CASE NOTES

---

Pythiosis, ('Swamp Cancer' in horses) - Emerging in Cattle in Australia?

Jocelyn Todd, District Veterinarian, North Coast Local Land Services, South Grafton; Erika Bunker, Veterinary Pathologist, Belinda O'Rourke, Technical Officer and Sophia Callaghan, Plant Pathologist, NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle

Posted Flock & Herd February 2022

Introduction

Pythium spp are common plant pathogens. The organism belongs to the Oomycetes group, most closely related to brown algae, but with a superficial resemblance to fungi. Pythium insidiosum is the only species causing disease in humans and animals (pythiosis). The condition has most frequently been reported in Brazil but occurs worldwide. Horses are most affected, and cutaneous pythiosis (swamp cancer) is the most common form.

In Australia, pythiosis has been reported in horses in several states but not NSW. To our knowledge, this is the first report of pythiosis in cattle in Australia.

History and clinical findings

In February 2021, on a property near Grafton on the North Coast of NSW, after prolonged rainfall, seven of 150 cattle grazing in swamps developed deep circular granulomatous ulcers on the lower legs.

The lesions occurred in the mob of mixed-age home-bred Brahman composite breed cows. The property is located on a coastal flood plain, and these cows regularly graze in swampy paddocks during summer months. Usually, weather conditions allowed them to move in and out of wet areas at will, but weather events during the summer of 2020 - 2021 meant low lying paddocks were much wetter, and for a longer period than usual, as well as being inaccessible for extended periods due to flooding.

Lesions were associated with varying degrees of lameness from mild to non-weight bearing. Inspection and treatment of cattle was made extremely difficult by ongoing wet weather conditions and minor flooding. The owner initially treated with topical Stockholm tar, but then progressed to treating with long-acting oxytetracycline and anti-inflammatories. Treatment was variably successful.

Figure 1. Lower leg lesions, Black Cow. Photograph: Tanya Jacques

Figure 2. Lower leg lesion, White Cow. Photograph: Tanya Jacques

Biopsies from the skin lesions of two cows were submitted for histopathology taken by the attending District Veterinarian and fixed and fresh samples were sent to the laboratory.

Histopathology

H&E-stained sections from formalin-fixed skin biopsy samples from the two cows were examined microscopically. Both cows had a severe chronic ulcerative, granulomatous and eosinophilic dermatitis. In one cow occasional small blood vessels at the base of ulcerated areas had undergone fibrinoid necrosis. Both had extensive proliferation of fibrovascular tissue in the dermis, and extensive infiltration by viable and degenerate polymorphs (eosinophils and neutrophils), lymphocytes, macrophages and multinucleated giant cells, with formation of granulomas. Fragments of hyphae could sometimes be distinguished within giant cells or located centrally in a granuloma and accentuated by Splendore Hoepli-like material.

Figure 3. Skin, dermis. Sparse hyphae fragments accentuated by Splendore Hoepli-like material and surrounded by eosinophils, epitheloid and multinucleated macrophages (H&E stain, 200x). Photograph: Erika Bunker

Based on history and histological lesions, it was considered that pythiosis was the likely cause of the skin lesions in these cows.

As the cell walls of Oomycetes don't contain chitin (unlike fungi), they don't stain with PAS, so GMS stain was selected to further assess the fungal-like elements.

Figure 4. Skin, dermis demonstrating hyphae (GMS stain, 200x). Photograph: Erika Bunker

The appearance of the hyphae was consistent with P. insidiosum, having a diameter of about 5-8 µm, with nearly but not perfectly parallel thick walls, slight dilations and some curving, occasional septa and branching. The hyphae had a tendency to disintegrate. They cannot be distinguished from fungi on histological sections.

Microbiology

Fungal culture of samples from fresh skin lesions was set up. On initial routine aerobic bacterial culture on tissue samples from the leg lesions, various bacteria had previously been cultured that were considered secondary invaders or contaminants. After six days of incubation on Sabouraud Dextrose Agar colonies consistent with Pythium spp were not seen, and it was decided to consult with EMAI Plant Health Diagnostic Laboratory who routinely perform Pythium culture on plant samples, and who provided Pythium-selective media (potato carrot basal medium amended with Pimaricin and Rifampicin) to the Veterinary Microbiology Laboratory. Skin lesion material was incubated on the selective plates at 30 degrees, and growth was detected from day two. On day eight of culture the plates were returned to the Plant Health Laboratory where Pythium-like organisms were identified microscopically and set up in subcultures.

Molecular Biology

Molecular testing of material from the subcultures on pythium-selective media was undertaken by the Plant Health Laboratory and the PCR results were analysed by both Plant Health and Veterinary Molecular Biology teams. DNA consistent with Oomycete was detected by PCR, and further sequencing revealed P. insidiosum as closest match (at least 99.75% sequence identity).

Discussion and literature review

Epidemiological, clinical and laboratory findings in this case are consistent with those described in several reports of cutaneous pythiosis in cattle from South America, particularly Brazil, and fewer reports from cattle in the US.

