Appendix III: National Wildlife Health Center Pathology Reports

Preliminary Diagnoses:

1. Oral discs: Depigmentation of jaws or toothrows in 8 of 85 tadpoles from Welaka NFH, 6 of 20 tadpoles from Warm Springs NFH, and 9 of 23 tadpoles from Orangeburg NFH.

2. Gall bladders:  Myxozoan parasite (Myxidium sp.) in 24 of 27 tadpoles from Welaka NFH, 5 of 7 tadpoles from Warm Springs NFH, 6 of 10 tadpoles from Edenton NFH, and 1 of 3 tadpoles from Orangeburg NFH.

3. Skin ectoparasite:  Gyrodactylus sp (monogean trematode) in 11 tadpoles from Welaka NFH, 5 tadpoles from Warm Springs NFH, no tadpoles from Edenton NFH, and no tadpoles from Orangeburg NFH.

4. Intestinal pinworms:  Gyrinicola batrachiensis in 9 of 85 tadpoles from Welaka NFH, 1 of 20 tadpoles from Warm Springs NFH, and no tadpoles from Edenton and Orangeburg NFH.

5. Bacterial culture result: Aeromonas hydrophila was isolated from the intestines only of 7 of 8 tadpoles from Welaka NFH, 1 of 3 tadpoles from Warm Springs NFH, 1 of 4 tadpoles from Edenton NFH, and 1 of 6 tadpoles from  Orangeburg NFH.

Background:

One hundred fifty larval anurans of 11 species were collected from 4 national fish hatcheries in the months of May and June, 2005, for health screening (diagnostic examinations). The main purposes of this study were to determine whether any diseases of fish could be detected in amphibians from fish hatcheries and whether hatchery-associated amphibians had any important amphibian diseases that could be transported to new sites when hatchery-raised fish were shipped. The 4 national fish hatcheries, amphibian species and numbers of amphibians are shown above.

Preliminary Results:

This preliminary report includes results of virus cultures, bacterial cultures, external parasite examinations (for ectoparasites) and partial parasitology results. Histological examinations are in progress and will be reported later.

Virus cultures were done on 105 tadpoles. Fathead minnow cell lines were used for all virus cultures in order to increase chances of isolating fish viruses. No viruses were isolated in cultures.

Bacterial cultures were done on 42 tadpoles. Salmonella spp. were not isolated from any tadpoles. Recognized bacterial fish pathogens, such as Yersinia spp, Edwardsiella sp., Flavobacterium spp, Vibrio spp. and Aeromonas salmonicida were not isolated from these tadpoles. Coliform (sewage) bacteria such as Escherichia coli were not isolated from these tadpoles. The bacterium, Aeromonas hydrophila, which may cause secondary or opportunistic infections in fish and amphibians, was isolated from the intestines only (not from livers) of 10 of 21 tadpoles; this bacterium was isolated from the intestines of at least one tadpole from each of the 4 fish hatcheries. Because A. hydrophila was not isolated from the livers of any tadpoles, it is likely the organism was a normal, non-pathogenic inhabitant of the amphibian gut. Aeromonas hydrophila is ubiquitous in wetlands and has been isolated frequently from the intestines of normal-appearing amphibians nationwide.

Parasite examinations were done on the gall bladders of 47 tadpoles and many intestinal nematodes and ectoparasites. Thirty-six (76.6%) of 47 tadpoles had myxozoan parasites in their gall bladders; these myxozoa were identified as Myxidium sp. At least one tadpole from each fish hatchery had myxozoan parasites in their gall bladders. Very little is known about the myxozoan parasites of amphibians; for example, it is unknown whether fish and amphibians share this parasite, and the complex life cycle of all amphibian myxozoans has not been investigated. Illness and death have not been associated with infection by this parasite, but it is important to note that the intermediate life stages and route of infection to the gall bladder remain unknown. Furthermore, it is likely that a second common and presumably innocuous myxozoan parasite of the mesonephroi ("kidneys") of amphibians, Leptotheca ohlmacheri, will be detected in these tadpoles in histological examinations at a later date.

