First Report of Madurella Mycetomi Isolate That Induced Systemic Pulmonary and Myocardium Mycosis with Verminious Enteritis in Dugong Dugon, of Mersing, Johore

MALAYSIAN JOURNAL OF VETERINARY RESEARCH pages 30-39 • Volume 9 No. 2 July 2018

FIRST REPORT OF MADURELLA MYCETOMI ISOLATE THAT INDUCED SYSTEMIC PULMONARY AND MYOCARDIUM MYCOSIS WITH VERMINIOUS ENTERITIS IN DUGONG DUGON, OF MERSING, JOHORE

NORINA.L1*, AIDA M.1, ZAIDIN A.1, SYED ABDULLAH S.A.K.2, TAMIMI M.A.A.2, SAROL K.3, HANAFI H.3, NOORDIN M.M.4 AND MAZLINA M.4 1 Department of Veterinary Services State of Johor, Aras 4, Bangunan Dato’ Mohamad Ibrahim Munsyi 79630 Kota Iskandar, Nusajaya, Johor 2 Fishery Research Institute (FRI) Rantau Abang,Turtle and Marine Endangered Species (TUMEC) Research, Jabatan Perikanan Malaysia, 23050 Dungun Terengganu 3 Regional Veterinary Laboratory Johor Bahru,2Department of Veterinary Services, Lot PTB 11098, Jalan Taruka, Off Jalan Datin Halimah, 80350 Johor Bahru, Malaysia. 4 Faculty of Veterinary Medicine, University Putra Malaysia, Serdang, Selangor, Malaysia * Corresponding author: [email protected]

ABSTRACT. The major causes of the decline have the presence of big mast cells which of the Dugong population along the urban formed capsules, indicating presence of coast of Mersing, Johor includes gill netting, fungal spores causing systemic mycoses subsistence hunting, habitat loss from where the macrophages invade and engulf extreme weather events that are likely to the spores. The shrinkage of myocardium be exacerbated by climate change, human myocyte and myocardium necrosis with mild settlement, breach of the bots and sea water vasculitis indicates heart failure. Groucott’s pollution. In March 2017, a male Dugong stain confirmed Madurella mycetomi dugon, estimated age of 10 to 20 years, was infection that induced systemic pulmonary incidentally found dead near Pulau Tinggi, and myocardium mycosis in Dugong dugon. Mersing, Johor by a fisherman. The entire Keywords: Dugong dugon, Paradujardinia body was found to have old and new scars halicoris, Madurella mycetomi, zoonotic, Mersing, at the anterior part of the abdomen. The Johor right eye was protruded out and bleeding which indicated that the eye was pierced INTRODUCTION by a sharp object. Necropsy revealed the upper small intestine and the stomach Dugongs, also known as sea cows, were compartments were semi-impacted with a considered rare in Malaysia until a dugong massive helminth burden (more than 1,000 stranding incident was reported in nematodes). Paradujardinia halicoris worms Johor, Peninsular Malaysia, in 1999 which were identified based on morphological generated significant media and public characteristics. Zoonotic fungus named interest (Marsh et al., 2002; Rajamani et al., Madurella mycetomi were isolated from 2006). This incident created the awareness heart and lung after incubation for 14 days. on the status of the dugong in Malaysia Histologically, the lung was confirmed to which led to renewed interest in conducting

30 Volume 9 No. 2 July 2018 MALAYSIAN JOURNAL OF VETERINARY RESEARCH research on Dugong, especially with respect In Malaysia, along its 4,800 km to post-mortem and disease findings from coastline, stretching along the Malay stranded dugongs. Dugongs can be found Peninsula, Sabah and Sarawak, which in the shallow waters of shoals, reefs, sand consists of the southern part of the South flats, and seagrass beds throughout Johor, China Sea, are environments with coastal Sabah and Sarawak. The Islands of Mersing habitats — mangroves, coral reefs and – Pulau Tinggi, Pulau Besar and Pulau Sibu seagrasses. There are 78 seagrass beds with lush vegetation, golden beaches and scattered throughout the Peninsular and East rich underwater flora and fauna. Their crystal Malaysia. Dugong dugon (status: vulnerable) clear waters are known to house the only and Chelonia mydas (green turtle, status: habitat for the endangered dugong. Mersing endangered) are found to be associated and waters, Tanjung Leman and the eastern both species feed on seagrasses. Dugongs islands off Mersing (Pulau Tinggi, Pulau Besar became rare because they were hunted for and Pulau Sibu) showed that Pulau Sibu and meat and hide (Holttum, 1954). Presently, Pulau Tinggi have the highest concentration dugongs are found in areas with abundant of Dugong. According to statistics provided seagrasses such as Pulau Sibu, Pulau by the Department of Fisheries Malaysia, Tengah, Pulau Besar and Pulau Tinggi on the dugong population in waters off Johor the east coast and around Tanjung Adang- was estimated at 50 heads, while Sabah Merambong shoals of Sungai Pulai, Johore and Sarawak have about 50. The population (Japar Sidik and Muta Harah, 2002). Seagrasss of dugong in Malaysia is threatened with species with low fibre and high nitrogen, and extinction, taking into account the average are easy to digest, seem to be the favourite death rate every year of between three to food of dugongs. Two species of seaweed five per year. favoured are Halophila ovalis (Figure 1) and Halodule uninervis (Figure 2).