Lisanka et al. reported cases in cattle in Northeastern Brazil, both from semiarid regions when water accumulated in ponds during the dry season and vegetation around remained green throughout the year, and coastal tropical regions, where cattle grazed around irrigation channels in an area flooded during the rainy season¹. In another report from Southern Brazil calves grazing next to a river with extensive marshy regions were affected². Interestingly, in a case from the Pantanal region of Brazil hyphae were found in dilated hair follicles³; this finding could reflect the attraction of Pythium zoospores to hair as a route of entry into the dermis. In an outbreak in flooded Savanna regions of Venezuela young calves from several farms were more affected than mature animals; in this study several serological assays were used; the ELISA detected 100% of affected animals and also some apparently healthy animals were positive suggesting that animals in enzootic areas can be in contact with P. insidiosum without developing disease⁴.

There is a report of bovine pythiosis in a Red Brangus beef calf from Texas, USA where immunotherapy was tried, and it was suggested that recovery with therapy is more speedy than spontaneous recovery⁵. It must be noted here that, as Pythium spp are not fungi, the response to antimycotic therapeutics is poor.

Pythiosis in sheep (cutaneous form⁶, oronasal form^7,8 and alimentary form⁹) and goats (cutaneous form¹⁰) has only been reported in Brazil. Conidiobolus rhinitis, which has been reported in sheep in NSW¹¹, is an important differential diagnosis for nasal pythiosis¹². Oesophagus, forestomachs and abomasum were affected in sheep with digestive tract pythiosis, with transmural lesions, involvement of peritoneum and occasional vascular fibrinoid necrosis and thrombosis⁹. It is most likely that the digestive tract infection occurred through the consumption of water or plants with infective zoospores. The presence of sheep in the vicinity of water ponds during the hot dry season when forage is not available elsewhere seems to be the main predisposing factor for the occurrence of pythiosis in sheep in the Brazilian semiarid region. Carmo et al. (2021) provide a comprehensive review of pythiosis in sheep and goats¹³. As in cattle, disease occurs sporadically, but in sheep it is more often lethal.

Pythiosis has been reported in various other species including humans and birds, with organ systems in addition to those described here also affected¹⁴. Martins et al. compare histological changes in equine, canine and bovine pythiosis¹⁵.

In Australia, apart from sporadic human cases—for example keratitis in a three-year-old girl from the Northern Territory after swimming in pools¹⁶—pythiosis (cutaneous form, 'swamp cancer') has only been reported in horses, but not in NSW. In Northeast Queensland, various 'phycomycosis' forms in horses from 266 cases from five different labs were studied, the majority caused by Phycomyces destruens (now P. insidiosum). Most were observed between March and July, after the monsoonal wet summer, originating from the wet season between November and May¹⁷.

Epidemiology and life cycle

It is important to note that to date there is no evidence of animal-to-animal or animal-to-human transmission.

Transmission occurs from the environment (water, plants, soil) where the organism is maintained and replicates under favourable conditions (stagnant water with vegetation and warm temperatures).

Contact with zoospores in water (or on wet plant material, or in wet soil) is needed for infection. Zoospores in water are attracted to animal hair, injured skin and plants. When they find a suitable host they secrete sticky material, adhere to animal or plant tissue, germinate, and form hyphae that invade the host. Germ tubes on zoospores settle on hair pointing towards the hair follicle. Hyphae on animal tissue penetrate the dermis and provoke an inflammatory response that varies between species. On host plant tissue colonised by hyphae these develop sporangia that burst and release many zoospores into water. The motile zoospores 'whiz around' in the water for about 20 minutes before settling on animal or plant tissue, perpetuating the cycle. Water and plants are sufficient to maintain the cycle; an animal host is not needed. Zoospores that don't find suitable substrate encyst and form resistant spores to stay in the environment^18,19.

Diagnosis

Pythiosis should be on the list of differential diagnoses in ulcerative skin lesions in cattle but also in sheep—where nasal or alimentary tract lesions may be seen—as a possible emerging disease in areas where it has not been diagnosed before and where the right environmental conditions are present. The skin lesions are usually on the lower legs, however any part of the body that may have prolonged contact with stagnant water, wet plant material, or moist soil could be affected.

It is possible that the disease in cattle is underreported; it could be mistaken for bacterial pododermatitis and, with spontaneous recovery in cattle, lesions may not be investigated by producers as often as they would be in horses.

An aetiological diagnosis is important for management of the condition.

Together with a good history that describes environmental conditions, samples required are fixed and fresh tissues from the skin lesions including dermis, if possible, from more than one animal, and blood if serology is to be used. The best testing protocol is histology in combination with one or several of the following: culture and molecular testing, immunohistochemistry, and/or serology. Availability of immunohistochemistry and serology in Australia may be limited. Culture in the present case was difficult as the organism failed to grow on routine fungal media. Culture on Sabouraud media is generally recommended and usually successful^3,4,14. Cornmeal agar² and potato dextrose agar⁹ have also been used successfully. Failure to grow may be related to the low number of organisms in bovine lesions, disintegration of the hyphae in granulomas, suboptimal storage of the sample (refrigerated instead of room temperature), delayed set up of culture and/or contamination. According to Gastra et al. in their overview of P. insidiosum 'kunkers' from horses are more likely to yield a positive culture result than biopsied tissues¹⁴. Hyphae don't form zoospores in culture media but do so in water culture when inoculated onto grass leaves. Microscopic examination of a wet preparation of lesion material treated with potassium hydroxide is used in horses and could be tried but may not be reliable in cattle as fewer organisms are present. It is sometimes difficult to discern the sparse, sometimes disintegrating, hyphae on H&E stained histological sections, but the lesions are relatively specific and alert the pathologist to look further.