Intestinal parasites in these tadpoles were limited to the common and geographically widespread amphibian pinworm, Gyrinicola batrachiensis. Nine tadpoles from Welaka NFH had pinworms and only one tadpole from Warm Springs NFH had pinworms. It is likely additional small or immature pinworms will be found in these tadpoles in histological examinations. Pinworms are considered innocuous, even in massive infections of the intestine. Cestodes and adult trematodes were not found in these tadpoles, but it is possible these parasites will be found in histological examinations. Immature trematodes (metacercariae) were found at dissections in a few tadpoles (n= 11) from these fish hatcheries; this suggests that few aquatic snails (the first intermediate host for metacercaria that infect amphibians) were present at the hatcheries and sites where the tadpoles were collected and that the final hosts of these parasites (usually fish- and amphibian-eating birds and mammals) also were present. Metacercariae were observed in the skin, muscles, body cavity and mesonephroi ("kidneys") of 11 tadpoles. Because most encysted metacercaria are too small to be seen with the naked eye, it is likely more tadpoles will be found to have metacercaria in histological examinations.

The skin of many tadpoles was examined microscopically with the animal submerged in water in order to detect minute ectoparasites. Protozoa resembling Epistylis and Trichodina were found in many tadpoles, but because these protozoa tend to increase in numbers in captive situations, and because many tadpoles were held alive for up to 10 days before being euthanized and examined, the significance of these protozoa is uncertain. Illness and death in amphibians have not been linked to these two genera of protozoa. However, an uncommon monogean ectoparasite of amphibians was detected in 11 tadpoles from Welaka NFH and 5 tadpoles from Warm Springs NFH. These numbers do not represent infection rates in tadpoles from these 2 hatcheries, because examinations for the parasites usually were halted when 1-3 tadpoles in each group were found to be infested. The monogean trematodes in a few tadpoles were identified by a parasitologist as Gyrodactylus sp. This genus is principally an ectoparasite of fish, but it is infrequently detected in aquatic amphibians in southeastern USA and California. Light or mild infestations, as were found in these tadpoles, probably are innocuous, but heavy infestations can cause skin irritation and may result in secondary or opportunistic bacterial or fungal skin infections. It is unclear why this fish-associated ectoparasite was not detected in tadpoles from Orangeburg and Edenton NFHs.

Oral discs of these tadpoles were carefully examined and the toothrows were sketched. Special attention was given to the black, keratinized portions of the jaw sheaths and toothrows because loss of black pigment may indicate infection by the pathogenic amphibian chytrid fungus, Batrachochytrium dendrobatidis. Other causes for loss of black pigment (depigmentation) are possible, but oral chytridiomycosis is probably the most common cause of depigmentation of the oral disc. A total of 23 tadpoles from 3 of 4 fish hatcheries had partial depigmentation of their oral discs. Histological examinations will be necessary to confirm whether any of these abnormal oral discs were due to infection by Batrachochytrium dendrobatidis.

Discussion & Conclusions:

Viruses. All virus cultures are complete. No viruses were isolated from tadpoles in this study. It is unlikely but possible that viral infections will be detected by histological examinations at a later date. But at present, there is no evidence for common or widespread viral infections in amphibians from southeastern national fish hatcheries.

Bacteria. All bacterial cultures are complete. Important bacterial diseases of fish, such as those caused by Yersinia spp, Vibrio spp, Edwardsiella sp, Streptococcus iniae, and Aeromonas salmonicida were not detected in these tadpoles. In addition, coliform bacteria were not isolated from the intestines and internal organs of the tadpoles. Routine and special bacterial cultures for Salmonella spp. also were negative in all tadpole specimens. Absence of bacteria ("no growth") in the livers of all sampled tadpoles indicates there was no evidence of bacterial septicemias in the amphibians and that appropriate sterile technique was maintained during the dissections of these frequently tiny tadpoles.

Fungi. There was no overt external evidence of watermold infections in the tadpoles, hence, no specimens were submitted for watermold cultures. Cultures for chytrid fungi are not done at our Center. Histological examinations will be completed at a later date and may provide confirmed diagnoses of oral chytridiomycosis in some tadpoles. Other fungal infections also may be detected in histological examinations.

