In February 2016, a group of 155 Eastern Dwarf Tree Frogs (Litoria fallax) experienced a mass mortality event over the course of one week, from which only 35-45 individuals survived. These wild frogs were collected 48 hours before the first mortality was recorded from Kooragang Island, Hunter River, Newcastle, NSW. Deaths occurred over a period of one week from all nine holding tanks. Generally the frogs were asymptomatic, with some animals exhibiting a slight redness of the abdomen prior to death. Dorsal and oral swabs were taken from each animal (live and dead), and water and faecal samples were taken from each tank.
Two carcasses (TARZ-11050.1, TARZ11050.2) were examined by a local veterinarian before being forwarded to The Australian Registry of Wildlife Health for histology. A third frog (TARZ-11050.3) was transported live to The Registry for examination prior to euthanasia and sample collection. Samples from an additional five animals were sent to Associate Professor David Phalen, University of Sydney.
This is a summary of the findings.
TARZ-11050.3: No external lesions. Hydration was good, muscle mass fair, but fat deposits were absent, and the frog looked thin. Internally, no coelomic fat pads were evident. Blood pooling in the coelomic cavity after the apex of the heart was cut appeared thin and watery. The gastrointestinal tract was devoid of ingesta. There were miliary white foci throughout the parenchyma of the testes (see image).
The histopathology is similar for all three animals.
Throughout each tissue examined there are large numbers of round to elongate protozoa that have a defined nucleus and kinetoplast (a network of circular DNA inside a large mitochondrion that contains many copies of the parasite’s mitochondrial genome). The elongate organisms often have what appears to be an undulating membrane. In some organisms a discrete flagellum is evident. Multifocally in the subcutaneous tissues there appear to be more round forms of these organisms, but there are also scattered flagellate forms. Reticuloendothelial cells within the bone marrow contain cytoplasmic clusters of large round protozoa that have a visible nucleus and often a kinetoplast. These structures are amastigotes, the life cycle phase without flagellae. Similar clusters of what appear to be amastigotes are present in the sinusoids of the liver and myocardial interstitium.
The number of flagellated organisms in the cardiac chambers, coronary vessels and epicardium is astounding.
FIGURE 1: Bone Marrow TARZ-11050.1 (H&E) M: Monocyte containing numerous amastigotes, the intracellular stage of the trypanosome spp. A: Cluster of amastigotes within the bone marrow.
FIGURE 2: TARZ-11050.2 Multiple flagellated organisms within the myocardium (H&E)
FIGURE 3: TARZ-11050.1 Numerous epimastigotes and trypomastigotes within myocardium and circulating blood (H&E)
Large numbers of organisms are also evident in the perineurium, meninges and periosteum (TARZ-11050.2 and .3), but additional lesions are not evident within the bone/cartilage, liver, lung, heart, brain, eye, liver or pancreas.
The epidermis is extensively coated with bacterial colonies that penetrate into the subcutaneous tissues and muscle.
There is a focal cluster of what appear to be dilated renal tubules containing cross sections of helminth parasites that have no cuticle, no pseudocoelom and no obvious gastrointestinal tract. The organisms are surrounded by a thin layer of amorphous material and a thin layer of desquamated cells. In cases TARZ-11050.2 and .3, rare refractile, oval structures, as described in the bone marrow are evident within the renal interstitium.
Splenic tissue is difficult to distinguish due to the prevalence of large reticuloendothelial cells. Many of these cells contain individual or multiple round protozoa with an eccentric nucleus and visible kinetoplast. The tissue also contains scattered cells with pyknotic or karyorrhectic nuclei.
The small intestine of TARZ-11050.2 and .3, and the large intestine of all three cases contain many holociliate parasites.
The lacrimal glands contain rare structures that resemble protozoa. In TARZ-11050.1, there is a small oval body containing numerous elliptical zoites, consistent with a coccidian parasite. In TARZ-11050.2 and .3, the mucosa contains multifocal coccidial gametocytes, and rare oocysts are evident within epithelium.
FIGURE 4: Lacrimal gland containing a coccidial oocyst (TARZ-11050.2)(H&E)
Approximately 60-70% of the testicular volume is replaced with thin walled cysts containing clusters of small oval protozoa. Organisms range from basophilic, to transparent, and refractile. Multiple oval polar bodies are evident within most organisms. Spermatogenesis is prevalent within the remnant spermatic cords. A testicular impression smear was collected from TARZ-11050.3. It is highly cellular, with large numbers of spermatids. Many oval, refractile protozoa containing pyriform polar bodies are evident throughout the smear.
