Wim Gaastra

Len J A Lipman
email: l.j.a.lipman@iras.uu.nl

Arthur W A M De Cock

Tim K Exel

Raymond B G Pegge

Josje Scheurwater

Raquel Vilela

Leonel Mendoza

Keywords: Pythium insidiosum, pythiosis, oomycete, immunotherapy

Pythium insidiosum is an oomycete pathogenic in mammals. The infection occurs mainly in tropical and subtropical areas, particularly in horses, dogs and humans. Infection is acquired through small wounds via contact with water that contains motile zoospores or other propagules (zoospores or hyphae). The disease, though described as emerging has in fact already been described since 1884. Depending on the site of entry, infection can lead to different forms of pythiosis i.e. a cutaneous, vascular, ocular, gastrointestinal and a systemic form, which is rarely seen. The infection is not contagious; no animal-animal or animalhuman transmission has been reported so far. Therapy includes radical surgery, antifungal drugs, immunotherapy or a combination of these therapies. The prevention to contract the disease in endemic areas is difficult. Avoiding stagnant waters could be of help, although the presence of P. insidiosum on grass and soil in enzootic areas renders this practice useless.

Pythium insidiosum is the only etiologic agent of pythiosis in mammals. Most cases of pythiosis have been reported in dogs, horses and humans. Only sporadic cases in other animals, such as calves [START_REF] Pérez | Epizootic cutaneous pythiosis in beef calves[END_REF], cats [START_REF] Miller | Cutaneous pythiosis in beef calves[END_REF][START_REF] Thomas | Pythiosis in dogs and cats[END_REF][START_REF] Rakich | Gastrointestinal pythiosis in two cats[END_REF], sheep [START_REF] Miller | Cutaneous pythiosis in beef calves[END_REF][START_REF] Tabosa | Outbreaks of pythiosis in two flocks of sheep in northeastern Brazil[END_REF], Santurio et al., 2008), a bird [START_REF] Pesavento | Cutaneous pythiosis in a nestling white faced ibis[END_REF] and tropical animals held in captivity [START_REF] Camus | Granulomatous pneumonia caused by Pythium insidiosum in a central American jaguar, Panthera onca[END_REF][START_REF] Wellahan | Pythiosis in a dromedary camel (Camelus dromedaries)[END_REF][START_REF] Buergelt | Abdominal pythiosis in a Bengal tiger (Panthera tigris tigris)[END_REF] are known. Pythiosis is a rarely occurring, nontransmissible disease traditionally found in tropical, subtropical and temperate regions [START_REF] De Cock | Pythium insidiosum sp. Nov., the etiologic agent of pythiosis[END_REF][START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. Recently however, pythiosis was also observed in California and Arizona, where the climate does not fit this description. These observations might indicate that the environmental niche for P. insidiosum is expanding, probably as a consequence of environmental changes like deliberate flooding of rice fields or irrigated landscape development [START_REF] Berryessa | Gastrointestinal Pythiosis in 10 Dogs from California[END_REF][START_REF] White | Cutaneous pythiosis in a California nontravelled horse[END_REF]. In Thailand pythiosis is considered to be endemic. Pythiosis in humans is life-threatening with high rates of morbidity and mortality, especially in regions with a lack of tools for early diagnosis and effective treatment. While pythiosis is often described as an emerging disease [START_REF] Laohapensang | Vascular pythisosis in a thalassemic patient[END_REF], the disease was already described in 1884 by British veterinarians working with horses in India [START_REF] Smith | The pathology of bursattee[END_REF].

The agent causing the disease (at that time named Hyphomycosis destruens equi) was isolated for the first time in 1901 by Dutch scientists working with horses in Indonesia and A c c e p t e d M a n u s c r i p t 3 again in 1924 by another Dutch (de Haan andHoogkamer, 1901, Witkamp 1924). The disease has been known under various other names: bursattee or bursatte (derived from the Indian word Burus, Bursator or Bausette which means rainy season), espundia (Latin America), equine phycomycosis (Australia, USA), granular dermatitidis (Japan), hyphomycosis destruens equi (Indonesia), leeches (USA), swamp cancer (Australia, USA) and summer sores (Australia, Latin America, USA) [START_REF] Kerr | On the disease to which horses are subject to the East Indies, termed baursette[END_REF][START_REF] Fish | Leeches: a histological investigation of two cases of equine mycosis with a historical account of a supposed similar disease,called bursatte, occurring in India[END_REF][START_REF] Witkamp | Bijdrage tot de kennis van de Hyphomycosis destruens[END_REF][START_REF] Gonzalez | Espundia equina: Etiologia y patogenesisi de una ficomicosis[END_REF][START_REF] Ichitani | Pythium gracile isolated from the foci of granular dermatitis in the horse (Equus caballus)[END_REF].

The fungus-like nature of the causal agent of the disease was probably first reported by [START_REF] Smith | The pathology of bursattee[END_REF] and [START_REF] Drouin | Sur une nouvelle mycose du cheval[END_REF]. By lack of sporulation the agent could not be identified and it did not get a name until 1961 when [START_REF] Bridges | A phycomycosis of horses caused by Hyphomyces destruens[END_REF] named the organism Hyphomyces destruens. This name was derived from the name of the disease Hyphomycosis destruens which was introduced by de Haan and Hoogkamer (1901) and later extended to Hyphomycosis destruens equi by De Haan (1902). However, the name H. destruens was not validly published and lacked a Latin description and the designation of a type. Bridges and Emmons called the disease phycomycosis because they thought the organism was a zygomycete, probably Mortierella. [START_REF] Austwick | Swamp cancer[END_REF] observed zoospore development when cultures grown on Sabouraud dextrose agar were transferred to an aqueous medium. They concluded that the H. destruens actually belonged to the Oomycete genus Pythium. Based on this discovery, [START_REF] Chandler | A color atlas and textbook of the histopathology of mycotic diseases[END_REF] proposed the term pythiosis for the disease. In 1980, [START_REF] Ichitani | Pythium gracile isolated from the foci of granular dermatitis in the horse (Equus caballus)[END_REF] isolated a Pythium sp. from a diseased horse and found it to be morphologically similar to Pythium gracile Schenk [START_REF] Amemiya | Granular dermatitis in the horse, caused by Pythium gracile[END_REF]. However, P. gracile is a poorly described species of which the identity cannot be verified.

Moreover, it was isolated from algae in Germany, where pythiosis does not occur. The oomycete was formally described as Pythium insidiosum when sexual sporulation was observed by De Cock et al (1987). Almost simultaneously [START_REF] Shipton | Pythium destruens sp.nov., an agent of equine pythiosis[END_REF] proposed the binomial Pythium destruens for a strain isolated from an Australian horse with pythiosis.

Based on priority P. destruens is now considered a synonym of P. insidiosum [START_REF] Mendoza | Antigenic relationship between Pythium insidiosum de Cock et al.1987 and its synonym Pythium destruens Shipton[END_REF].

Pythium insidiosum mainly occurs in surface water amongst others in standing inland waters and occasionally in soil [START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF][START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF]. Not much is known about the ecological preference of P. insidiosum, but the presence of water which induces the formation of zoospores seems to be a prerequisite [START_REF] Supabandhu | Isolation and identification of the human pathogen Pythium insidiosum from environmental samples collected in Thai agricultural areas[END_REF].

Since P. insidiosum usually occurs under wetland conditions, more cases are seen after heavy rain or floods [START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF][START_REF] Miller | Experimental pythiosis in rabbits[END_REF][START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF]. Other risk A c c e p t e d M a n u s c r i p t factors for developing pythiosis have not yet been identified. Floods after heavy rain have been incriminated as one of the natural resources used by P. insidiosum to expand its ecological niche to new areas [START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF][START_REF] Supabandhu | Isolation and identification of the human pathogen Pythium insidiosum from environmental samples collected in Thai agricultural areas[END_REF].

Phylogenetic analysis has shown that Pythium spp. are closer related to diatomeae and algae than to true fungi [START_REF] Kwon-Chung | Phylogenetic spectrum of fungi that are pathogenic to humans[END_REF], Hudspeth et al., 2000, Martin;[START_REF] Martin | Phylogenetic relationships among some Pythium species inferred from sequence analysis of the mitochondrially encoded cytochrome oxidase II gene[END_REF]. Pythium spp.

belong to the kingdom Stramenopila, the Phylum Oomycota, the order Peronosporales and the family Pythiaceae [START_REF] Alexopoulos | Introductory Mycology[END_REF]. Pythium spp have also been classified by others in the kingdom Chromista, the Phylum Pseudofungi, the class Oomycetes, the order Pythiales and the family Pythiaceae [START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF]. In the most recent classification [START_REF] Dick | Straminipilous fungi: systematics of the peronosporomycetes including accounts of the marine straminipilous protist, the plasmodiophorids and similar organisms[END_REF], P. insidiosum as a member of the genus Pythium is classified in the kingdom Straminipila, class Peronosporomycetes (= Oomycetes), order Pythiales and family Pythiaceae. This classification, however, was not yet based on DNA sequence data and may change in the near future.

The Agent

Microscopically P. insidiosum develops mycelium like fungi, but it is not a true fungus, since its cell walls do not contain chitin but are composed of cellulose and ß-glucans, its cytoplasmic membrane lacks ergosterol, the thallus is diploid and coenocytic, the sexual process is oogamy and the organism develops biflagellate zoospores in wet environments [START_REF] Alexopoulos | Introductory Mycology[END_REF]. Zoospores are single nucleated cells without a cell wall that can swim with the help of two flagella; a tinsel (anterior) flagellum and a whiplash (posterior) flagellum. The posterior flagellum is thought to be responsible for the movement of the zoospore through water, the antherior flagellum functions as a rudder. Zoospores swim in a helical or spiral pattern interrupted by random changes of direction. Zoospores cannot divide or multiply [START_REF] Walker | Zoospore development in the oomycetes[END_REF]. The zoospores are considered to be the infective propagules, they show chemotaxis and become encysted once they come in contact with either decaying or injured plant tissue (there is no evidence that healthy plant tissue has a similar effect). Injured tissue of a mammalian host that enters the ecosystem of P. insidiosum exerts the same effect. A glycoprotein secreted on the surface of the encysted zoospores allows adhesion to the injured tissue [START_REF] Estrada-Garcia | Monoclonal antibodies to the adhesive cell coat secreted by Pythium aphanidermatum zoospores recognize 200x300Mr glycoproteins stored within large peripheral vesicles[END_REF][START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF][START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF].