Protozoa.  No specific tests for protozoa were attempted on these tadpoles. While many tadpoles were examined microscopically for ectoparasites while submerged in water, most of the observed protozoa were considered innocuous. About 35% of tadpoles had Epistylis-like protozoa attached to their skin; however, this number probably is the minimum infection rate because once the parasite was detected in 1 to 3 tadpoles of each species from a site, submerged examinations on remaining individuals from the group were not done. About 22% of the 105 tadpoles from Welaka and Warm Springs NFHs had mild numbers of Trichodina-like protozoa on their skin (and in their mouths and spiracles). Again, this protozoa is generally considered innocuous. Additional protozoa, such as intestinal coccidia, may be detected later in histological examinations.

Myxozoa. Perhaps the most unexpected finding in these tadpoles was the high infection rates by the myxozoan parasite, Myxidium sp. in the gall bladders. A total of 36 (76.6%) of 47 tadpoles had myxozoan parasites in their gall bladders. At least one tadpole from each of the 4 fish hatcheries had this parasite. Because so little is known about amphibian myxozoans, the significance of this parasite to the amphibian hosts and the susceptibility of fish at each hatchery are unknown. While the parasite probably is innocuous in amphibians, the effects of heavy or massive infections remain unknown. It is likely a second common and generally innocuous myxozoan parasite, Leptotheca ohlmacheri, will be found in the mesonephroi ("kidneys") of some tadpoles in histological examinations at a later date.

Helminths. Encysted metacercariae were present in various body regions in these tadpoles, and a few had innocuous pinworms in their intestines. It is likely that 3 or 4 different genera or species of metacercaria were found in these tadpoles, since each species usually has a preferred body region or organ. Hence, the metacercaria in the mesonephroi probably are Echinostoma sp. (or Family: Echinostomatidae) while the very tiny metacercaria in the skin of the ventrum, vent tube and hindlimbs probably are Ribeiroia sp. The identity of the metacercaria in the skeletal muscles and body cavity is unknown. It is possible additional helminthic parasites will be detected later in histological examinations.

While all helminthic parasites appeared to be present in sparse or very mild numbers, it should be noted that until recently most metacercariae were considered innocuous. Recent experimental studies with metacercariae of the malformation-inducing parasite, Ribeiroia sp., indicate that as few as 5 metacercaria may kill very young tadpoles.

Ectoparasites. At least 3 types of parasites were found on the skin of these tadpoles. Two were protozoa and were previously discussed. The third ectoparasite was the monogean trematode, Gyrodactylus sp. All infested tadpoles had mild numbers of these parasites and were from Welaka and Warm Springs NFHs. Again, rates of infection in each species of amphibian and each hatchery were not determined, because examinations were halted after 1-3 tadpoles in each group were found to be infested. It is not clear whether these ectoparasites are host-specific for amphibians or are generalists capable of infesting fish and amphibians. Identifications of some preserved monogeans to species may be attempted later. Although pathology reports generally avoid lists of diseases and organisms that were not found, it should be noted that anchorworms (Lernaea sp.) and leeches were not detected in any tadpoles.

Summary

Viruses were not isolated from any tadpoles from the 4 national fish hatcheries. Well known bacterial diseases of fish were not detected in cultures in any tadpoles. Salmonella spp and E. coli were not isolated from any tadpoles. In external examinations under a dissecting microscope, several tadpoles had loss of pigment (depigmentation) on their oral discs; while these abnormalities may have multiple causes, the most common cause is infection by the pathogenic chytrid fungus, Batrachochytrium dendrobatidis. Histological examinations are still in progress and will be necessary to confirm chytridiomycosis. A few ectoparasites were found in tadpoles from Welaka and Warm Springs NFHs; it is possible the monogean trematode, Gyrodactylus sp., represents transmission of a fish parasite to amphibians, but this ectoparasite has been detected in amphibians in other states and may be a fairly common parasite of aquatic amphibians. Although a high rate of infection by the myxozoan parasite, Myxidium sp., was found in the gall bladders of tadpoles from all 4 fish hatcheries, the significance of this parasite to fish and amphibian health is uncertain. Additional parasites and infectious diseases may be detected in these tadpoles in histological examinations that are still in progress. 

     ___________________________
     Pathologist: D. Earl Green, D.V.M.
               Diplomate, Amer. Coll. Vet. Pathol.

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