Figure 1. Halophila ovalis (rumput dayung) Figure 2. Halodule uninervis

31 MALAYSIAN JOURNAL OF VETERINARY RESEARCH Volume 9 No. 2 July 2018

However, the ecosystem for seaweed with black-grain mycetomas (Cortez K.J. et located along the beach, which is shallow al., 2008; Ahmed A. et al., 2002). and protected, face pollution and Thus, the aim of the study was to destruction as a result of reclamation, rapid report the ﬁndings of Madurella mycetomatis development and trawling, thus, a risk to the infection which infected the myocardium survival of dugong. Many studies showing and systemic pulmonary tissues of Dugong a decrease in population of dugongs in an dugon and its zoonotic implications. In area, can be attributed to damage of habitat carrying out this investigation, the common especially seaweed depletion. In addition, infections found in wild dugong is elucidated dugongs are also at risk of being trapped adding to current information on the causes in fishing equipment like drift nets used by of death in the dugong community which fisherman, breach of the boats and sea water will eventually help to conserve the dugong pollution. population by effective mitigation. Mycetoma has a worldwide distribution but is restricted to specific climate zones. MATERIALS AND METHOD Mycetomas are chronic, granulomatous, subcutaneous infections caused by In April 2017 at 5.30pm, a male Dugong either eumycetes fungi or actinomycetes dugon, estimated to be between 10 to 20 bacteria, giving rise to eumycetomas and years of age, 1.3 m length, and weighing actinomycetomas, respectively. The disease 150 kg, was found dead near Pulau Tinggi, is endemic in many tropical countries, and Mersing, Johor by a fisherman. The Dugong is characterised by slow progression with dugon was declared as an endangered risks of bone and visceral involvement. species by the International Union for The organisms are present in the soil, plant Conservation of Nature (IUCN) (2010). The material, polluted aquatic environments, carcass was brought to the jetty of the salt water and air (Ahmed et al., 2004) Fisheries Development Authority of Malaysia and may enter the subcutaneous tissue by (Lembaga Kemajuan Ikan Malaysia, LKIM), traumatic inoculation or by a thorn prick and the cosmetic necropsy was done in-situ. (Ahmed et al., 2004) or other lesions of the Cosmetic post-mortem was done, whereby skin. The microorganisms can be identified the internal organs such as liver, lung, spleen, by fungal culture using media such as heart, stomach and intestine were removed Sabourad’s agar, Brain Heart Infusion agar but the outer body parts were maintained. and Malt extract agar (Ahmed et al., 2004). A cosmetic post-mortem allows full access Tropical eumycetoma is frequently caused to all tissues required by the pathologist, by the fungus Madurella mycetomatis. There but does not take significantly more time to are at least two dozen species of fungi- carry out and allows for the preservation of causing eumycetoma throughout the world, skin and skeleton for museum based studies but the most prevalent causative specie or preservation for artistic purposes. The (approximately 70% of reported cases) is internal cavity of the carcass was cleaned Madurella mycetomatis, which is associated with water and 10 rolls of cotton were