Pythium insidiosum infections occur mostly in apparently healthy humans and animals [START_REF] Thianprasit | Human pythiosis[END_REF][START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF][START_REF] Thomas | Pythiosis in dogs and cats[END_REF][START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. Pythiosis occurs in regions of Southeast Asia (India, Indonesia, Japan, A c c e p t e d M a n u s c r i p t 5 Korea, New Guinea, and Thailand) (Ichitani and Amemiya, 1982[START_REF] Thianprasit | Fungal infection in Thailand[END_REF][START_REF] Thianprasit | Human pythiosis[END_REF][START_REF] Sohn | Enteric pythiosis in a Jindo dog[END_REF], eastern coastal Australia and New Zealand [START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF][START_REF] Triscott | Human subcutaneous pythiosis[END_REF][START_REF] Murdoch | Pythium insidiosum keratitis[END_REF], South America (Argentina, Brazil, Colombia, Venezuela) [START_REF] Gonzalez | Espundia equina: Etiologia y patogenesisi de una ficomicosis[END_REF]Ruiz, 1975, Mendoza, 2005), Costa Rica (Mendoza and[START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF][START_REF] Alfaro | Four cases of equine bone lesions caused by Pythium insidiosum[END_REF], Guatemala [START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF], Haiti [START_REF] Virgile | Human corneal ulcer caused by Pythium insidiosum[END_REF], Panama and Nicaragua [START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF] and North America, Mexico and the United States [START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF][START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. In the USA it occurs most often in the Gulf coast states but has also been identified in other states [START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF][START_REF] Grooters | Pythiosis and Zygomycosis[END_REF]. The first case of pythiosis from Africa (Mali) was reported in a dog [START_REF] Rivierre | Pythiosis in Africa[END_REF]. This case was confirmed by molecular sequencing, placing the African strain in a separate taxon from the American and Asian clusters reported by Schurko et al., (2003a).

Pythium insidiosum is well adapted to the body temperature of its mammalian hosts. It has an optimum and maximum temperature for growth of 34-36° and 40-45° respectively (De Cock et al., 1987). It grows well on various artificial media when incubated at 25° or 37°C.

Sabouraud agar [START_REF] Witkamp | Bijdrage tot de kennis van de Hyphomycosis destruens[END_REF][START_REF] Witkamp | Het voorkomen van metastasen in de regionaire lymphlieren by Hyphomycosis destruens[END_REF][START_REF] De Cock | Pythium insidiosum sp. Nov., the etiologic agent of pythiosis[END_REF], vegetable extract agar, peptone yeast glucose agar and potato flakes agar all can be used [START_REF] De Cock | Pythium insidiosum sp. Nov., the etiologic agent of pythiosis[END_REF][START_REF] Shipton | Pythium destruens sp.nov., an agent of equine pythiosis[END_REF], Grooters et al., 2002b). On Sabouraud agar P. insidiosum grows in submerged, white to colourless colonies, which have an irregular radiate pattern [START_REF] De Cock | Pythium insidiosum sp. Nov., the etiologic agent of pythiosis[END_REF][START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF], 1996) (Fig. 1A). Zoospores are only developed in water cultures; they are stimulated by the presence of ions such as K + , Ca 2+ , Mg 2+ and chemically attracted by plant material, animal hairs or pieces of animal tissue [START_REF] Austwick | Swamp cancer[END_REF][START_REF] Shipton | Pythium destruens sp.nov., an agent of equine pythiosis[END_REF][START_REF] Mendoza | A method to obtain rapid zoosporogenesis of Pythium insidiosum[END_REF][START_REF] Chaiprasert | Induction of zoospore formation in Thai isolates of Pythium insidiosum[END_REF][START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF]. While in some oomycetes, zoospore formation can occur within minutes and is considered one of the fastest developmental processes in any biological system [START_REF] Walker | Zoospore development in the oomycetes[END_REF], in P.insidiosum zoospore formation can take one hour or more [START_REF] Mendoza | A method to obtain rapid zoosporogenesis of Pythium insidiosum[END_REF]. P. insidiosum does not need a susceptible mammalian host for its survival or propagation since it is able to survive and multiply in its natural environment on decaying plants [START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF].

Molecular Phylogeny of Pythium insidiosum.

The first reports showing that P. insidiosum is a unique oomycetous pathogen of mammals came from molecular phylogenetic studies of several Pythium species [START_REF] Martin | Phylogenetic relationships among some Pythium species inferred from sequence analysis of the mitochondrially encoded cytochrome oxidase II gene[END_REF][START_REF] Lévesque | Molecular phylogeny and taxonomy of the genus Pythium[END_REF]). [START_REF] Lévesque | Molecular phylogeny and taxonomy of the genus Pythium[END_REF] divided Pythium species in at least 10 different phylogenetic groups (designated A to K). P. insidiosum is found in A c c e p t e d M a n u s c r i p t group C together with P. grandiosporangium. Grouping of P. insidiosum together with this marine saprotrophic microbe is intriguing since P. insidiosum is a fresh water organism.

Moreover, the two species are significantly different with regard to morphology and growth/temperature relationships. The clustering of the two in one clade is more likely to be the consequence of long range attraction then of a genuine close relationship. Schurko et al (2003a, b) The ability of P. insidiosum to develop appressoria and the chemotaxis of its zoospores to plant tissue suggests that this pathogen took advantage of these ancestral features and adapted them to a lifestyle as a mammalian parasite. When P. insidiosum acquired the pathogenic attributes to invade mammals, hopefully can be answered after completion of its complete genome sequence. Several genome sequencing projects of plant pathogenic oomycetes are already finished and some others are underway. To our knowledge this is not yet the case for P. insidiosum.

Epidemiology and pathogenesis

Zoospores show a marked chemotaxis towards animal hair, wounds, other damaged skin parts or intestinal mucosa [START_REF] De Cock | Pythium insidiosum sp. Nov., the etiologic agent of pythiosis[END_REF][START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF]. Upon microscopy, adhesion of zoospores to the cut edges of skin but hardly to undamaged tissue was observed [START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF][START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF]. The location of the lesions is therefore directly related to the parts of the body that were in direct contact with water containing zoospores of P. insidiosum. For example, in horses lesions are seen especially on the legs and ventral parts of the abdomen. Lesions caused by punctures and insect bites can also be a "port d'entree" for Pythium insidiosum [START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF][START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF][START_REF] Rees | Disorders of the skin[END_REF]. Insect bites are of interest from an epidemiological point of view since Schurko et al., (2003a) identified an isolate from an infected larva of Culex quinquefasciatus (a widespread tropical mosquito) in India as P. insidiosum. This suggests that P.insidiosum has the ability to invade Once the zoospores are in contact with mammalian or plant tissue (in wet environments) they encyst on the surface of the injured tissue(s). The encysted zoospores secrete a sticky amorphous glycoprotein that mediates the adhesion of zoospores to tissue before they enter the tissue [START_REF] Mendoza | Life Cycle of the human and animal oomycete pathogen Pythium insidiosum[END_REF][START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF]. The encysted zoospores stimulated by the host's body temperature develop a germ tube (hypha) that extends from the zoospores into the infected tissue and later can also infiltrate blood vessels (humans), which makes spreading within the body tissues easier. The disease can also be acquired through traumatic lesions and contact with hyphae of P. insidiosum [START_REF] Alfaro | Four cases of equine bone lesions caused by Pythium insidiosum[END_REF]. The invasion of blood vessels can lead to thrombosis and invasion of large arteries [START_REF] Imwidthaya | Human pythiosis in Thailand[END_REF][START_REF] Thitithanyanont | Use of an immunogenic therapeutic vaccine to treat a lifethreatening human arteritic infection caused by Pythium insidiosum[END_REF][START_REF] Krajaejun | Clinical and Epidemiological Analysis of Human Pythiosis in Thailand[END_REF][START_REF] Pupaibool | Human Pythiosis[END_REF][START_REF] Laohapensang | Vascular pythisosis in a thalassemic patient[END_REF]. In addition to P. insidiosum host colonization by invasive growth, the secretion of proteases and the exertion of mechanical force by the tips of the elongating hyphae have been implicated as putative virulence factors [START_REF] Shipton | Pythium destruens sp.nov., an agent of equine pythiosis[END_REF][START_REF] Ravishankar | Mechanics of solid tissue invasion by the mammalian pathogen Pythium insidiosum[END_REF]. For P. insidiosum this force has been measured and compared with the resistance of human and equine skin to needle insertion. The data show that a significant reduction in tissue strength has to be obtained by the action of proteases, before penetration of the tissue by hyphae can take place [START_REF] Ravishankar | Mechanics of solid tissue invasion by the mammalian pathogen Pythium insidiosum[END_REF][START_REF] Macdonald | Biomechanical interaction between hyphae of two Pythium species (Oomycota) and host tissues[END_REF][START_REF] Davis | Relationship between temperature optima and secreted protease activities of three Pythium species and pathogenicity toward plant and animal hosts[END_REF].

All three tested strains of P.insidiosum secrete three or more proteases of different molecular weight. Two of them were present in the three strains. The specificity of the proteases is not known, but inhibition experiments identified them as serine proteases [START_REF] Davis | Relationship between temperature optima and secreted protease activities of three Pythium species and pathogenicity toward plant and animal hosts[END_REF].

Secretion of proteases seems to be a general feature of pathogenic oomycetes [START_REF] Bangyeekhun | Molecular cloning and characterization of two serine proteinase genes from the crayfish plague fungus, Aphanomyces astaci[END_REF][START_REF] Torto-Alalibo | Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors[END_REF]. Pythiosis progresses rapidly and if not treated in the early stages can become life threatening in both humans and animals. So far zoonotic properties have not been demonstrated for P. insidiosum.

