**SANJUAN RIVER FISH HEALTH SURVEYS (1992 - 1999)**

Submitted by Fish Health Biologists 

Jerry Landye, Beth McCasland, Carol Hart andKarl Hayden

&

John C. Thoesen, Director, Pinetop FishHealth Center 

                                                    U.S.Fish and Wildlife Service 

Pinetop Fish Health Center 

P.O. Box 160 

Pinetop, Arizona 85935 

November 5, 1999 

Prepared for the San Juan River Biological Committee 

** **

**Table of Contents**

Introduction.........................................................................................1

Materials andMethods.........................................................................1 

Results.................................................................................................2

Fish species withabnormalities................................................2

Abnormality types...................................................................3

Fish health inspectiondata...........................................3

Table1...........................................................3

Fishery biologistdata...................................................4

Table2............................................................4

Abnormality incidences versus rivermile...................................4

Fish health inspectiondata............................................4

Fishery biologistdata....................................................4

Bacterial isolates of San Juan RiverFish...................................5

Table3............................................................5

Electrofishingdamage...............................................................6

Asian tapeworm and otherparasites...........................................7

Viruses......................................................................................7

Histopathologyfindings.............................................................7

Discussion..............................................................................................8

Flannelmouth suckers as an indicatorspecies..............................8

Cause of lesions and otherabnormalities.....................................8

Bacterial isolates of San Juan Riverfish.....................................8

Electrofishing damage...............................................................9 

Asian tapeworm and otherparasites...........................................9

Viruses......................................................................................9

Severity of disease in San Juan River andSummary...................9

Conclusions........................................................................................10

ExecutiveSummary............................................................................10

References..........................................................................................11

**INTRODUCTION**

Fish health surveys on the San Juan River fromFarmington, New Mexico, to Clay Hills, Utah, were initiated in October 1992 by PinetopFish Health Center (PFHC) personnel. The goal of the survey was to determine the cause oflesions and other abnormalities observed in native and nonnative fish. This reportsummarizes the fish health findings in San Juan River fish from October 1992 through May1999.

The investigations focused on the flannelmouth sucker, *Catostomuslatipinnis*, as it is the most prevalent fish species sampled and had been the mostsusceptible species to abnormalities, including lesions. Gaufin, Smith, and Dotson (1960)had reported a similar situation with *C. latipinnis* in the Green River, Wyoming.For these reasons, *C. latipinnis* was used as an indicator species ofenvironmental stressors or diseases. When present, these stressors also may affect theendangered Colorado pikeminnow (squawfish), *Ptychocheilus lucius*, razorbacksucker, *Xyrauchen texanus*, and other fish species in the San Juan River.

Fish health sampling included: 1) field observations(e.g., ectoparasite and endoparasite identifications, and noting the incidence of internaland external abnormalities), and 2) subsequent laboratory investigations including viralassays, bacterial identifications, and histological examinations. Fish also were examinedfor electro-induced damage including spinal deformities and damaged kidney tissue.

**MATERIAL AND METHODS**

Fish health personnel accompanied fishery biologists onSan Juan River adult monitoring trips, Hogback, New Mexico, to Mexican Hat, Utah.Additional trips were made to sample fish from Mexican Hat to Clay Hills, Utah, andFarmington to Hogback, New Mexico. All fish were collected using rafts mounted withelectrofishing apparatus. All fish species with abnormalities were noted, including butnot limited to: bluehead sucker (*Catostomus discobolus*), flannelmouth sucker (*C.latipinnis*), razorback sucker (*X. texanus*), channel catfish (*Ictaluruspunctatus*), Colorado pikeminnow (*P. lucius*), roundtail chub (*Gilarobusta*), common carp (*Cyprinus carpio*), red shiner (*Cyprinellalutrensis*), fathead minnow (*Pimephales promelas*), and speckled dace (*Rhinichthysosculus*). Fishery biologists noted and saved fish with lesions, tumors, and missingor deformed eyes for subsequent observations by fish health personnel. For purposes ofthis report, a lesion is defined as an abnormal change in skin structure due to injuryand/or infectious disease. Disease, defined as lack of ease or as a stressed condition,encompasses all abnormalities in this report. Other stressors including leeches andobvious predator-inflicted wounds were noted by biologists, but were not consideredabnormalities. 

Field notes were obtained from the fishery biologists andanalyzed for number of each taxa collected and what types of abnormalities were observed.This provided a river mile by river mile accounting of observations for a large number offish. 