32 Volume 9 No. 2 July 2018 MALAYSIAN JOURNAL OF VETERINARY RESEARCH placed in the thorax and abdominal region which indicated that the eye was pierced by and the incision was sewed up using catgut a sharp object (Figure 5). Upon observation strings sized 4/0. Approximately 1,500 ml of the thoracic and abdominal cavity filled of 10% formalin were injected in-between with serous fluid. The animal had a large the stitches until it reached the cotton for amount of brown, serosanguinous fluid and fixation. Then, the carcass was brought a moderate amount of fibrin in its peritoneal back to the Fishery Research Institute (FRI) cavity. There were multifocal paintbrush in Rantau Abang, Terengganu for further and ecchymotic haemorrhages on the fixation. Examination of lesion on fresh lung, visceral and parietal pleura. The skeletal liver, and heart were done and samples were musculature was oedematous. All internal taken and sent for bacteriology and fungus organs were congested while spleen and isolation; and also were fixed in 10% buffered kidney were autolysed. Congested trachea, formalin for histopathology study. They were severe pulmonary congestion and hepatic processed according to routine laboratory congestion were observed. The dugong’s procedures. A large quantity of nematode upper small intestine and the stomach worms were found in the stomach and compartments were semi-impacted with a anterior part of its duodenum and jejunum massive helminth burden (more than 1,000 of the intestine. The worms were preserved nematodes) (Figure 6). Cause of death was uncompressed in 70% GL ethanol and not determined but was possibly associated were sent for parasite identification. All the with the parasite burden of Paradujardinia samples were transported to the Regional halicoris. Veterinary Laboratory, Johor Baharu, for further examination. Laboratory Finding

RESULTS AND DISCUSSION Parasitology

Details of results from the investigation Paradujardinia halicoris worms were are shown as in figures below identified using stereomicroscope to differentiate between the male and female. Necropsy finding The worm has three lips with interlabia, intestinal caecum and ventriculus (Figure In general, the carcass was in good 7 and Figure 8). The male worm showed condition. Gross observation showed that presence of two spicules alae in the middle entire body had presence of old scars at the shaft (Figure 10) while the female showed anterior part of the abdomen and also the presence of vulva (Figure 9). presence of ocular and oral bleeding (Figure 3 and Figure 4). A new cut was found on Bacteriology and Fungus the left abdomen and around the mouth region. The right eye protruded out and Pasteurella haemolytica var ureae was isolated bleeding around the eye region was noted, from lung, liver and spleen, Bordetella

33 MALAYSIAN JOURNAL OF VETERINARY RESEARCH Volume 9 No. 2 July 2018

Figure 3. Carcass with the presence of Figure 4. Presence of old and new cuts on ocular and oral bleeding. the left abdomen.

Figure 5. Protruding right eye and bleeding Figure 6. Presence of high burden of around the eye region suspected of being nematodes in the stomach and intestine. pierced by a sharp object and noted presence of cut around mouth region.

Figure 7. The anterior part of Paradujardinia Figure 8. The mouth part of Paradujardinia halicoris. halicoris with presence of three lips, interlabia, intestinal caecum and ventriculus.

34 Volume 9 No. 2 July 2018 MALAYSIAN JOURNAL OF VETERINARY RESEARCH

vulva

spicule

Figure 9. Posterior end of Paradujardinia Figure 10. Posterior end of Paradujardinia halicoris showing vulva region indicating halicoris showing spicule region indicating female worm. male worm.

Figure 11. Wet mount from culture stained with lactophenol cotton blue (LCB) (×10 magification) showing brown pigmented septate hyphae with intercalary chlamydospores (arrow) characteristic of Madurella mycetomi. bronchiseptica from lung and Madurella pigmented septate hyphae with intercalary mycetomi fungus from heart and lung. The chlamydospores characteristic of Madurella culture of discharged granules from heart mycetomi and lung in Sabouraud’s dextrose agar (SDA) grew Madurella mycetomi after incubation Histology for 14 days. Confirmation the SDA culture by Wet mount stained with Lactophenol Cotton Microscopic examination on lung tissue Blue (Figure 11) with presence of brown revealed fibrous tissues and inflammation

35 MALAYSIAN JOURNAL OF VETERINARY RESEARCH Volume 9 No. 2 July 2018

Figure 12. Mast cell with surrounding Figure 13. Fig.13: Pulmonary mycosis with cartilage indicating fungus (systematic presence of black spores indicating fungal mycotic) [HE×10]. infection [Groucott’s stain ×20].