Clinical signs in animals

Pythiosis has been reported in several species, but most cases occur in otherwise immunocompetent horses and dogs. In animals the infection develops in the form of a cutaneous or intestinal disease, although cases of localized lung and bone infection, as well as systemic dissemination through lymph nodes have been also described [START_REF] Witkamp | Het voorkomen van metastasen in de regionaire lymphlieren by Hyphomycosis destruens[END_REF][START_REF] Goad | Pulmonary pythiosis in a horse[END_REF], Mendoza et al., 1988[START_REF] Alfaro | Four cases of equine bone lesions caused by Pythium insidiosum[END_REF][START_REF] Reis | Disseminated pythiosis in three horses[END_REF].

Horses

A c c e p t e d M a n u s c r i p t

In horses there seems to be no predisposition for breed, age or sex of the animals [START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF][START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF][START_REF] White | Cutaneous pythiosis in a California nontravelled horse[END_REF]. In this species the cutaneous form is more prevalent whereas the intestinal form is rarely recorded [START_REF] Brown | Intestinal pythiosis in a horse[END_REF][START_REF] Morton | Chronic eosinophilic enteritis attributed to Pythium sp. in a horse[END_REF], Purcel et al., 1994). The Dutch authors [START_REF] Bubberman | Twee uitgebreide gevallen van Hyphomycosis destruens equi[END_REF][START_REF] Witkamp | Bijdrage tot de kennis van de Hyphomycosis destruens[END_REF] describe for all their equine patients that they were apparently suffering from itching, since they were rubbing the infected area of the body against the wall of the stable or biting the wound when allowed. In some cases this lead to auto-mutilation (Mendoza et al., 1986). These authors also describe the awful smell associated with horses infected with this pathogen. Lesions in the cutaneous form often consist of large, rounded, granulomatous nodular ulcerative tissue. The lesions are tumour-like and consist of necrotic tissue, containing eosinophils and hyphae of P. insidiosum [START_REF] Headley | Cutaneous pythiosis in a slaughtered horse: a case report[END_REF][START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF][START_REF] Miller | The comparative pathology of equine cutaneous phycomycosis[END_REF][START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF][START_REF] Chaffin | Cutaneous pythiosis in the horse[END_REF]. The tissue and the draining fistulas contain cores of necrotic yellow-gray material referred to as "kunkers".

Kunkers can vary in size, from a grain of rice, to various centimetres and can sometimes be found in bandage material (Fig. 2). Kunkers are specific for equine pythiosis and absent in other affected species [START_REF] Leal | Pitiosis eqüina no Pantanal brasileiro: aspectos clínico-patológicos de casos típicos e atípicos[END_REF][START_REF] Mendoza | Infections caused by the oomycetous pathogen Pythium insidiosum[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. Kunkers in horses are formed by degranulation of eosinophils over the invading hyphae of P. insidiosum.

New eosinophils degranulate over the old ones and the structural mass grows in size. In horses with chronic pythiosis the only place where the hyphae of P. insidiosum can be found is within kunkers. The clinical signs of a horse with pythiosis may include in addition to skin and bone lesions, lameness and enlargement of regional lymph nodes, anemia and hypoproteinemia. Both hypoproteinemia and anemia occur as a result of blood loss and loss of exudates consisting of serum and cations through large ulcerated skin lesions (Miller,1983, Mendoza and[START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF]) (Fig. 3). Intestinal pythiosis in horses is characterized by stenotic fibrous and disseminated gastrointestinal lesions (Brown and Roberts,1988[START_REF] Allison | Enteric pythiosis in a horse[END_REF][START_REF] Purcell | Jejunal obstruction caused by a Pythium insidiosum granuloma in a mare[END_REF].

Dogs

In dogs, in contrast to horses, the gastrointestinal form of pythiosis occurs more often than the subcutaneous form (Fig. 4). The clinical symptoms include vomiting, weight loss, intermittent diarrhoea and palpable masses in the abdomen [START_REF] Fischer | Gastrointestinal pythiosis in Missouri dogs: eleven cases[END_REF]. Extension of the infection to the pancreas, mesenteric lymph nodes and bile ducts can occur [START_REF] Thomas | Pythiosis in dogs and cats[END_REF][START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF][START_REF] Berryessa | Gastrointestinal Pythiosis in 10 Dogs from California[END_REF]. Lesions may involve legs and face or tail [START_REF] Thomas | Pythiosis in dogs and cats[END_REF]. Infection by P. insidiosum in dogs is more frequent in young immunocompetent adults [START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF], Grooters et al., 2003).

A c c e p t e d M a n u s c r i p t

Cats Pythiosis in cats is rare and usually the lesions are confined to the skin and subcutaneous tissues (Fig, 5) (Thomas andLewis, 1998, Grooters, 2003,). Gastrointestinal infections in cats were reported only recently [START_REF] Rakich | Gastrointestinal pythiosis in two cats[END_REF]. There is no predisposition for breed, age or sex [START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF].

Cattle

Pythiosis in cattle usually occurs during the rainy season in subtropical areas. It is considered a sporadic disease in this species. However, an epizootic event involving more than 60 calves was reported in Venezuela [START_REF] Pérez | Epizootic cutaneous pythiosis in beef calves[END_REF]. The disease occurs more frequently on the limbs with pruritus and claudication. Tumor-like masses with fistules and ulcerated tissue of the limbs is common. The hyphae are usually localized at the centre of eosinophilic granulomas. The affected areas are extremely painful and most animals cannot stand up which usually leads to dehydration and death. Secondary bacterial contamination with anaerobes and other bacteria is common (Fig. 6) [START_REF] Miller | Cutaneous pythiosis in beef calves[END_REF][START_REF] Santurio | Cutaneous Pythiosis insidiosi in calves from the Pantanal region of Brazil[END_REF], Perez et al. 2005).

It has been suggested that in tropical countries the disease in cattle termed "infectious pododermatitis" usually attributed to anaerobic bacteria, maybe caused by P. insidiosum, which could open the door for anaerobic bacteria causing pododermatitis as a secondary infection (Perez et al., 2005).

Sheep

Pythiosis in sheep has been reported to cause cutaneous lesions in different anatomical areas and as a rhinopharyngeal disease [START_REF] Tabosa | Outbreaks of pythiosis in two flocks of sheep in northeastern Brazil[END_REF][START_REF] Riet-Correa | Outbreak of rhinopharyngeal zygomycosis in sheep in Paraiba, Northeastern Brazil[END_REF]. Eosinophilic granulomatous lesions are usually reported on the limbs and rhinopharingeal areas. Clinical signs involving the rhinopharinge include bilateral serosanguineous nasal discharge, swelling of nostrils, and the skin of the face (Fig. 7) [START_REF] Riet-Correa | Outbreak of rhinopharyngeal zygomycosis in sheep in Paraiba, Northeastern Brazil[END_REF]. Involvement of the lungs has also been reported [START_REF] Tabosa | Outbreaks of pythiosis in two flocks of sheep in northeastern Brazil[END_REF].

Birds

Only a single report on P. insidiosum affecting birds has been recorded [START_REF] Pesavento | Cutaneous pythiosis in a nestling white faced ibis[END_REF]. The infected animal was a Californian nestling white-faced ibis (Plegadis chihi) with multiple ulceration of its wings, neck, head and limbs. P. insidiosum hyphae were found at the centre of necrotizing eosinophilic granulomas. This report shows that P. insidiosum can also affect birds and thus veterinarians dealing with birds should investigate similar skin lesions for the presence of this oomycete.

Captive animals

Spectacled Bears

How often the disease occurred in this species or the number of cases has yet to be officially reported. So far all cases have come from a zoo in Columbia, South Carolina. Several adult spectacled bears (Tremarctos oronatus) were seen with lesions involving the preputial glands, other cutaneous areas and the gastrointestinal tract (A.A. Padhye, personal communication).

The disease has been reported at least twice in this species from the same zoo. The presence of P. insidosum in the affected tissue was confirmed by microscopy and culture.

Camels

Only a single case of pythiosis involving a 4.5 years-old male captive dromedary camel (Camelus dromedarious) in a Florida zoo has been recorded (Wellehan et al., 2004). The animal developed a granulomatous mass on the right side of the face and the diagnosis pythiosis was confirmed by culture. Despite immunotherapy and iodine treatment the camel died six months after the initial diagnosis. At necropsy hyphae of P. insidiosum were also found in the third compartment of the stomach. Two unpublished cases of pythiosis in camels from a zoo in Tennessee were recently confirmed (Videla, R. and Mendoza, L. unpublished).

Big cats

P. insidiosum has been reported causing an unusual primary pulmonary infection in a seven months old Central American jaguar (Panthera onca) that later died of the infection [START_REF] Camus | Granulomatous pneumonia caused by Pythium insidiosum in a central American jaguar, Panthera onca[END_REF]. In addition, an adult captive Bengal tiger (Panthera tigris tigris) was diagnosed with abdominal pythiosis in Florida [START_REF] Buergelt | Abdominal pythiosis in a Bengal tiger (Panthera tigris tigris)[END_REF]). The tiger later died of the infection. At necropsy several intestinal tumor-like masses were found. The diagnosis was confirmed by serology.