In May 1994, fish health personnel initiated disease"inspections" of randomly selected *C. latipinnis* from allelectrofishing boats. The purpose of the inspections were 1.) To supplement diseasesamples collected by fishery biologists; 2.) To ascertain health status of apparentlyhealthy and moribund fish in regular intervals throughout the sampling region; 3.) To use *C.latipinnis* as an indicator species of potential environmental hazards affecting otheranimal and plant life in the San Juan River. Inspections were performed on a randomfive-fish sample every designated mile (every fifth river mile).

Fish health personnel performed complete necropsies onall inspection fish as well as fish with abnormalities saved by other biologists. Necropsyincluded: external and internal inspection for gross pathology (lesions, organdeformities, unusual color, excess peritoneal fluid, etc.), bacterial sampling fromkidneys and lesions using sterile techniques (disinfecting the leading edge of lesionswith 70% isopropyl alcohol), and chemical fixation of various organs for histopathologicalexamination. Electrical power for light microscopic observation of skin and gill wet-mountpreparations on selected fish for ectoparasites was provided by a backpack electrofishinggenerator. 

Bacterial samples were isolated on one of two standardbacterial media: brain-heart infusion agar (BHIA) or trypticase soy agar (TSA). Bacterialisolates were identified in the laboratory using the Minitek™ classification system.A fish was considered bacteremia-positive when bacteria was cultured from kidney tissue.Bacteremia was defined as presence of bacteria in the blood; this is considered a diseasestate because blood and kidneys are sterile in healthy fish.

Tissue fixation for histology was performed in the fieldusing Bouin's fixative or 10% formalin for 48 hours with subsequent transfer to 70%isopropyl alcohol. Preserved tissued were sent to the National Fish Health Laboratory,Biological Resources Division (USGS), Kearneysville, WV for histopathological examination.Potential electrofishing-induced, damage was measured by noting gross physiological traumato kidneys and by radiographing (X-ray) randomly collected fish to identify vertebraldamage. Asian tapeworms, *Bothriocephalus acheilognathi* were observed bydissecting fish gastrointestinal tracts using stereo microscopes in the field. Virologytissues were stored in minimal essential medium (MEM) at 4� C and returned to the PFHCfor viral tissue culture assays. 

**RESULTS**

**Fish species with abnormalities:**

Even though *C. latipinnis* comprised between50-60 percent of all fish sampled during each fish survey, it accounted for 70-90 percentof all abnormalities observed. Depending on the survey, *C. carpio, C. discobolus*,or *I. punctatus* ranged from 1 percent to 15 percent of the abnormalities. 

**Abnormality types:**

Lesions comprised a majority of the abnormality types,but missing or deformed eyes, scoliosis or lordosis, and a few tumors were found duringthe fourteen sampling periods. Since the May 1996 survey, there has been a decrease oflesions observed that continued through May 1999. Most of the abnormalities noted in thelatter period were damaged eye tissue. Spinal deformities continued to decrease eachsampling period.

**Percentage of fish with abnormalities:**

**Fish health inspection data**:

Randomly selected *C. latipinnis* (inspectionfish) from each designated mile are represented in Table 1. Each of these fish representsa complete necropsy procedure and all abnormalities were noted by fish health biologists.

**Table 1. San Juan River *C. latipinnis*fish health biologists' inspection data for abnormalities, 10/94 - 5/99.**

| Date  | # of fish sampled | # of fish with abnormalities | % of fish with abnormalities |
|-------|-------------------|------------------------------|------------------------------|
| 10/94 | 95                | 5                            | 5                            |
| 5/95  | 99                | 8                            | 8                            |
| 10/95 | 100               | 5                            | 5                            |
| 5/96  | 100               | 19                           | 19                           |
| 10/96 | 100               | 4                            | 4                            |
| 5/97  | 100               | 1                            | 1                            |
| 10/97 | 110               | 1                            | 0.9                          |
| 5/98  | 80                | 2                            | 2.5                          |
| 5/99  | 80                | 3                            | 3.8                          |

During the May 1996 survey, the health of the suckerpopulations were in poor condition compared to all other survey periods. Fish healthbiologists not only sampled the randomly selected *C. latipinnis*, but alsonecropsied an additional 231 fish. These fish were mostly suckers and were selected due toabnormalities. Of these 331 fish examined, 145 had lesions. Comparing this effort to allother survey periods where the fishery biologists were saving fish with abnormalities andgiving them to the fish health biologist, no other survey had so many fish withabnormalities. During the last three years, fish health biologists have observed only anoccasional lesion, with no concentration of abnormalities in a specific area of the river.