Figure 14. Macrophage engulfing fungal Figure 15. Myocardium with black spores spores in the lung [×40 HE]. indicating fungal infection [Groucott’s stain ×20] in bronchioles. Presence of big mast cells which may have caused some loss in detail which form capsules, indicate presence in tissues due to autolysis. The shrinkage fungal spores which cause systemic mycoses of myocardium myocyte a mild degree (Figure 12 and Figure 13). Blood vessels of myocardium necrosis was also seen. were surrounded by inflammatory cells Myocardium mycosis was present with black which line the vessel but no evidence of spores fungal infection after staining with vasculitis. Some macrophages are seen to Groucott’s (Figure 15). Hepatic infiltration of invade and engulf the spores (Figure 14). neutrophils and inflammatory cells between Presence of mild vasculitis and necrosis was sinusoids was observed. Piknotic nucleus also noted which indicates heart failure. and hepatocytes became disarrangement Furthermore, the case was attended to more and some area of hepatocytes were seen than 24 hours after discovering the dugong,

36 Volume 9 No. 2 July 2018 MALAYSIAN JOURNAL OF VETERINARY RESEARCH to be hollow or empty which indicates activities. It is likely that human activities autolysis. have contributed to the substantial decline in dugong numbers along the urban coast of CONCLUSION Mersing especially in the Pulau Sibu-Tinggi area. Human activities, such as trawling The dugong habitat did not fall within the and dredging and water pollution, have a protected zones of the Johor state marine negative effect on sea-grass ecosystems, as parks and the Sultan Iskandar Marine Park have natural events, such as cyclones and (TLSI). According to the research by MareCet flooding (Heinsohn and Spain, 1974; Marsh since 2010, the female dugong and their et al., 2000; Greenland and Limpus, 2007). calves in the Pulau Sibu-Tinggi area are all Several dugong mortalities each year occur outside the protected zone. Johor has 13 because of boat strikes and netting which marine parks, including the Pulau Besar, is in agreement with previously published Pulau Tinggi and Pulau Sibu Marine Parks data (Greenland and Limpus, 2007), human (Anon,2010). The TLSI which was opened in activities were identified as a significant July 2017, is operated by the Johor National cause of dugong mortality Parks Corporation. TLSI covers a zone of up Little is known about Dugong‘s to three nautical miles as it is the norm with common disease processes and causes of other marine parks. Fishing and all kinds of natural mortality. The carcass was found activities that damage the marine biological to have a massive helminth (Paradujardina ecosystem are prohibited at all the marine halicoris) burden in its stomach. Little is parks concerned. The survey also showed known about the pathogenicity of helminth that Johor’s dugong and sea grass habitats parasites in sirenians (Bossart, 2001), and were situated outside the protected zones Paradujardina halicoris were often found of its marine parks, thus the state authorities in large numbers in dugong stomachs, have decided to establish a sanctuary to including those in apparently good body protect their natural terrain. condition. The burden of worms may have As seagrass is the main source of caused physical effects on gastric function, nourishment for dugong, protecting the or less likely, perhaps, parasite-induced underwater seagrass beds is of paramount appetite suppression, similar to that which importance to ensure the continued survival occurs with some intestinal parasites in of these creatures. Seagrass ecosystems domesticated animals (Dynes et al., 1998). are very sensitive and are readily destroyed Eumycetoma is a subcutaneous fungal by anything that increases turbidity and infection in which the etiological agent deprives them of light or causes nutrient occurs in the form of more or less compact enrichment and increased epiphytic growth mycelial grains Tropical eumycetoma is Dugongs’ dependence on seagrasses mostly frequently caused by the fungus Madurella restricts them to shallow, nearshore waters, mycetomatis. The disease is characterised by habitats that are increasingly being occupied extensive subcutaneous masses, usually by humans and influenced by human with sinuses draining pus, blood, and