FIGURE 5: Replacement of normal testicular tissue with refractile Myxobolus-like organisms (TARZ-11050.1)(H&E)
FIGURE 6: Testis - multiple oval polar bodies are evident within most organisms, some of which are highly refractile (TARZ-11050.1)(H&E)
TARZ-11050.1: The gastric lumen contains a small number of round structures that appear similar to coccidial oocysts. Cellular debris is prevalent throughout the mucosa, but no coccidial organisms are evident. A cross section of a nematode (cuticle, pseudocoelom, gastrointestinal tract and lateral alae) can be seen in the intestine.
TARZ-11050.1: Multifocal intra-vascular mononuclear cells are filled with cytoplasmic bacteria. This seems most obvious in the renal vasculature. Within skeletal muscle, multifocal myocytes have fragmented myofibrils and contraction band formation. Large numbers of organisms are evident within capillaries and within the endomysium.
TARZ-11050.2 and .3: The gall bladder contains a poorly cellular amorphous organism that lacks a cuticle and pseudocoelom, and has no gastrointestinal tract. The structure may contain calcareous corpuscles and also multiple transparent oval structures with bipolar round bodies. These structures also have an annular undulation of the central zone of the capsule.
FIGURE 7: Gall bladder FROM TARZ-11050.2 showing two myxosporidia-like organisms.
Trypanosomiasis - multisystemic, severe, chronic
Cutaneous bacterial invasion
Testicular myxozoa - multifocally extensive, severe, chronic - presumptive Myxobolus sp.
Renal myxozoa (TARZ-11050.2 and .3) - multifocally extensive, severe, chronic - presumptive Myxobolus sp.
Renal helminths - focal, mild, chronic
Lacrimal gland coccidiosis, suspect (TARZ-11050.1)
Gall bladder parasite (TARZ-11050.2, TARZ 11050.3) - focal, subacute
Septicaemia – acute (TARZ-11050.1)
Myodegeneration – acute (TARZ-11050.1)
Intestinal nematodiasis - mild, subacute (TARZ-11050.1)
Suspected anaemia (TARZ-11050.3)
Trypanosomes are not uncommon in amphibians from Queensland. However they have not been identified in amphibians from New South Wales examined through the Registry.
Overseas, T. rucosae, T. hosei, T. ambystomae, are reported in amphibians. T. inopinatum has been reported in amphibians in association with haemorrhage, swelling, anaemia and death associated with damage to the reticuloendothelial system (Brumpt 1924, Bardsley & Harmsen 1973). T. rotatorium can be pathogenic in tadpoles or in heavy infections, with many organisms visible in the kidneys (Bardsley and Hamsen 1973). T. pipientis has been associated with splenic enlargement, but rarely associated death (Flynn, 1973). Given the unusual nature of these organisms in NSW and the intensity of infection in these animals, characterisation is warranted. Fresh or ethanol fixed tissues could be used.
TARZ-11050.1 appears to have died with extensive an external coating of bacteria which have invaded the subcutaneous tissues. The presence of bacteria within the cytoplasm of macrophages supports the diagnosis of septicaemia. In the five animals examined at the University of Sydney, Dr Phalen also noted a massive bacterial infection of the skin, muscle and subcutaneous tissues, as well as enteritis. He considered it consistent with the pure cultures of Aeromonas sp. cultured from other frogs, which supports a diagnosis of bacterial septicaemia. If fresh tissues from animals can be located, bacterial culture might help to determine whether the amphibians were infected by one probable primary pathogen, or by a variety of opportunistic invaders.
All frogs have severe, multisystemic Trypanosome sp. infections. The organisms consume a considerable portion of the volume of many capillaries and the cardiac chambers. The severity of the infection seems suggestive that it may have played a role in the mortality event. Perhaps the combined effects of capture, captive housing, systemic bacterial infection and this severe underlying infection worked collectively to incite the mass mortality event.
The frogs also had considerable testicular myxosporidian parasites, most likely Myxobolus sp. These organisms were probably an incidental finding, as they are not uncommon in some amphibian populations. The parasites consumed a considerable portion of the testicular parenchyma and may have decreased overall spermatogenic output. Normal spermatogenesis was evident in the remnant tissue, so the impact of infection on reproductive fitness is uncertain.
The renal helminth infections appear to be a localised and incidental finding.
Degeneration of skeletal muscle (TARZ-11050.1) in amphibians is a common finding. This change can occur post mortem as a result of fixation artefact rather than as the result of a capture myopathy.
Interestingly TARZ-11050.2 and TARZ-11050.3 seemed to have a very mild, incidental infection of a periocular gland with coccidial organisms. Perhaps the coccidial structure seen in TARZ-11050.1 also originated from this location. Extra-intestinal coccidial infections are uncommon, but have been reported in several species.
TARZ-11050.2 and TARZ-11050.3 also had a structure in the gall bladder that looked like a cestode, but may have been a large n plasmodium. Within the parenchyma of the organism there were transparent oval structures with bipolar round bodies, and there were annular undulations of the capsule, consistent with myxozoan parasites.