Pythiosis in Humans

Although it has long been observed in animals, the disease in humans was only recently described in Thai patients [START_REF] Thianprasit | Fungal infection in Thailand[END_REF][START_REF] Thianprasit | Human pythiosis[END_REF]. In humans too, pythiosis affects apparently healthy individuals. Susceptible hosts become infected after contact with zoospores through contact of a skin wound with contaminated water. Agricultural related activities or water associated leisure activities are considered to be predisposing factors for human pythiosis [START_REF] Sathapatayayongs | Human pythiosis associated with thalassemia hemoglobinopathy syndrome[END_REF][START_REF] Moraes | Human pythiosis, Brazil[END_REF], Supabandhhu J., 2008). Thalassemia is also considered to be a predisposing factor in Thailand A c c e p t e d M a n u s c r i p t [START_REF] Sathapatayayongs | Human pythiosis associated with thalassemia hemoglobinopathy syndrome[END_REF]), but in a population with a high incidence of Thalassemic patients this claim has been questioned (Mendoza et al., 2003b). The majority of human cases (80%) of pythiosis have been reported in Thailand [START_REF] Sathapatayayongs | Human pythiosis associated with thalassemia hemoglobinopathy syndrome[END_REF][START_REF] Thianprasit | Human pythiosis[END_REF][START_REF] Imwidthaya | Human pythiosis in Thailand[END_REF], Vanittakanakom et al., 2004). Other countries where human pythiosis cases have been reported include: Australia, Brazil, Haiti, Malaysia, New Zealand, and the USA, [START_REF] Rinaldi | Pythium insiidosum causes a severe disease in a healthy boy[END_REF], Triscot et al., 1993[START_REF] Virgile | Human corneal ulcer caused by Pythium insidiosum[END_REF][START_REF] Badenoch | Pythium insidiosum keratitis confirmed by DNA sequence analysis[END_REF][START_REF] Moraes | Human pythiosis, Brazil[END_REF]. In a report on more than 100 cases of pythiosis in Thailand, four different forms of pythiosis were described: a) the (sub) cutaneous form (infecting the face or limbs as a granulomatous and ulcerating lesion), found in 5% of the cases, b) the vascular type (affecting arteries and resulting in arterial occlusion and aneurysm), found in 59% of the cases, c) the ocular and orbital form in which corneal ulcers are formed, is found in 33% of the cases and d) pythiosis at unusual places (i.e. disseminated pythiosis and infection of internal organs, observed in 3% of the cases [START_REF] Krajaejun | Clinical and Epidemiological Analysis of Human Pythiosis in Thailand[END_REF]. From a Brazilian study, in which the morphological and molecular characteristics of a new equine isolate were compared with a human isolate, it was clear that both isolates are 99% similar [START_REF] Moraes | Morphological and molecular characterization of an equine isolate of Pythium insidiosum and comparison with the first human isolate from the same geographic region[END_REF]. The isolates were obtained from the same region. This indicates that both humans and animals are sensitive for the same agent and host specificity of different genotypes seems not very likely. Since all American P. insidiosum isolates are included in a single phylogenetic taxon indicating their similarity (Schurko et. al., 2003b), this observation was not unexpected. In the USA human cases of pythiosis are seldom reported and only a small number of cases have been officially published [START_REF] Rinaldi | Pythium insiidosum causes a severe disease in a healthy boy[END_REF][START_REF] Shenep | Successful medial therapy for deeply invasive facial infection due to Pythium insidiosum in a child[END_REF]. However, it is likely that pythiosis in the past has been misdiagnosed as a fungal infection (Mendoza et al., 2003). In Australia and the USA children seem more susceptible to the orbicular form [START_REF] Rinaldi | Pythium insiidosum causes a severe disease in a healthy boy[END_REF][START_REF] Triscott | Human subcutaneous pythiosis[END_REF][START_REF] Shenep | Successful medial therapy for deeply invasive facial infection due to Pythium insidiosum in a child[END_REF], Mendoza et al., 2003b), whereas adults develop the subcutanous form.

Experimental pythiosis

Rabbits

The susceptibility of rabbits to experimental infection with P. insidiosum was demonstrated almost 100 year ago by [START_REF] Witkamp | Bijdrage tot de kennis van de Hyphomycosis destruens[END_REF][START_REF] Witkamp | Het voorkomen van metastasen in de regionaire lymphlieren by Hyphomycosis destruens[END_REF] and has been used ever since as an experimental model to test the effectiveness of immunotherapy and antimycotic agents [START_REF] Amemiya | Isolation of a fungus of the Mortierellaceae from an equine granular dermatitis III[END_REF][START_REF] Ichitani | Pythium gracile isolated from the foci of granular dermatitis in the horse (Equus caballus)[END_REF][START_REF] Miller | Experimental pythiosis in rabbits[END_REF][START_REF] Patino-Meza | Role of the zoospores of Pythium insidiosum in the experimental reproduction of pythiosis in susceptible species[END_REF], Santurio et al., 2003a[START_REF] Pereira | Caspofungin in vitro and in vivo activity against Brazilian Pythium insidiosum strains isolated from animals[END_REF], 2008). It is believed that 17,500 zoospores A c c e p t e d M a n u s c r i p t are needed to generate the disease in rabbits (Santurio et al., 2003a). However, Patino-Meza (1988) induced the infection using only 500 or less zoospores. The inoculation with P. insidiosum in any other species has been consistently unsuccessful. [START_REF] Patino-Meza | Role of the zoospores of Pythium insidiosum in the experimental reproduction of pythiosis in susceptible species[END_REF] reported no results on the experimental inoculation of P. insidiosum in species such as cattle, dogs, and horses. The author used a container with water containing hundreds of P. insidiosum zoospores and submerged a horse limb with small skin wounds, without success. [START_REF] Witkamp | Bijdrage tot de kennis van de Hyphomycosis destruens[END_REF] mentions that in guinea pigs a similar syndrome as in rabbits develops after experimental infection, but the guinea pigs apparently were suffering much less and their general health was much better. Oddly, natural infection of rabbits with P. insidiosum has yet to be documented [START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. In a bizarre report, inoculation of native people and equines with kunkers (from horses with pythiosis) without developing the disease was mentioned [START_REF] Smith | The pathology of bursattee[END_REF]. In summary, experimental pythiosis is only possible in rabbits.

Immunology of Pythium insidiosum infections

In an early report by veterinarians on equine pythiosis in Indonesia (the former Netherlands India) it was mentioned that infected animals develop anti-P. insidiosum antibodies easily detected using antigens extracted from the pathogen in an immunodifussion test [START_REF] Witkamp | Bijdrage tot de kennis van de Hyphomycosis destruens[END_REF]. This was confirmed by serological studies in humans and animals suffering from pythiosis, showing that P. insidiosum antigens trigger a humoral immune response upon contact with the host (Miller and Campbell, 1982, Mendoza et al., 1986[START_REF] Imwidthaya | Immunodiffusion test for diagnosing human pythiosis[END_REF], Mendoza et al., 1992a, Grooters et al., 2002a[START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF]. It seems, however, that the presence of this type of immunity does, not clear infections in humans or animals (Miller and Campbell, 1982[START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF][START_REF] Triscott | Human subcutaneous pythiosis[END_REF][START_REF] Thitithanyanont | Use of an immunogenic therapeutic vaccine to treat a lifethreatening human arteritic infection caused by Pythium insidiosum[END_REF]. The cellular immunity, provided by activated macrophages, mast cells, eosinophils and other inflammatory cells, seems directly involved in the extensive tissue damage observed in infected hosts (Miller andCampbell, 1982, Mendoza and[START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF]. Degranulation of eosinophils and mast cells at the infected sites causes the intensive pruritus reported in horses and other species; and is also responsible for the bacterial contamination occurring after development of necrosis and ulcerating tissue with the classical strong odour reported in equine pythiosis of the skin [START_REF] Bubberman | Twee uitgebreide gevallen van Hyphomycosis destruens equi[END_REF].

Based on these data it was postulated that the humoral immunity observed in infected hosts with pythiosis only triggers precipiting and agglutining anti-P. insidiosum antibodies related to a T helper 2 (Th2) immune response (Mendoza et al., 2003a, Mendoza and[START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF]. These antibodies are not protective, but can be used for the diagnosis of the disease. It was further postulated that the cellular immunity triggered by P. insidiosum locks the immune system in a Th2 mode which contributes to a worsening of the condition and eventually leads to death. It was suggested that treatment of the disease, using antigenic proteins extracted from P. insidiosum (see Immunotherapy below), might nevertheless work if these immunogens were presented to the immune system in a different fashion. The observation that high levels of interferon gamma (IFN-γ) and Interleukin 2 (IL2) (indicators of a Th1 mediated immunity) and a decrease of Th2 mediated interleukins are present in cured patients supports this idea (Thitianyanont et al., 1998, Mendoza and[START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF].

Of interest with respect to the immunology of pythiosis is a report that anti-P.

insidiosum antibodies developed by different host species seem to detect different P. insidiosum antigens. Antibodies present in cattle, horses and humans with pythiosis were seen to bind to different hyphal cell compartments upon immuno-electron microscopy and protein-A colloidal gold -labeling [START_REF] Garcia | Mapping of Pythium insidiosum hyphal antigens and ultrastructural features using TEM[END_REF]. This suggested that different hosts recognize different P. insidiosum antigens, which can have implications for the variable response to immunotherapy in some species (see Immunotherapy below). Western blot analysis with sera from bovine, feline, canine, equine and human patients, also showed the detection of different antigens depending on the serum of the species tested [START_REF] Chindamporn | Antibodies in the sera of host species with Pythiosis recognize a variety of unique immunogens in geographically divergent Pythium insidiosum strains[END_REF].

Recognition of different dominant P. insidiosum antigens by antibodies present in cattle sera, but not by horse and rabbit antibodies has likewise been reported (Leal et al., 2005).

Diagnostics

Equine pythiosis should not be mistaken for habronemiasis (also named swamp cancer, a disease of horses caused by the nematodes Habronema muscae, H. majus (H. microstoma and Draschia megastoma), skin fungal infections caused by Conidiobolus and Basidiobolus spp., extreme granulation tissue, bacterial granulation tissue or an invasive squamous cell carcinoma [START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF]. Early diagnosis of pythiosis is very important for a successful therapy. Clinical expertise of a veterinarian or physician with the various clinical forms of the disease in different species is crucial for an early diagnosis.

Sample Collection

For equine pythiosis the collection of several kunkers and their transportation to the laboratory in water or a saline solution with antibiotics (streptomycin and ampicillin) is recommended. Biopts and scrapings should be transported to a laboratory as soon as possible.

The samples have to be washed with distilled water and transported at room temperature in water or saline solution. For long trips (two days or more) the clinical samples (including A c c e p t e d M a n u s c r i p t kunkers) should be transported in saline solution with a few drops of broad spectrum antibiotics such as chloramphenicol or tetracycline. However, it was shown that specimens can also be refrigerated and P. insidiosum can be recovered from specimens that have been refrigerated for up to 5 days (Grooters et al., 2002b). In our experience however, cooling at 4˚C (shipping on ice) inhibits growth of P. insidiosum from about 20% of the clinical samples.

A positive diagnosis for P. insidiosum infection can be obtained in three ways: a) determination of the presence of the agent by wet mount examination in 10% KOH followed by culturing, b) detection of anti-P. insidiosum antibodies using serological assays , and c) detection of DNA of the infectious agent in the infected tissue by PCR and sequencing.

