathead below the south fork of the Flathead River, Jefferson, Madison, Missouri above the Marias River, and Yellowstone above the Bighorn River. The Beaverhead, Clark Fork above the Bitterroot River, Jefferson, Madison, and Missouri above the Marias River have the greatest number of waters containing fish infected with M. cerebralis. Comparisons of infection levels (number of pooled samples that contain fish infected with M. cerebralis) between species among these drainages show significantly lower levels of infection in brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss in the Missouri above the Marias River drainage and significantly higher levels of infection of rainbow trout in the Jefferson. Comparisons of differences in infection levels between drainages among species show that, in the Beaverhead, Clark Fork above the Bitterroot River, and Madison, infection levels in brown trout are significantly higher than in rainbow trout. This is partially attributed to losses of juvenile rainbow trout because of M. cerebralis infection, leading to biased samples. Histopathologic studies showed lesions were consistently less severe in brown trout than other species and occurred in a different location (gill arches versus ventral calvarium). In six of the nine affected drainages (Beaverhead, Blackfoot, Clark Fork above the Bitterroot River, Flathead below the South Fork, Jefferson, and Madison), infected fish were found at or near the time that intensive sampling was initiated in the spring of 1995. In the three remaining affected drainages (Missouri above the Marias River, Yellowstone above the Bighorn River, and the Big Hole), infected fish were not identified until at least 15 months after the initiation of widespread testing. This indicates that in the first six drainages listed above, the infection was well established prior to 1995 but spread into the last three drainages in the ensuing months. Methods of transmission and the sources of infection are unknown, although the absence of infected fish in state, private, and federal hatcheries in Montana indicates hatchery fish from these sources are not likely to be responsible.