Pathology in Practice

Catherine G. Lamm Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, OK 74078.

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 DVM, DACVP
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Eileen M. Johnson Department of Veterinary Pathobiology, Center For Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078.

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Stephen K. Smith Department of Veterinary Pathobiology, Center For Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078.

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Sandra E. Morgan Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, OK 74078.

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 DVM, MS, DABVT

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History

A dead adult largemouth bass (Micropterus salmoides), along with several other dead bass and shad (Alosa sapidissima), was found floating in a pond. The pond was 1 of 3 ponds located on a large native grass range used by 800 beef cattle. No fish deaths were detected in the other 2 ponds. On the day the fish were found dead, the ambient air temperature had increased sharply to > 38°C (100°F). The grass range had no areas of shade, and cattle were seen standing in the pond in which the dead fish were found both before and at the time the fish were found dead. The owner had reported 3 stillbirths among the cattle during the preceding week, and 2 of the 3 stillborn calves had been submitted to the Oklahoma Animal Disease Diagnostic Laboratory for postmortem examination. The owner was concerned that the fetal deaths were related to the pond water in some manner given that the dead fish were found floating in the pond in which the cattle were seen standing. The herd manager was uncertain as to the exact number of dead fish in the pond but had noted that at least 2 types of fish were affected. The herd manager submitted 1 bass and samples of water from 3 locations within the pond for postmortem examination and evaluation of water quality, respectively.

Necropsy of 1 of the 2 stillborn calves revealed that it died as a result of trauma to the head. The other calf underwent necropsy; serum samples were assessed for antibodies against Leptospira spp, bovine viral diarrhea virus, Brucella abortus, and bovine herpesvirus 1 and for fluorescent antibody testing (ie, antibodies against bovine viral diarrhea virus and bovine herpesvirus 1), and tissue samples were collected for histologic examination and aerobic bacterial and Campylobacter spp cultures. On the basis of results of these assessments, an infectious cause of the stillbirth of that calf could not be identified.

Clinical and Gross Findings

At necropsy, the adult male bass weighed 1.6 kg (3.5 lb); it was 45 cm long and was in good body condition. Within the coelomic cavity, there were dozens of 1-cm-long, coiled, thin nematodes embedded within the serosa of coelomic organs (Figure 1). Rare, flat, white 3- to 5-cm-long cestodes were also identified free within the coelomic cavity. The intestinal loops were loosely adhered together with fibrin. There were approximately 12 white areas (pinpoint to 2 mm in diameter) within the liver. The appearance of coelomic organs was otherwise considered normal. No gross abnormalities were evident in the gills or brain.

Figure 1—
Figure 1—

Photographs of the coelomic cavity (A) and liver (B) of a dead adult largemouth bass (Micropterus salmoides) that was found floating, along with several other dead bass and shad (Alosa sapidissima), in a pond. In panel A, numerous larvae are present (arrows) in the coelomic cavity. In panel B, notice the multifocal, white areas (pinpoint to 2 mm in diameter [arrows]) within the liver, which extended into the parenchyma on section.

Citation: Journal of the American Veterinary Medical Association 238, 6; 10.2460/javma.238.6.703

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page

Histopathologic Findings

Tissue samples of liver, gills, intestines, heart, and brain were processed routinely for histologic examination. Nematode larvae that were at various stages of development as well as miniature cestodes were embedded within the mesentery and in the wall of the intestine of the fish (Figure 2). Within the wall of the intestine, granulomas composed of necrotic cellular debris with a rim of epithelioid macrophages surrounding small (100 μm in diameter) nematode larvae were detected. There were occasional areas of mineralization within the granulomas. The larger nematode larvae embedded within the mesentery were approximately 500 μm in diameter and had a pseudocoelomic body cavity, a digestive tract, lateral chords, excretory gland cells, and coelomyarian musculature. Reproductive structures were not observed. The nematode larvae within the mesentery were surrounded by necrotic cellular debris, degenerate neutrophils, and large foamy macrophages.

Figure 2—
Figure 2—

Photomicrographs of a section of the muscular wall of the intestine of the fish in Figure 1 in which large anisakid larvae are present within the mesentery (large arrow) and small anisakid larvae are embedded in the wall (large arrowheads; main image) and a cross-sectional view of a large anisakid larva (inset). The cross-sectional view of the larva reveals a distinct body cavity with a digestive tract (small arrowhead), excretory gland cell (EC), and lateral chords (small arrows), but a reproductive tract is not present. H&E stain; in main and inset images, bar = 500 and 250 μm, respectively.

Citation: Journal of the American Veterinary Medical Association 238, 6; 10.2460/javma.238.6.703

The cestodes that were free within the coelomic cavity were 1 mm in width, lacked a body cavity or digestive tract, and had calcareous corpuscles within the parenchyma (Figure 3). There was no inflammation associated with the cestodes. Throughout the liver, there were randomly scattered multifocal granulomas composed of necrotic cellular debris surrounded by epithelioid macrophages. Rarely, parasitic remnants were present within the center of the granulomas.

Figure 3—
Figure 3—

Photomicrograph of a section of the mesentery in the fish in Figure 1. In this view, a cestode is free within the mesentery. The cestode has a tegument (arrowhead), lacks a body cavity and a digestive tract, and has calcareous corpuscles embedded within the parenchyma (asterisk). H&E stain; bar = 100 μm.