Cytology and histology, may help in the diagnosis of pythiosis, but do not allow differentiation between pythiosis and infections caused by the zygomycetes Connidiobolus and Basidiobolus [START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF][START_REF] Grooters | Pythiosis and Zygomycosis[END_REF].

a) Culturing and Wet Mount Examination

Wet mount examination in 10% KOH can be performed directly on samples taken from the infected individual. Wet mount preparations are a rapid way to microscopically detect the presence of sparsely septate hyphae, which may suggest the presence of P. insidiosum and pythiosis (Fig. 8). Next, a positive diagnosis can be made by culturing followed by sporulation of the pathogen in liquid cultures (Mendoza andPrendas, 1986, Chaiprasert et al., 1990).The production of zoospores alone is not sufficient for a positive diagnosis as other oomycetes produce similar zoospores. Kunkers collected from horses are more likely than biopsied tissues to yield a positive culture. Care should be taken to avoid bacterial contamination upon culturing by the addition of antibiotics and careful processing of samples [START_REF] Grooters | Pythiosis and Zygomycosis[END_REF]. Usually, the collected biopsy samples, kunkers (horses) or tissue, are washed three times with sterile saline solution before culture. The tissue is then cut into 5 to 10 mm in diameter blocks, implanted into 2% dextrose Sabouraud agar plates and incubated at 37 o C. A beaker with distilled water to increase the humidity inside the incubator is recommended. Positive samples are detected after 24 to 48 hours of incubation as small radiate growing colonies coming from the inoculated blocks (L. Mendoza, unpublished data) (Fig. 1A).

For an accurate diagnosis, a tissue biopsy or tissue deep scrapings can be taken from cutaneous and subcutaneous pythiosis cases; clinical samples can be cultured and microscopically examined for the presence of sparsely septate hyaline hyphae (Fig. 1B). Due to the fact that P. insidiosum in culture does not develop sporangia on the commonly used A c c e p t e d M a n u s c r i p t agar media, the induction of a sporangium producing motile biflagellate zoospores should usually be performed in order to identify the pathogen as P. insidiosum (Mendoza andPrendas, 1988, Grooters et al., 2002b) (Fig. 9). All Pythium species develop zoospores in wet cultures in the presence of calcium and magnesium iones. Since P. insidiosum was recognized as the only oomycete pathogenic for mammals for a long time, induction of zoosporogenesis was considered enough for a presumptive diagnosis of pythiosis. With the description of an emerging oomycosis in 2003, that still awaits confirmation this might no longer be the case [START_REF] Grooters | Clinicopathologic findings associated with Lagenidium sp. infection in 6 dogs: initial description of an emerging oomycosis[END_REF]. However, further identification of the culture as P. insidiosum by serology or using molecular tools, including sequencing is recommended in any case [START_REF] Schurko | Development of a species specific probe for Pythium insidiosum and the diagnosis of Pythiosis[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF].

Histopathology

In contrast to zygomycetes in the order of the mucorales (Absidia, Mucor, Rhizopus, Saksenaea, and others) and in the order of the entomophthorales (Basidiobolus and Conidiobolus) Pythium insidiosum hyphae do not stain well in Hematoxylin and Eosin (H&E) [START_REF] Ribes | Zygomycetes in human disease[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. Histopathologically a pyrogranulomatous inflammatory infiltration with large quantities of eosinophilic granulocytes is often seen [START_REF] Rees | Disorders of the skin[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF][START_REF] Grooters | Pythiosis and Zygomycosis[END_REF]. Although the visualization of hyphae is difficult in H&E staining, the presence of necrotic eosinophilic granulomas should lead to consideration of the presence of entomophthoromycetous fungi (Conidiobolus and Basidiobolus species), P. insidiosum, and/or putative parasitic infections such as habronemiasis in horses (Fig. 10). To visualize P. insidiosum hyphae in tissue, samples can be stained with PAS (periodic acid-Schiff) or Gomori methenamine silver staining of which the latter is to be preferred [START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. P. insidiosum hyphae are present as 2.6 to 6.4 μm wide (sometimes as large as >10.0 μm), irregular sparsely septate hyaline filaments with a thick cell wall. Occasionally branches are formed at angles of 90 o degrees [START_REF] Miller | Granulomatous and neoplastic disease of equine skin[END_REF][START_REF] Miller | The comparative pathology of equine cutaneous phycomycosis[END_REF][START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF] (Fig. 10).

b) Serodiagnosis.

Serodiagnosis of pythiosis can be performed by immunodiffusion. The test is very specific but unfortunately has a low sensitivity (Mendoza et al., 1986, Prachartam et al., 1991). Other tests based on detection of antibodies, like an Enzyme Linked Immuno-Sorbent Assay (ELISA), an immunochromatographic assay or a Western blot were developed later to increase sensitivity and specificity (Mendoza et al., 1992a[START_REF] Mendoza | Serodiagnosis of human and animal pythiosis using an enzyme-linked immunosorbent assay[END_REF], Grooters et al., 2002a[START_REF] Krajaejun | Development and evaluation of an in-house enzyme-linked immunosorbent assay for early diagnosis and monitoring of human pythiosis[END_REF][START_REF] Chindamporn | Antibodies in the sera of host species with Pythiosis recognize a variety of unique immunogens in geographically divergent Pythium insidiosum strains[END_REF], Krajaejun et al., 2009). Because these tests are difficult to perform in rural areas, [START_REF] Jindayok | Hemagglutination test for rapid serodiagnosis of human pythiosis[END_REF] developed a haemagglutination A c c e p t e d M a n u s c r i p t 16 test in which agglutination of sheep red blood cells coated with a P. insidiosum extract is tested against serum samples of patients suspected of pythiosis. The test was found to be simple, rapid and reliable for serodiagnosis of vascular and cutaneous pythiosis. P. insidiosum can also be identified in fixed tissues by immunofluorescence (Mendoza et al., 1987) or by the immunoperoxidase staining technique (Brown and Roberts,1988).

c) Molecular diagnosis, PCR.

Molecular techniques have been developed to identify P. insidosum in the clinical laboratory in the absence of culture. A specific diagnostic PCR using the internal transcribed spacer (ITS) of the rRNA locus of P.insidiosum has been used by several laboratories [START_REF] Grooters | Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum[END_REF][START_REF] Reis | Disseminated pythiosis in three horses[END_REF][START_REF] Vanittanakom | Identification of Emerging Human-Pathogenic Pythium insidiosum by serological and molecular assay based methods[END_REF][START_REF] Moraes | Morphological and molecular characterization of an equine isolate of Pythium insidiosum and comparison with the first human isolate from the same geographic region[END_REF]. Since most skin and intestinal lesions are contaminated with environmental microbes of which DNA sequences are not yet available from the data base, diagnostics based solely on the specific molecular weight of amplicons has to be interpreted with caution. A species specific DNA probe, based on a 530 bp fragment of the ribosomal intergenic spacer (IGS), was developed by [START_REF] Schurko | Development of a species specific probe for Pythium insidiosum and the diagnosis of Pythiosis[END_REF]. Although these methodologies are not yet available on a large scale, they can be of help in cases of where fixed tissue is submitted for histopathology (see also serodiagnosis).

Management of pythiosis

The infections caused by P. insidiosum poorly respond to therapy. In addition to wide surgical excision, antimicrobial agents and immunotherapy have been used with some success in the treatment of pythiosis. Independent of the chosen therapy, it is of utmost importance that treatment starts as early as possible.

Radical surgery, including amputation, still is the most used and effective treatment for this infection in humans and animals [START_REF] Mcmullan | Amphothericin B for the treatment of localized subcutaneous phycomycosis in the horse[END_REF][START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF][START_REF] Thomas | Pythiosis in dogs and cats[END_REF][START_REF] Krajaejun | Clinical and Epidemiological Analysis of Human Pythiosis in Thailand[END_REF]. Surgical debridement of skin lesions in dogs and horses with the disease is very popular as well but a high rate of reoccurrence is seen (45%). Some investigators have recommended immunotherapy as an important alternative for the treatment of equine pythiosis .It has to be taken into account that the reported cure rates of different therapies most often are calculated from a small number of patients and that therapy is often a combination of actions like surgical therapy, immunotherapy or administration of antimycotic agents.

Antimycotic agents

A c c e p t e d M a n u s c r i p t Since P. insidiosum was believed to be a fungus several antimycotic agents were used to treat the infection [START_REF] Mcmullan | Amphothericin B for the treatment of localized subcutaneous phycomycosis in the horse[END_REF]. However, Stramenopilan microbes such as P. insidiosum lack ergosterol in their cytoplasmic membrane and thus should not be susceptible to antimycotic agents [START_REF] Sekhon | In vitro sensitivity of Penicillum marneffei and Pythium insidiosum to various antifungal agents[END_REF][START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF][START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF].

Agents interfering with ergosterol biosynthesis like the azoles (itraconazole, ketoconazole, miconazole, fluconazole etc.) and terbinafine and amphotericin B change the permeability of the cell membrane, causing fungal cell lysis and thus can be expected to have little effect on P. insidiosum. Despite this drawback there are reports of clinical success using these drugs [START_REF] Bissonnette | Nasal and retrobulbar mass in a cat caused by Pythium insidiosum[END_REF][START_REF] Triscott | Human subcutaneous pythiosis[END_REF][START_REF] Shenep | Successful medial therapy for deeply invasive facial infection due to Pythium insidiosum in a child[END_REF][START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF]. Several reports on the sensitivity of P. insidiosum for combinations of ergosterol biosynthesis inhibitors and caspofungin (an inhibitor of β-glucan synthesis, (Deresinski and Stevens, 2003) have appeared recently [START_REF] Grooters | Pythiosis, lagenidosis and zygomycosis in small animals[END_REF][START_REF] Pereira | Caspofungin in vitro and in vivo activity against Brazilian Pythium insidiosum strains isolated from animals[END_REF], 2008, Argenta et al., 2008[START_REF] Brown | In vitro susceptibility of Pythium insidiosum and a Lagenidium sp to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam[END_REF], Cavalheiro et al., 2009a, 2009b[START_REF] Argenta | In vitro paradoxical growth of Pythium insidiosum in the presence of caspofungin[END_REF]. The oomycete cell wall mainly contains cellulose and β-glucan, which is an essential component of the cell wall [START_REF] Hendrix | Sterol induction of reproduction and stimulation of growth of Pythium and Phytophtora[END_REF]. Consequently, in these reports inhibitors of β-glucan synthesis (like caspofungin) have been tested.