37 MALAYSIAN JOURNAL OF VETERINARY RESEARCH Volume 9 No. 2 July 2018 fungal grains. Madurella mycetomi (black myocardium necrosis with black spores grain mycetoma) is the most common indicate fungal infection after staining with eumycetoma and worldwide, followed by Groucott’s. Scedosporium apiosperma, Scedosporium Unfortunately, there has been prolificans, and Madurella grisea (McGinnis no previous reported case of Madurella and Fader, 1988). It is a slow growing fungal mycetomatis in marine animal especially infection, which initially involves the skin in Dugong dugon. This is the first case of and subcutaneous tissues and progresses to Madurella mycetomatis infection which leads to involve deeper structures. Upon examination systemic pulmonary and myocardium mycosis it was found that the surface of the carcass with verminious Paradujardinia halicoris was present with the old and new scars infestation in Dugong. indicating that the dugong was injured In conclusion, the post-mortem found previously with sharp objects, without any possible diseases that could infest wild wound treatment. A possible pathogeneses dugong and it gives an indication in future may include direct introduction of an for pathologists and veterinarians on what infectious agent through a penetrating to look for in cases of dead dugongs. The wound or the hematogenous spread of findings of fungal and parasitic infestations infection from some other focus. The wound are important and significant findings in progressively healed naturally probably by future conservation work that needs to being traumatically inoculated with plant be carried out. These efforts will help to materials or soil contaminated by these fungi. maintain the dugong population in the The infection is classically characterized coastal waters of Malaysia. by the presence of subcutaneous nodules, increasing in size, which may suppurate REFERENCES and drain a serous, serosanguineous or purulent discharge through multiple sinus 1. Ahmed A., Adelmann D. and Fahal A., Verbrugh H., van Belkum A. and de Hoog S. (2002). Environmental tracts (Ahmed et al., 2004). Although the occurrence of Madurella mycetomatis, the major agent disease incubation time remains unclear, of human eumycetoma in Sudan, J Clin Microbiol, 40(3): 1031-1036. symptoms may appear several months to 2. Ahmed A.O., van Leeuwen W. and Fahal A., van de years’ post infection (Ahmed et al., 2004). Sande W., Verbrugh H. and van Belkum A. (2004). Microscopically, the lung revealed fibrous Mycetoma caused by Madurella mycetomatis: a neglected infectious burden, Lancet Infect Dis, 4(9): tissues and inflammation in bronchioles. 566-574. There was also presence of big mast cells 3. Anonymous (2010). Sanctuary plans for sea cows in Johor. In: PressReader–The Borneo Post (Sabah). https:// which formed capsules, indicating presence www.pressreader.com/malaysia/.../281818578623653. of fungal spores causing systemic mycoses Accessed 7 Mac 2017 where the macrophages invade and engulf 4. Bossart G. (2001). Manatees. In: CRC Handbook of Marine Mammal Medicine. 2nd Edition, Dierauf L. and Gulland the spores. Presence of mild vasculitis and F. (eds.). CRC Press LLC, Boca Raton, Florida, pp. 939– no bend necrosis indicates the heart failure 960. 5. Cortez K.J., Roilides E. and Quiroz-Telles F. et al. (2008). was evident. The shrinkage of myocardium Infections cause by Scedosporium spp, ClinMicrobiol Rev, myocytes and presence of mild degree of 21(1): 157-197.

38 Volume 9 No. 2 July 2018 MALAYSIAN JOURNAL OF VETERINARY RESEARCH

6. Dynes R., Poppi D., Barrell G. and Sykes A. (1998). 12. Marsh H., Penrose H., Eros C., Hughes J. (2002). Dugong Elevation of feed intake in parasite-infected lambs by status reports and action plans for countries and central administration of a cholecystokinin receptor territories. UNEP early warning and assessment report antagonist. British Journal of Nutrition, 79: 47-54. series. IUCN Publication. 162 pp. 7. Greenland J. and Limpus C. (2007). I: Dugong In: Marine 13. Marsh H., Eros C., and Webb R. (2000). Dugongs in wildlife stranding and mortality database annual report health and disease. In: Proceedings 335, Proceedings 2007, Queensland Environmental Protection Agency (University of Sydney Postgraduate Foundation in Conservation technical and data report, Brisbane, Veterinary Science, Marine Wildlife: The Fabian Fay Course Queensland, Australia, 23 pp. of Veterinarians, Sydney, Australia, pp. 301-317 8. Heinsohn G. and Spain A. (1974). Effects of a tropical 14. McGinnis M.R., Fader R.C. (1988). Mycetoma: a cyclone on littoral and sub-littoral biotic communities contemporary concept. Infect Dis Clin North Am. 2: 939- and on a population of dugongs (Dugong dugon 954. (Müller)). Biological Conservation 6: 143-152. 15. Rajamani L., Cabanban A.S., Ridzwan A.R. (2006). 9. Holttum R.E. (1954). Plant life in Malaya. Longman, Indigenous use and trade of dugong (Dugong dugon) London. in Sabah, Malaysia. Ambio 35: 266-268. 10. International Union For Conservation Of Nature (IUCN) (2010). IUCN Red List of Threatened Species. Version ACKNOWLEDGMENTS. We acknowledge pathologists, 2010.2. www.iucnredlist.org. Accessed July 2010. veterinarians, and staffs from Fishery Research Institute 11. Japar Sidik Bujang, Muta Harah Zakaria (2002). Dungun, Terengganu, staffs from Regional Veterinary Seagrasses in Malaysia. In: World Atlas of Seagrasses. Laboratory Johor Baharu and those who involved directly Green E.P., Short F.T., Spalding M.D. (eds.), California and indirectly in performed or assisted with the post- University Press. Chapter 14, pp 166-176. mortem examinations.