Citation: Journal of the American Veterinary Medical Association 238, 6; 10.2460/javma.238.6.703

The other organs examined histologically, including the gills, were considered normal. Routine applications of acid-fast stains were performed on all tissue samples, and acid-fast organisms, such as Mycobacterium spp, were not detected within the sections examined microscopically.

Parasitological and Water Quality Findings

Nematodes were collected from the coelomic cavity of the fish and placed in Beltsville fixative for microscopic examination. The parasites each had 3 lips, an esophageal ventriculus, an anteriorly directed intestinal appendix, and a lack of reproductive organs. On the basis of these findings and the host species, the parasites were identified as anisakid larvae, most likely Contracaecum sp.

The pH of each of the 3 water samples that were collected from the pond in which the fish were found dead was measured by use of a pH meter,a and the values ranged from 7.07 to 7.21. Sulfate concentration was measured by use of a pocket colorimeter,b and values ranged from 11 to 36 mg/L. Total salts concentration and conductivity were measured by use of a conductivity meter.c The total salts concentration in the 3 water samples ranged from 134 to 169 mg/L, and conductivity values ranged from 283 to 351 millisiemens/cm. The 3 water samples were also tested for nitrates and nitrites by use of semiquantitative test strips; no nitrates or nitrites were detected in any sample. Direct microscopic examination of drops from each sample did not reveal any algae within the water. On the basis of these findings, the water quality of all 3 samples was considered acceptable for drinking by cattle. Unfortunately, the temperature and the concentrations of dissolved oxygen in the pond water were not measured on-site at the time of the fish mortality event.

Morphologic Diagnosis

Granulomatous coelomitis, hepatitis, and enteritis with intralesional nematodes and free cestodes within the coelom.

Comments

Parasitism is one of the most common infections in freshwater and marine fish. Parasite infections may be associated with no clinical signs through severe, debilitating disease and death.1,2 The types of parasites, host factors, and environmental conditions influence disease severity in fish. Parasites of fish are widely diverse, including both protozoan and metazoan parasites.1,2 Although nematode and cestode infections are common in fish, these organisms are less likely to be associated with disease in fish, compared with the effects of protozoal or trematode infections.1,2

Contracaecum spp, Anisakis spp, and Porrocaecum spp are nematodes in the family Anisakidae and can infect a wide variety of host species.3 Some species of anisakids can infect people.2,4–6 There are more than 200 known species within the family of Anisakidae, and although morphological descriptions of many of those species have been reported, little is known of the life cycle of the organisms.7 For most species of anisakids, the eggs are passed into the environment from the host during defecation. The first-stage larvae develop within the eggs. Upon release from the eggs, the larvae molt into free-living second-stage larvae within an aquatic environment, where they are eaten and infect the intermediate host. Although information regarding third-stage larvae is limited, researchers suspect that second-stage larvae molt and become third-stage larvae in protostomate invertebrate hosts, such as shrimp, scallops, and adductor mussels.7 Fish can act as both paratenic and intermediate hosts for anisakids, depending on the species of anisakid and species of host. If a fish is a compatible host, third-stage larvae in the intestine can penetrate the intestinal wall and mature within the coelomic cavity or liver. Anisakid larvae can also be found in the muscle, spleen, stomach, reproductive system, and gills of fish. The third- and fourth-stage molts often occur in the stomach of the definitive host (eg, fish, birds, reptiles, and mammals).7,8

The development of granulomas within the coelomic cavity in fish are certainly not specific to a particular disease process, and differential diagnoses include parasitic and bacterial infections.5 The most common cause of disseminated granulomas in fish is infection with Mycobacterium spp.5,9 The 3 most common species of Mycobacterium that infect fish are Mycobacterium marinum, Mycobacterium chelonae, and Mycobacterium fortuitum.9,10 Infection with Mycobacterium spp is typically ruled out histologically by use of acid-fast stains on tissue sections. Other bacterial infections that can induce granulomatous lesions in fish include but are not limited to infection with Pasteurella piscicida, Renibacterium salmoninarium, or Nocardia spp.5,9

Infection with largemouth bass virus (an iridovirus) is a differential diagnosis for large fish die-offs because infection can result in high morbidity and mortality rates in this species.11 On gross examination, affected largemouth bass have peritonitis with small pale foci in the liver and a bright red spleen.11 Histologically, areas of inflammation and necrosis can be observed in the superficial portions of the liver, stomach, intestines, and spleen.11 However, the swim bladder appears to be the target of infection, which results in extensive hemorrhage.5 In the fish of this report, largemouth bass virus infection was unlikely given the lack of consistent gross and histologic findings and the fact that 2 species of fish were affected.

The anisakid larvae and cestodes in the coelomic cavity of the fish were considered incidental findings. The cause of death of the fish was not determined. Evaluation of several other dead fish as well as measurement of the water temperature and dissolved oxygen on-site might have provided useful information, as would evaluation of the environment, water, and fish in each of the other 2 ponds on the property. Given the clinical history, high ambient temperatures and low oxygen tension in the pond where the dead fish were found were suspected to have contributed to the deaths. The stillbirths in the cattle were also attributed to high ambient temperature, lack of shade on the range, and recent exertion related to vaccination of the dams.

a.

Accumet Research AR50 pH meter, Fisher Scientific, Pittsburg, Pa.

b.

HACH pocket colorimeter II, HACH, Loveland, Colo.

c.

ORION conductivity meter, model 115, Thermo Fisher Scientific, Schaumburg, Ill.

References

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