Caspofungin when tested in vivo in a model such as the rabbit, generates a reduction in the growth of the lesions in treated animals. Growth of the lesions resumes when therapy is stopped [START_REF] Pereira | Caspofungin in vitro and in vivo activity against Brazilian Pythium insidiosum strains isolated from animals[END_REF]. Combinations of terbinafine and itraconazole or voriconazole performed better against a number of isolates of P. insidiosum than each agent apart [START_REF] Argenta | In vitro activities of voriconazole, itraconazole, terbinafine, alone or in combination against Pythium insidiosum isolates from Brazil[END_REF]. An observation also made for combinations of terbinafine and amphotericin B, metronidazole, rifampicin, ibuprofen and fluvastatin (Carvalheiro et al., 2009a(Carvalheiro et al., , 2009b)). It is of concern that a high variability in susceptibility of the different P. insidiosum strains used in these studies was observed (Cavalheiro et al., 2009a). An antimycotic agent that awaits further study of its usefulness against P. insidiosum infection is the phenylamide compound mefenoxam, an inhibitor of RNA polymerase that has been used against plant pathogenic oomycetes [START_REF] Brown | In vitro susceptibility of Pythium insidiosum and a Lagenidium sp to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam[END_REF]. Unfortunately, the extensive testing of new antimycotic drugs in animals is often hampered by the costs involved.

Immunotherapy

Immunotherapy for the treatment of pythiosis in horses has been used for more than 20 years and more than 600 animals have been treated today [START_REF] Miller | Treatment of equine phycomycosis by immunotherapy and surgery[END_REF][START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF][START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF]. The first vaccine, developed in 1981 and derived from killed ultrasonicated mycelium, was effective in half of the equine patients and clinical improvement was observed in another 33% [START_REF] Miller | Treatment of equine phycomycosis by immunotherapy and surgery[END_REF]. A second vaccine containing antigens secreted by P.insidiosum and obtained from a broth culture after precipitation was tested in a number of horses from Costa Rica [START_REF] Mendoza | Equine pythiosis in Costa Rica:Report of 39 cases[END_REF]. The efficacy of this vaccine was not much different from the efficacy of the vaccine developed by Miller, but it is more stable and easier to prepare (Mendoza et al., 2003). In a clinical study involving 18 infected horses and 6 infected dogs which were treated with P. insidiosum-vaccine 72% of the horses and 2 dogs were cured. It is important to note that the cure rates in this study were obtained by combination therapies using either surgery or antimycotical agents. Most animals that were treated developed a mild reaction at the injection site [START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF].

After a week of immunotherapy it became evident that the hyphae of P. insidiosum in the infected tissue were damaged. In dogs this therapy was also applied with some success [START_REF] Hensel | Immunotherapy for treatment of multicentric cutaneous pythiosis in a dog[END_REF]. Immunotherapy in dogs has a lower cure rate than in horses and in cats it has only has been tested three times with no response whatsoever [START_REF] Thomas | Pythiosis in dogs and cats[END_REF]. In cattle immunotherapy worked in the majority of the treated cases, about 65 animals have been treated with success. This variation in success rate between species is intriguing and deserves further study (see also Immunology of Pythium insidiosum infections). Vaccines prepared from extracts of P. insidiosum cultures in liquid medium with ultrasound or by vortexing have also been used for immunotherapy. The vaccine prepared by ultrasound treatment showed no effect on the lesions while the vortexed vaccine reduced the lesions in experimental rabbits by 71% (Santurio et al., 2003a).

In humans, immunotherapy was first successfully used in 1998 in a Thai boy who had a P. insidiosum vascular infection where surgery and antimycotic therapy did not work adequately. The boy was given a dose of P. insidiosum vaccine twice, with an interval of 2 weeks and within a year healing occurred [START_REF] Thitithanyanont | Use of an immunogenic therapeutic vaccine to treat a lifethreatening human arteritic infection caused by Pythium insidiosum[END_REF]. In a clinical study in people suffering from the vascular form of pythiosis, immunotherapy was used as a last resort [START_REF] Wanachiwanawin | Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans[END_REF]. After two administrations of the vaccine with a two week interval, four patients reacted positively to the vaccine, two patients moderately and two patients showed no response. The last two deceased within a short time. It is assumed that the success of this therapy is better if it is applied as soon as the disease is diagnosed. The mortality increases especially when people suffer from chronic lesions older than two months [START_REF] Wanachiwanawin | Efficacy of immunotherapy using antigens of Pythium insidiosum in the treatment of vascular pythiosis in humans[END_REF]. Until now ~60 Thai patients have been treated with immunotherapy against P. insidiosum infection with about 55% cure rate. A likely explanation for the mechanism by which immunotherapy against P.insidiosum infection works is that a switch in the host's immune response occurs from a Th2 (during infection) to a Th1 immune response (after immunotherapy) (Mendoza et al. [START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF]. This is supported by the detection of an increase in interleukins related to Th1 or Th2 immune responses [START_REF] Thitithanyanont | Use of an immunogenic therapeutic vaccine to treat a lifethreatening human arteritic infection caused by Pythium insidiosum[END_REF]. For instance, the immune response to P. insidiosum infection in humans appears in all cases to be characterized by an elevated response in interleukin 4 and 5 production, with high IgE titers, and inflammatory cells such as mast cells and eosinophils present in large amounts. This response is consistent with a Th2 response and suggests that the pathogen has developed an evolutionary strategy to present antigens to the immune system that may trigger such a response. In contrast, in vaccinated cured patients a response with high titres of interleukin 2 and INF-γ which induce a mononuclear immune response, typical of a Th1 response is triggered [START_REF] Thitithanyanont | Use of an immunogenic therapeutic vaccine to treat a lifethreatening human arteritic infection caused by Pythium insidiosum[END_REF], Mendoza et al., 2003a). Details of the immune response triggered during infection and after immunotherapy are shown in (Fig. 11). It has been argued that an effective treatment of P. insidiosum in the future should include immunotherapy and a combination of both immunotherapy and surgery or immunotherapy and antifungal drugs [START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF]. A rapid diagnosis and an early start of the treatment are essential in the management of this infection [START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF]. Prevention P. insidiosum infection occurs after exposure of hosts with an open skin to environments containing propagules (zoospores or hyphae) of the pathogen. Tropical wetland environments have been implicated as important infection sites including ponds of water, soil and grass in the endemic areas. People and animals in contact with zoospores (incriminated as the infecting units) from the environment are at risk of contracting the infection. Besides zoospores, hyphae and resting oospores are also seen as a possible source of infection.

Acquiring the infection from infected animals has not yet been reported [START_REF] Mendoza | Topley and Wilson's Microbiology and Microbial Infections[END_REF][START_REF] Mendoza | Immunology and immunotherapy of the infections caused by Pythium insidiosum[END_REF]. In fact, Arvis (1916) mentions explicitly that a foal touching an infected area beside the udder with its nose and lips for weeks stayed completely healthy.

Nevertheless, physicians and medical personnel should handle cases of the disease carefully wearing protective gloves during treatment of wounds in humans and animals suffering from pythiosis.

Conclusions

Pythium insidiosum is the cause of pythiosis, a rare non-transmissible disease that currently occurs in the tropics, subtropics and some temperate regions. However, in view of the global warming it can be expected that this disease could spread to more temperate areas A c c e p t e d M a n u s c r i p t 20 in the world [START_REF] Mendoza | Pythium insidiosum and mammalian hosts[END_REF]. Relatively few cases of pythiosis are reported yearly, irrespective of the species infected, but this is very likely an underestimation as the disease mainly occurs in rural areas in developing countries. Pythium insidiosum has the appearance of a fungus, but is closely related to algae and diatomaea. Infection occurs mainly in horses, dogs and humans. Infections are caused by motile zoospores that are present in surface water and infect an apparently healthy individual through small skin lesions. The zoospores are attracted to these wounds or lesions by a chemo-attractant. No individual-individual transmission occurs. Extensive surgery is in many cases the only way to treat patients. This is not always successful and death of patients occurs regularly. Immunotherapy is also relatively successful but a marked difference in response to immunotherapy from one species to another is observed. The meaning of the immunogenic preferences within different hosts is not known, but ongoing investigations in this area hopefully will soon shed some light on this peculiar finding.

Since P. insidiosum is not a true fungus it does not react well to treatment with antifungal agents. The search for a chemical therapy for this disease therefore has to continue unabated. Little is known about potential virulence factors of the agent. Hopefully knowledge of this aspect of P. insidiosum pathogenesis will increase once the complete nucleotide sequence of its genome becomes available. In general the molecular biology of P.insidiosum pathogenesis is not yet well developed in comparison to other infectious agents. Until then, P. insidiosum will remain a fascinating microbe for those who study it and a devastating one for those who suffer from it. A five days old culture of Pythium insidiosum at 37 o C grown on 2% Sabouraud dextrose agar (Panel A). P. insidiosum from plate cultures showing sparsely septate hyphae in lactophenol blue (20 X) (Panel B). Figure 2 Fresh kunkers collected from a horse with cutaneous pythiosis. Kunkers are stony masses found only in horses with pythiosis. Note the different shapes developed in horses with pythiosis. Similar masses have been reported in equine habronemiasis. A differential diagnosis of habronemasias and pythiosis can be made by demonstration of sparsely septate hyaline hyphae within kunkers in the case of pythiosis. Figure 3 Equine pythiosis can affect different areas of the skin (Panel A and B) and in chronic cases bones can also be invaded (Panel C). Figure 11.