**Fishery biologist data:**

Fishery biologist data included all fish speciescollected and abnormalities observed. Utilizing the data for *C. latipinnis* and *C.discobolus*, survey periods were compared (Table 2). This provided a large number ofsuckers in the data base and changes in frequencies of abnormalities from each river mileprovided trends of probable problem areas of the San Juan River. The discrepancy in valuesbetween fishery biologist and fish health biologist data is explained by the fish healthbiologists' ability to observe pathology more carefully with more time allotted to fishhealth. The lower fishery biologist abnormality incidence values may be attributed in partby the inclusion of *C. discobolus* values which lowered the overall abnormalityincidence rate. However, the same trend in abnormality incidences was observed in bothdata sets from each type of survey.

**Table 2. Six year review of fishery biologists'San Juan river *C. latipinnis* and *C. discobolus* abnormality data (10/91 -10/97)**Date# of fish sampled% of fish with abnormalities% of abnormalities that are lesions10/9116062.6546/9230183.66510/9234130.33010/9329590.4555/9418783.07410/9495240.6305/9512660.12510/9522610.8255/9625573.47210/9628881.6335/9752700.41910/9774400.332
**Abnormality incidences versus river mile (RM):**

**Fish health inspection data:** Duringyears of high abnormalities, most were found in sections RM 121-156 and RM 76.

**Fishery biologist data:** Most of theabnormalities observed by fishery biologists consistently were found on fish in RM126-156. As with fish health inspection data, fewer abnormalities were found in the Fall(October) compared to Spring (May) surveys. Few abnormalities were found from Lake Powell(RM 0) to the head of the canyon (RM 78). In this area, bite marks caused by fishpredators being the most common. From the head of the canyon to Farmington, NM (RM 178),wounds from bird predators were common. Both of these wound types were easilydistinguished from lesions. 

**Bacterial isolates of San Juan River fish:**

Bacterial isolates from fish from all survey trips areidentified in Table 3. Bacteremia, as indicated by bacterial growth from kidney tissuetended to follow the abnormality trends on each of the surveys.

**Table 3. Bacteria isolated from San Juan Riverfish, 1994-1999.**Taxa lesionkidney*Acinebacter anitranus* X*A. lwoffi*XX*Actinobacillus lignieresii*X *Aeromonas* sp.XX*Aeromonas hydrophila*XX*Citrobacter freundi* X*Enterobacter agglomerans*X *E. cloacae* X*E. sakazaki*X *Flavobacterium* sp.X *F. miningosepticum* X*Hafnia alveri*X *Klebsiella arizonae* X*K. pneumoniae* X*Pleisiomonas shigelloides*XX*Proteus mirabilis*X *P. vulgaris*X *Pseudomonas* sp.XX*P. aeruginosa*X *P. cepacia*XX*P. fluorescens*X *P. maltophilia*XX*P. multifili* X*P. pseudoalcaligenes*XX*P. putrefaciens*XX*P. stutzeri* X*P. vesicularis* X*Salmonella arizonae*X *Serratia liquifaciens*X *S. odorifera*XX*S. rubidaea*XX*Shigella* sp.XX*Staphylococcus* sp.XX*Streptococcus* sp. X*Vibrio alginolyticus*XX*V. cholerae* (non-human)X *V. fluvialis*XX*Yersinia entercolitica*X *Y. psuedotuberculosis*X Gram positive rodsXX
**Electrofishing damage:**

Damaged kidney tissue was noted in fish necropsies andspinal deformities were noted from both fishery and fish health biologists' observations.Randomly collected *C. latipinnis* from each electrofishing raft were radiographedon vertical and lateral planes and analyzed for spinal damage of any type (Alcumbrac 1994,pers. comm.). Both compression and step-down fractures were found in less than 10% of thefish. Of the three rafts surveyed, there was no correlation with manufacture of the unit;however, those rafts with a larger cathode surface area had the least amount of spinaldamage to fish. Since the radiograph survey was completed, rafts have been modified, whichprobably has accounted for fewer spinal abnormalities. 

**Asian Tapeworm and other parasites:**

Asian tapeworms were first detected in *C. carpio*in the San Juan River near Bluff, UT (RM 82) in October 1994 (Landye and McCasland 1997).The invasion of this tapeworm to other sections of the river and cyprinid species wasdocumented in subsequence surveys from Mexican Hat (RM 52.8) to Fruitland Diversion, NM(RM 166). This included infections found in older stocked*P. lucius*, but notyoung of the year stocked fish. No Asian tapeworms have been found in other families offish in the San Juan River. 