Conclusion

Emergence of the disease in NSW cattle provides some evidence of geographic expansion of P. insidiosum and the disease it causes, which has been observed in other countries. As reports from Brazil and USA show, cases now occur in semiarid areas, in addition to tropical and subtropical areas, and during the dry season, in addition to the rainy season. Changes in environmental conditions may be responsible; climate change leading to extreme weather conditions with floods after heavy rain not only provide ideal conditions but also flush organisms to new areas; manipulation of water systems by humans (irrigation) can have a similar impact¹³.

It appears that disease occurs when either the animals don't have a choice but to graze in flooded areas in wet times, or they choose to do so because the only green feed available is in swampy areas in dry times.

All cattle at the Grafton property eventually recovered (over 3-4 months), although some have ongoing mild lameness and joint inflammation due to suspected secondary bacterial infection with joint involvement. They remain mobile enough to maintain good body condition and breed successfully.

This report intends to increase awareness of pythiosis as a potential emerging disease in ruminants in Australia, highlight the environmental conditions under which it can be expected and list the diagnostic tools available to make a definitive diagnosis.

Acknowledgements

We wish to thank the Histology Lab at EMAI for supplying histological sections and special stains, EMAI Plant Health, Microbiology and Molecular Biology Labs for performing selective culture and molecular testing, the EMAI Veterinary Pathology team for their support preparing the report.

References

1. Lisanka AM, Souto EPF, Frade MTS, Pimentel LA, Azevedo EO, Kommers GD, Riet-Correa F, Dantas AFM. Pythiosis in cattle in Northeastern Brazil. Pesq. Vet. Bras. 2020; 40 (5): 340-345
2. Konradt G et al. Cutaneous Pythiosis in calves: An epidemiologic, pathologic, serologic and molecular characterization. Medical Mycology Case Reports 2016; 14: 24-26
3. Santurio JM et al. Cutaneous Pythiosis insidiosum in calves from the Pantanal region of Brazil. Mycopathologia 1998; 141: 123-125
4. Perez RC et al. Epizootic cutaneous pythiosis in beef calves. Veterinary Microbiology 2005; 109: 121-128
5. Grant D et al. Cutaneous pythiosis in a Red Brangus beef calf cured by immunotherapy. Medical Mycology Case Reports 2016; 14: 1-3
6. Tabosa IM et al. Outbreak of Pythiosis in Two Flocks of Sheep in Northeastern Brazil. Veterinary Pathol. 2004; 41: 412-415
7. Bernardo FD et al. Pythiosis in sheep from Parana, Southern Brazil. Pesq. Vet. Bras. 2015; 35 (6): 513-517
8. Santurio JM et al. Granulomatous rhinitis associated with Pythium insidiosum infection in sheep. The Veterinary Record 2008; 163: 276-277
9. Riet-Correa F et al. Pythiosis of the digestive tract in sheep. Journal of Veterinary Diagnostic Investigations 2012; 24 (6): 1133-1136
10. Carmo PMS et al. Cutaneous Pythiosis in a Goat. Journal of Comparative Pathology 2015; 152: 103-105
11. Carrigan MJ et al. Ovine nasal Zygomycosis caused by Conidiobolus incongruous. Australian Veterinary Journal 1992; 69 (10): 237-240
12. Ubiali DG et al. Pathology of Nasal Infection caused by Conidiobolus lamprauges and Pythium insidiosum in Sheep. Journal of Comparative Pathology 2013; 149: 137-145
13. Carmo PMS et al. Diseases caused by Pythium insidiosum in sheep and goats: a review. J. Diagn. Vet. Invest. 2021; 33 (1): 20-24
14. Gaastra W et al.. Pythium insidiosum: An overview. Veterinary Microbiology 2010; 146: 1-16
15. Martins TB et al. A Comparative Study of the Histopathology and Immunohistochemistry of Pythiosis in Horses, Dogs and Cattle. Journal of Comparative Pathology 2012; 146: 122-131
16. Badenoch P et al. Pythium insidiosum keratitis in an Australian child. Clinical and Experimental Ophthalmology 2009, 37 (8): 806-809
17. Miller RI et al. Clinical Observations on Equine Phycomycosis. Australian Veterinary Journal 1982; 58 (June): 221-226
18. Mendoza L et al. Life cycle of the Human and Animal Oomycete Pathogen Pythium insidiosum. J Clin Microb 1993; 31 (11): 2967-2973
19. Mendoza L. Pythium insidiosum. 2016. www.researchgate.net Accessed online 15 June 2021