The right section of the figure shows the inflammatory response of a host naturally exposed to Pythium insidiosum. In this scenario, zoospores (or other propagules) of P. insidiosum attach to the injured skin of a host; the encysted zoospore stimulated by the host's temperature develops a germ tube that actively penetrates the host tissue causing the infection. As the hypha develops it releases exoantigens that are presented to the host immune system. The antigen presenting cells (APC) will process the immunogens and by releasing IL4 the Th0 naïve cells become Th2. The stimulated Th2 subset will then release IL4, IL5, and IL10, which in turn stimulates B cells to produce precipitating IgG, IgM and IgE molecules. IgE along with IL5 triggers the migration of mast cells and eosinophils to the site of infection that later degranulate over the P. insidiosum hyphae causing tissue damage in the infected host. The left side of the figure depicts the putative response to P. insidiosum immunogens after immunotherapy. Upon injection the immunogens are presented to APC in a different fashion than during natural infection. The Th0 naïve cells turn into Th1 releasing IFNγ and IL2 stimulating the cell mediate immunity (CMI) and cytotoxic lymphocytes (CTL) that eventually could eliminate the pathogen from the infected tissues. Experimental evidence suggests that this strategy could protect the host for short periods of time (one year) by stimulating B cells to produce protective IgG classes.

  s c r i p t insects and thus the possibility of transmission through infected mosquitoes is of concern, especially in the tropical regions where mosquitoes are prevalent.

  A c c e p t e d M a n u s c r i p t 10 In the last 10 years various reports on pythiosis in captive zoo animals have appeared. The animals involved include animals such as bears, camels and members of the Pantherae such as a tiger and a jaguar. Reports on the following species are available.

  ,

Figure 1

 1 Figure1A five days old culture of Pythium insidiosum at 37 o C grown on 2% Sabouraud dextrose agar (Panel A). P. insidiosum from plate cultures showing sparsely septate hyphae in lactophenol blue (20 X) (Panel B). Figure2Fresh kunkers collected from a horse with cutaneous pythiosis. Kunkers are stony masses found only in horses with pythiosis. Note the different shapes developed in horses with pythiosis. Similar masses have been reported in equine habronemiasis. A differential diagnosis of habronemasias and pythiosis can be made by demonstration of sparsely septate hyaline hyphae within kunkers in the case of pythiosis. Figure3Equine pythiosis can affect different areas of the skin (Panel A and B) and in chronic cases bones can also be invaded (Panel C). Figure 4. In Panel A and B the clinical manifestation of cutaneous pythiosis in dogs is shown. In dogs this type of infection is very insidious and life threatening. Ulcerate granulomatous tissues, usually contaminated with bacteria are the main characteristic of dog pythiosis. Intestinal pythiosis is the most common type of infection in this species. In Panel C a duodenal tumoral-like mass from a dog with chronic gastro-intestinal tract pythiosis is shown (Courtesy of Dr. Y. Perazzo).Figure 5. Pythiosis in cats occurs less frequent than in dogs and horses. The infection affects the subcutaneous tissue but rarely becomes ulcerate. The cat in the figure has a typical mass near the toraxic area caused by Pythium insidiosum (Courtesy of Dr. R.C. Thomas) Figure 6. In Panels A and B two beef cattle with ulcerate tissue caused by Pythium insidiosum are shown. Note the swelling of the affected limb and the presence of some fistulae (Panel A) (Courtesy of Drs. R.C. Perez and J.J. Luis-Leon).Figure 7. In Panel A a sheep with a strong inflammation of the face and bloody rhinorrhea caused by Pythium insidiosum is shown. In Panel B the location of the granulomatous tissue is inside the nostril passages (Courtesy of Drs. F. Riet-Correa and S.M.S. Silva).Figure 8. A 10% KOH wet mount preparation of a kunker from a horse. Panel A and B are the continuous microscopic field of the clinical sample. Note the long sparsely septate hyaline hyphae of Pythium insidiosum. The presence of numerous vesicles within the hyphae is usually observed.Figure 9. Water cultures containing positive ions and grass leaves inoculated with Pythium insidiosum trigger the formation of sporangia containing zoospores. Sporangia are not differentiated from vegetative hyphae and are present inside the grass blades. Only a hypha-like discharge tube grows out through which the protoplasma is released in a thinwalled vesicle at the tip. Inside the vesicle the zoospores are differentiated. Zoospores are released after rupture of the vesicle membrane. Panel A: the vesicles with zoospores that developed after three hours incubation. Panel B: a close-up of a vesicle before zoospore release. Below the vesicle the empty discharge tube is visible. Figure 10. Panels A and B are H&E histological preparations showing a granulomatous reaction with numerous eosinophils, giant cells, mast cells and other cells from an intestinal case of dog pythiosis. Panel B: unstained hyphae of Pythium insidiosum (arrow, 40X). Panels

  Figure1A five days old culture of Pythium insidiosum at 37 o C grown on 2% Sabouraud dextrose agar (Panel A). P. insidiosum from plate cultures showing sparsely septate hyphae in lactophenol blue (20 X) (Panel B). Figure2Fresh kunkers collected from a horse with cutaneous pythiosis. Kunkers are stony masses found only in horses with pythiosis. Note the different shapes developed in horses with pythiosis. Similar masses have been reported in equine habronemiasis. A differential diagnosis of habronemasias and pythiosis can be made by demonstration of sparsely septate hyaline hyphae within kunkers in the case of pythiosis. Figure3Equine pythiosis can affect different areas of the skin (Panel A and B) and in chronic cases bones can also be invaded (Panel C). Figure 4. In Panel A and B the clinical manifestation of cutaneous pythiosis in dogs is shown. In dogs this type of infection is very insidious and life threatening. Ulcerate granulomatous tissues, usually contaminated with bacteria are the main characteristic of dog pythiosis. Intestinal pythiosis is the most common type of infection in this species. In Panel C a duodenal tumoral-like mass from a dog with chronic gastro-intestinal tract pythiosis is shown (Courtesy of Dr. Y. Perazzo).Figure 5. Pythiosis in cats occurs less frequent than in dogs and horses. The infection affects the subcutaneous tissue but rarely becomes ulcerate. The cat in the figure has a typical mass near the toraxic area caused by Pythium insidiosum (Courtesy of Dr. R.C. Thomas) Figure 6. In Panels A and B two beef cattle with ulcerate tissue caused by Pythium insidiosum are shown. Note the swelling of the affected limb and the presence of some fistulae (Panel A) (Courtesy of Drs. R.C. Perez and J.J. Luis-Leon).Figure 7. In Panel A a sheep with a strong inflammation of the face and bloody rhinorrhea caused by Pythium insidiosum is shown. In Panel B the location of the granulomatous tissue is inside the nostril passages (Courtesy of Drs. F. Riet-Correa and S.M.S. Silva).Figure 8. A 10% KOH wet mount preparation of a kunker from a horse. Panel A and B are the continuous microscopic field of the clinical sample. Note the long sparsely septate hyaline hyphae of Pythium insidiosum. The presence of numerous vesicles within the hyphae is usually observed.Figure 9. Water cultures containing positive ions and grass leaves inoculated with Pythium insidiosum trigger the formation of sporangia containing zoospores. Sporangia are not differentiated from vegetative hyphae and are present inside the grass blades. Only a hypha-like discharge tube grows out through which the protoplasma is released in a thinwalled vesicle at the tip. Inside the vesicle the zoospores are differentiated. Zoospores are released after rupture of the vesicle membrane. Panel A: the vesicles with zoospores that developed after three hours incubation. Panel B: a close-up of a vesicle before zoospore release. Below the vesicle the empty discharge tube is visible. Figure 10. Panels A and B are H&E histological preparations showing a granulomatous reaction with numerous eosinophils, giant cells, mast cells and other cells from an intestinal case of dog pythiosis. Panel B: unstained hyphae of Pythium insidiosum (arrow, 40X). Panels

  Figure1A five days old culture of Pythium insidiosum at 37 o C grown on 2% Sabouraud dextrose agar (Panel A). P. insidiosum from plate cultures showing sparsely septate hyphae in lactophenol blue (20 X) (Panel B). Figure2Fresh kunkers collected from a horse with cutaneous pythiosis. Kunkers are stony masses found only in horses with pythiosis. Note the different shapes developed in horses with pythiosis. Similar masses have been reported in equine habronemiasis. A differential diagnosis of habronemasias and pythiosis can be made by demonstration of sparsely septate hyaline hyphae within kunkers in the case of pythiosis. Figure3Equine pythiosis can affect different areas of the skin (Panel A and B) and in chronic cases bones can also be invaded (Panel C). Figure 4. In Panel A and B the clinical manifestation of cutaneous pythiosis in dogs is shown. In dogs this type of infection is very insidious and life threatening. Ulcerate granulomatous tissues, usually contaminated with bacteria are the main characteristic of dog pythiosis. Intestinal pythiosis is the most common type of infection in this species. In Panel C a duodenal tumoral-like mass from a dog with chronic gastro-intestinal tract pythiosis is shown (Courtesy of Dr. Y. Perazzo).Figure 5. Pythiosis in cats occurs less frequent than in dogs and horses. The infection affects the subcutaneous tissue but rarely becomes ulcerate. The cat in the figure has a typical mass near the toraxic area caused by Pythium insidiosum (Courtesy of Dr. R.C. Thomas) Figure 6. In Panels A and B two beef cattle with ulcerate tissue caused by Pythium insidiosum are shown. Note the swelling of the affected limb and the presence of some fistulae (Panel A) (Courtesy of Drs. R.C. Perez and J.J. Luis-Leon).Figure 7. In Panel A a sheep with a strong inflammation of the face and bloody rhinorrhea caused by Pythium insidiosum is shown. In Panel B the location of the granulomatous tissue is inside the nostril passages (Courtesy of Drs. F. Riet-Correa and S.M.S. Silva).Figure 8. A 10% KOH wet mount preparation of a kunker from a horse. Panel A and B are the continuous microscopic field of the clinical sample. Note the long sparsely septate hyaline hyphae of Pythium insidiosum. The presence of numerous vesicles within the hyphae is usually observed.Figure 9. Water cultures containing positive ions and grass leaves inoculated with Pythium insidiosum trigger the formation of sporangia containing zoospores. Sporangia are not differentiated from vegetative hyphae and are present inside the grass blades. Only a hypha-like discharge tube grows out through which the protoplasma is released in a thinwalled vesicle at the tip. Inside the vesicle the zoospores are differentiated. Zoospores are released after rupture of the vesicle membrane. Panel A: the vesicles with zoospores that developed after three hours incubation. Panel B: a close-up of a vesicle before zoospore release. Below the vesicle the empty discharge tube is visible. Figure 10. Panels A and B are H&E histological preparations showing a granulomatous reaction with numerous eosinophils, giant cells, mast cells and other cells from an intestinal case of dog pythiosis. Panel B: unstained hyphae of Pythium insidiosum (arrow, 40X). Panels