The following endo- and ectoparasites were observedduring at least one fish health survey. The species name following the parasite nameindicates the fish from which the parasites were detected: *Ambiphyra (I. punctatus),Apiosoma (I. punctatus), Trichodina (Micropterus salmoides, I. punctatus, C. carpio, C.latipinnis,*and*C. discobolus), Tetrahymena (C. latipinnis), Henneguya (I.punctatus), Gyrodactylus (C. carpio, C. latipinnis,*and *C. discobolus),Ligictaluridus (I. punctatus), Hunterella (C. latipinnis), Corallobothrium fimbriatum (I.punctatus), B. acheilognathi (C. carpio, C. lutrensis, R. osculus,*and*P.lucius), Myzobdella (I. punctatus)*, and *Lernaea (C. latipinnis)*.**

Even though *Ichthyophthirius* ("Ich")was not detected during main river fish health surveys, samples of fish from backwaterareas were examined by PFHC staff and were found to be infected. 

Rainbow and brown trout were occasionally sampled andcranial tissue was taken for plankton centrifuge method of analysis for *Myxoboluscerebralis*, the causative agent for whirling disease. Even though this area is notsuitable for salmonid populations, the cold spring water runoffs allowed for a few troutto be present. A sample of the seasonal trout population was taken, but no *M.cerebralis* spores were detected. 

**Viruses**

All viral assays from San Juan River fish were negativefrom samples taken from May 1994 to May 1999.

**Histopathlogy findings**

Lesion samples were collected and shipped to Dr. VickiBlazer, National Fish Health Laboratory, Kearneysville, WV for histological analysis. Dr.Blazer presented three potential explanations for the lesions: "1) some mineraldeposit, possibly with sharp projections, gets into the dermis of bottom dwelling fish andcauses the reaction. 2) A metabolic disorder, possibly in response to some contaminant,which causes the body to produce the material or deposit it, abnormally in the dermis,which lead to a reaction to it. 3) Some interaction of a contaminant with UV radiationwhich leads to skin damage."(Blazer 1996, pers. comm.). Dr. Blazer's opinion on thepresence of "masses of bacteria" in the eroded epidermis and associated necroticmuscle is that they are secondary invaders, which concurs with the analysis.

**DISCUSSION**

**Flannelmouth suckers as an indicator species:**

*C. latipinnis* remained the best choice as anindicator species for environmental problems on the San Juan River throughout the study.This species has the highest proportion of abnormalities (in excess of 80%) than any otherfish species sampled, but was only about 60% of all fish sampled. This is consistent withthe abnormalities and fish species observed by Gaufin, Smith and Dotson (1960) in theGreen River.

**Cause of lesions and other abnormalities:**

We hypothesize that bacteria isolated from fish lesionsare secondary invaders-they are opportunistic organisms that cause lesions after initialtrauma due to contaminants, unsuccessful bird or fish predation, etc. The National FishHealth Laboratory concurs that the bacteria are secondary. Dr. Blazer also proposes (intwo of her three explanations for the cause of lesions) that a contaminant was presentwhich initiates the epidermal necrosis. Walker (1992) in a preliminary investigation onlesions from suckers in the Animas River, a tributary to the San Juan River, thought thatthe cause was a type of *Aeromonas salmonicida* bacterium. Gaufin, Smith, andDotson (1960) noted that most of the flannelmouth suckers with lesions were justdownstream of Green River, Wyoming where municipal sewage and oil discharge was takingplace.

Histological samples consistently have shown an unknownmaterial in micro quantities to be present in lesion sections. Herman and Lemm (1990)found this possible putative autoimmune reaction in striped bass. Until a micro-probe isavailable to analyze this material, the probable cause of these fish lesions will remainunknown. From all the fish health samples taken during this investigation, we caneliminate viral, bacterial, or parasitic agents as the primary cause of lesions in SanJuan River fish.

Gross pathology data from inspection fish and field notesof both fish health and fishery biologists demonstrated a decreasing abnormality incidencefrom 1993 to 1999. However, lesions increased in May 1996 to levels resembling thedisturbing May 1994 and June 1992 levels. It is unlikely that these spikes of lesions canbe attributed solely to spawning stress as this is an annual event. 

**Bacterial isolates of San Juan River fish:**

Of all the bacterial species isolated from all fishsampled, none are known primary fish pathogens, although *Aeromonas hydrophila*,the causative agent of Motile Aeromonas Septicemia (MAS), usually presents itself afterthe fish has been stressed. However, most would be considered opportunistic or secondaryfish pathogens. These taxa would affect fish that have been compromised by lesions,reproductive stress, and predator wounds. Fish carrying these bacteria are not animmediate threat to humans in contact with the San Juan River. Given the number ofbacterial taxa present in the system, and new evidence that species such as *Pseudomonasaeruginosa* can affect both plants and animals, humans with already compromised healthconditions should take precautions with water contact between Bluff, Utah and Farmington,New Mexico where most of these bacteria were recorded. 

**Electrofishing damage**: 

Possible electrofishing damage was noted by observationof spinal deformities in fishery biologists data and damaged kidney tissue during fishhealth surveys. The radiographs of randomly collected "healthy" *C.latipinnis*, demonstrated that most of the damage was in the form of compressionfractures while step-down fractures were poorly represented. These compression fracturesaccount for many of the spinal deformities found in the flannelmouth suckers.Modifications to cathodes of the electrofishing units during the study probably resultedin the reduction in spinal deformities noted during the course of the study. There was nocorrelation with electrofishing damage and manufacture of equipment.

**Asian tapeworm and other parasites:**

Asian tapeworms, *Bothriocephalus acheilognathi*willremain in the San Juan River as long as susceptible hosts are present. *B.acheilognathi* will pose a threat to native speckled dace, roundtail chub, and youngColorado pikeminnow. "Ich," *Ichthyophthirius multifiliis* is a potentialproblem to young of the year fish in backwaters during the warmer water temperatures.Leeches may provide areas that secondary bacteria can invade and young forms of *Lernaea*might have an effect on fish in backwaters during periods of warmer water. All otherparasites should have little or no effect on fish in the future unless water qualitydeteriorates.

**Virus:**

Since no evidence was found for virus being present inSan Juan River fishes, they are currently not a factor in fish populations.

**Severity of disease in the San Juan River andSummary:**

Since June 1992, San Juan River fish health data suggeststhat abnormality incidence in all fish species is low with the exception of"spikes" of lesions and other disease signs during the Spring hydrograph. Riversections with the highest abnormality incidence and bacteremia constantly have been miles136-156. Because of the inconsistency of spikes of lesions from year to year, it wouldsuggest that the source might be triggered by monsoon cyclonic storm activity from theprevious summer or occasional releases from a yet unknown source. 

Lesions remain the most appropriate abnormality for useas an indicator of stressful environmental conditions and resulting fish disease. Thereason for this is that the years when the percentage of fish with abnormalities is high,the percentage of abnormalities that are lesions also is high. Thus, incidences of lesionsin *C. latipinnis* will continue to be a useful tool as the primary indicator offish health in the San Juan River. 

Any future investigations should integratehistopathological methods with the use of a micro probe (when developed) or some of thenew techniques utilizing proteins as a method to detect fish stress.

**Conclusions:**- Fish pathogens are not the cause of the    majority of lesions observed on San Juan River fish.
- Asian tapeworm, *B. acheilognathi* was    discovered within a few months of its initial introduction and its infection of cyprinids    was documented both upstream and downstream as it spread.
- A large number of bacterial species were    present on and in San Juan River fish. Many of these taxa have the potential of secondary    infections in both fish and mammals.
- Reconfiguration of electrofishing equipment    (cathodes) can reduce the amount of injury to fish.
- Flannelmouth sucker, *C. latipinnis*remains    the best species for monitoring.
- Future research on fish lesions should    concentrate on the development of a nano probe to analyze the unknown material found in    histopathological samples of lesions.

**EXECUTIVE SUMMARY**

Fish health surveys on the San Juan Riverfrom Farmington, New Mexico, to Clay Hills, Utah, were initiated in October 1992 byPinetop Fish health Center personnel. The goal of the survey was to determine the cause oflesions and other abnormalities observed in native and nonnative fish. This reportsummarizes the fish health findings from October 1992 through May 1999. 

The investigation focused on theflannelmouth sucker, *Catostomus latipinnis* as it is the most common fish speciessampled and had been the most susceptible species to abnormalities, including lesions.Since this section of the San Juan River was part of the recovery area of the endangeredColorado pikeminnow (squawfish), *Ptychocheilus lucius* and threatened razorbacksucker, *Xyrauchen texanus,*concern about these abnormalities was high.

Fish health sampling included both fieldobservations and laboratory investigations. Fish also were examined for electro-inducedinjury.

As a result of the fish health survey theintroduced Asian tapeworm was discovered, a large number of bacterial species wereidentified, and modifications in electrofishing equipment were made to minimize damage tofish in the future. The exact cause of the lesions was not determined, but parasitic,bacterial, and viral agents have been eliminated. Future investigations should beundertaken when an appropriate nano probe is developed for the unidentified material foundin histopathological examinations.

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