  Figure1A five days old culture of Pythium insidiosum at 37 o C grown on 2% Sabouraud dextrose agar (Panel A). P. insidiosum from plate cultures showing sparsely septate hyphae in lactophenol blue (20 X) (Panel B). Figure2Fresh kunkers collected from a horse with cutaneous pythiosis. Kunkers are stony masses found only in horses with pythiosis. Note the different shapes developed in horses with pythiosis. Similar masses have been reported in equine habronemiasis. A differential diagnosis of habronemasias and pythiosis can be made by demonstration of sparsely septate hyaline hyphae within kunkers in the case of pythiosis. Figure3Equine pythiosis can affect different areas of the skin (Panel A and B) and in chronic cases bones can also be invaded (Panel C). Figure 4. In Panel A and B the clinical manifestation of cutaneous pythiosis in dogs is shown. In dogs this type of infection is very insidious and life threatening. Ulcerate granulomatous tissues, usually contaminated with bacteria are the main characteristic of dog pythiosis. Intestinal pythiosis is the most common type of infection in this species. In Panel C a duodenal tumoral-like mass from a dog with chronic gastro-intestinal tract pythiosis is shown (Courtesy of Dr. Y. Perazzo).Figure 5. Pythiosis in cats occurs less frequent than in dogs and horses. The infection affects the subcutaneous tissue but rarely becomes ulcerate. The cat in the figure has a typical mass near the toraxic area caused by Pythium insidiosum (Courtesy of Dr. R.C. Thomas) Figure 6. In Panels A and B two beef cattle with ulcerate tissue caused by Pythium insidiosum are shown. Note the swelling of the affected limb and the presence of some fistulae (Panel A) (Courtesy of Drs. R.C. Perez and J.J. Luis-Leon).Figure 7. In Panel A a sheep with a strong inflammation of the face and bloody rhinorrhea caused by Pythium insidiosum is shown. In Panel B the location of the granulomatous tissue is inside the nostril passages (Courtesy of Drs. F. Riet-Correa and S.M.S. Silva).Figure 8. A 10% KOH wet mount preparation of a kunker from a horse. Panel A and B are the continuous microscopic field of the clinical sample. Note the long sparsely septate hyaline hyphae of Pythium insidiosum. The presence of numerous vesicles within the hyphae is usually observed.Figure 9. Water cultures containing positive ions and grass leaves inoculated with Pythium insidiosum trigger the formation of sporangia containing zoospores. Sporangia are not differentiated from vegetative hyphae and are present inside the grass blades. Only a hypha-like discharge tube grows out through which the protoplasma is released in a thinwalled vesicle at the tip. Inside the vesicle the zoospores are differentiated. Zoospores are released after rupture of the vesicle membrane. Panel A: the vesicles with zoospores that developed after three hours incubation. Panel B: a close-up of a vesicle before zoospore release. Below the vesicle the empty discharge tube is visible. Figure 10. Panels A and B are H&E histological preparations showing a granulomatous reaction with numerous eosinophils, giant cells, mast cells and other cells from an intestinal case of dog pythiosis. Panel B: unstained hyphae of Pythium insidiosum (arrow, 40X). Panels

  Figure1A five days old culture of Pythium insidiosum at 37 o C grown on 2% Sabouraud dextrose agar (Panel A). P. insidiosum from plate cultures showing sparsely septate hyphae in lactophenol blue (20 X) (Panel B). Figure2Fresh kunkers collected from a horse with cutaneous pythiosis. Kunkers are stony masses found only in horses with pythiosis. Note the different shapes developed in horses with pythiosis. Similar masses have been reported in equine habronemiasis. A differential diagnosis of habronemasias and pythiosis can be made by demonstration of sparsely septate hyaline hyphae within kunkers in the case of pythiosis. Figure3Equine pythiosis can affect different areas of the skin (Panel A and B) and in chronic cases bones can also be invaded (Panel C). Figure 4. In Panel A and B the clinical manifestation of cutaneous pythiosis in dogs is shown. In dogs this type of infection is very insidious and life threatening. Ulcerate granulomatous tissues, usually contaminated with bacteria are the main characteristic of dog pythiosis. Intestinal pythiosis is the most common type of infection in this species. In Panel C a duodenal tumoral-like mass from a dog with chronic gastro-intestinal tract pythiosis is shown (Courtesy of Dr. Y. Perazzo).Figure 5. Pythiosis in cats occurs less frequent than in dogs and horses. The infection affects the subcutaneous tissue but rarely becomes ulcerate. The cat in the figure has a typical mass near the toraxic area caused by Pythium insidiosum (Courtesy of Dr. R.C. Thomas) Figure 6. In Panels A and B two beef cattle with ulcerate tissue caused by Pythium insidiosum are shown. Note the swelling of the affected limb and the presence of some fistulae (Panel A) (Courtesy of Drs. R.C. Perez and J.J. Luis-Leon).Figure 7. In Panel A a sheep with a strong inflammation of the face and bloody rhinorrhea caused by Pythium insidiosum is shown. In Panel B the location of the granulomatous tissue is inside the nostril passages (Courtesy of Drs. F. Riet-Correa and S.M.S. Silva).Figure 8. A 10% KOH wet mount preparation of a kunker from a horse. Panel A and B are the continuous microscopic field of the clinical sample. Note the long sparsely septate hyaline hyphae of Pythium insidiosum. The presence of numerous vesicles within the hyphae is usually observed.Figure 9. Water cultures containing positive ions and grass leaves inoculated with Pythium insidiosum trigger the formation of sporangia containing zoospores. Sporangia are not differentiated from vegetative hyphae and are present inside the grass blades. Only a hypha-like discharge tube grows out through which the protoplasma is released in a thinwalled vesicle at the tip. Inside the vesicle the zoospores are differentiated. Zoospores are released after rupture of the vesicle membrane. Panel A: the vesicles with zoospores that developed after three hours incubation. Panel B: a close-up of a vesicle before zoospore release. Below the vesicle the empty discharge tube is visible. Figure 10. Panels A and B are H&E histological preparations showing a granulomatous reaction with numerous eosinophils, giant cells, mast cells and other cells from an intestinal case of dog pythiosis. Panel B: unstained hyphae of Pythium insidiosum (arrow, 40X). Panels

  Figure1A five days old culture of Pythium insidiosum at 37 o C grown on 2% Sabouraud dextrose agar (Panel A). P. insidiosum from plate cultures showing sparsely septate hyphae in lactophenol blue (20 X) (Panel B). Figure2Fresh kunkers collected from a horse with cutaneous pythiosis. Kunkers are stony masses found only in horses with pythiosis. Note the different shapes developed in horses with pythiosis. Similar masses have been reported in equine habronemiasis. A differential diagnosis of habronemasias and pythiosis can be made by demonstration of sparsely septate hyaline hyphae within kunkers in the case of pythiosis. Figure3Equine pythiosis can affect different areas of the skin (Panel A and B) and in chronic cases bones can also be invaded (Panel C). Figure 4. In Panel A and B the clinical manifestation of cutaneous pythiosis in dogs is shown. In dogs this type of infection is very insidious and life threatening. Ulcerate granulomatous tissues, usually contaminated with bacteria are the main characteristic of dog pythiosis. Intestinal pythiosis is the most common type of infection in this species. In Panel C a duodenal tumoral-like mass from a dog with chronic gastro-intestinal tract pythiosis is shown (Courtesy of Dr. Y. Perazzo).Figure 5. Pythiosis in cats occurs less frequent than in dogs and horses. The infection affects the subcutaneous tissue but rarely becomes ulcerate. The cat in the figure has a typical mass near the toraxic area caused by Pythium insidiosum (Courtesy of Dr. R.C. Thomas) Figure 6. In Panels A and B two beef cattle with ulcerate tissue caused by Pythium insidiosum are shown. Note the swelling of the affected limb and the presence of some fistulae (Panel A) (Courtesy of Drs. R.C. Perez and J.J. Luis-Leon).Figure 7. In Panel A a sheep with a strong inflammation of the face and bloody rhinorrhea caused by Pythium insidiosum is shown. In Panel B the location of the granulomatous tissue is inside the nostril passages (Courtesy of Drs. F. Riet-Correa and S.M.S. Silva).Figure 8. A 10% KOH wet mount preparation of a kunker from a horse. Panel A and B are the continuous microscopic field of the clinical sample. Note the long sparsely septate hyaline hyphae of Pythium insidiosum. The presence of numerous vesicles within the hyphae is usually observed.Figure 9. Water cultures containing positive ions and grass leaves inoculated with Pythium insidiosum trigger the formation of sporangia containing zoospores. Sporangia are not differentiated from vegetative hyphae and are present inside the grass blades. Only a hypha-like discharge tube grows out through which the protoplasma is released in a thinwalled vesicle at the tip. Inside the vesicle the zoospores are differentiated. Zoospores are released after rupture of the vesicle membrane. Panel A: the vesicles with zoospores that developed after three hours incubation. Panel B: a close-up of a vesicle before zoospore release. Below the vesicle the empty discharge tube is visible. Figure 10. Panels A and B are H&E histological preparations showing a granulomatous reaction with numerous eosinophils, giant cells, mast cells and other cells from an intestinal case of dog pythiosis. Panel B: unstained hyphae of Pythium insidiosum (arrow, 40X). Panels

  ) and D (50X) are a Silver stain preparation of the same tissue. Note the presence of numerous none septate hyphae, some forming rounded shape structures.

  Figure 1

  Figure 2

Acknowlegements. We would like to thank Dr. Y. Perazzo, Dr. R.C. Thomas,, Dr. R. C. Perez , Dr. J.J. Luis-Leon, Dr. F. Riet-Correa and Dr. S.M.S. Silva for allowing us to use the pictures shown in Figs. 4, 5, 6 and 7.