Abstract

Systemic protothecosis was diagnosed in 17 Australian dogs between 1988 and 2005. There was a preponderance of young-adult (median 4 years), medium- to large-breed dogs. Females (12/17 cases) and Boxer dogs (7 cases, including 6 purebreds and one Boxer cross) were over-represented. Sixteen of 17 dogs died, with a median survival of four months. A disproportionate number of cases were from coastal Queensland. In most patients, first signs were referable to colitis (11/17 cases), which varied in severity, and was often present for many months before other symptoms developed. Subsequent to dissemination, signs were mostly ocular (12 cases) and/or neurologic (8 cases). Two dogs had signs due to bony lesions. Once dissemination was evident, death or euthanasia transpired quickly. Prototheca organisms had a tropism for the eye, central nervous system (CNS), bone, kidneys and myocardium, tissues with a good blood supply. Microscopic examination and culture of urine (5 cases), cerebrospinal fluid (CSF;1 case), rectal scrapings (4 cases), aspirates or biopsies of eyes (5 cases) and histology of colonic biopsies (6 cases) as well as skin and lymph nodes (2 cases) helped secure a diagnosis. Of the cases where culture was successful, P wickerhamii was isolated from two patients, while P zopfii was isolated from five. P zopfii infections had a more aggressive course. Treatment was not attempted in most cases. Combination therapy with amphotericin B and itraconazole proved effective in two cases, although in one of these treatment should have been for a longer duration. One surviving dog is currently still receiving itraconazole. Protothecosis should be considered in all dogs with refractory colitis, especially in female Boxers.

Introduction

Protothecosis is a rare disease of people and animals 1–8. In the veterinary literature, most reports concern cats 9–11, cattle 12 and dogs 13–40, although feline infections would appear to be the rarest. Protothecosis is caused by Prototheca spp., i.e., unicellular, achlorophyllic, aerobic algae 5, 6, closely related to members of the genus Chlorella35. Prototheca spp. have a world-wide distribution 1, 5–7 and are found in abundance in environmental niches including raw and treated sewage, trees, soil, flowing and standing water, food, cow's milk, and human and animal faeces 1, 5, 6, 8, 35.

Despite their abundance in nature and likely accidental ingestion, Prototheca spp. rarely cause disease in either people or animals. Since the first case was described in 1964, 107 human cases have been reported, mostly from North America and Asia 1, 5. Thirty-one canine cases have been described since the seminal report in 1969 by Van Kruiningen 14, predominantly as single case studies from North America.

Clinical features, response to therapy and prognosis for protothecosis differ greatly between host species. Disease in people is typically cutaneous or subcutaneous, while olecranon bursitis and systemic disease are reported less frequently 5, 6. Of the 107 reported human cases, 71 had cutaneous and/or subcutaneous manifestations 5, while 18 had systemic disease. Only two human deaths directly attributable to protothecosis have been reported 5, 40. In cats, only the cutaneous form has been described and wide surgical excision of lesions appears to be curative. 8, 10, 11, 41 Mastitis is the most common manifestation of protothecosis in cattle 1, 7, 8, 41, the organism presumably ascending via the teat canal.

In contrast, canine protothecosis is typically a widely disseminated disease with an insidious onset, relentless progression and inevitably fatal course 7, 8. Dogs generally show signs referable to the gastro-intestinal tract, particularly the colon, as well as ocular and/or neurologic symptoms. Common clinical signs include large bowel diarrhoea with fresh blood, blindness due to chorioretinitis and retinal detachment, deafness, seizures and ataxia. In many patients, colitis is present for many months prior to the onset of signs referable to dissemination. Short periods of remission, or a delay in progression, have been reported in some cases during treatment. However the majority of reported canine cases were euthanased or died soon after presentation. Necropsy confirms the disseminated nature of disease, with lesions in a variety of tissues including the colon, eyes, heart, brain, kidneys, skeletal muscle and liver 7, 8. Two dogs, each with cutaneous lesions, were reported to have survived the infection, including one dog that had organisms in urine 30, 31.

Of the three currently recognized Prototheca spp., only P.wickerhamii and P. zopfii have been incriminated as mammalian pathogens 1, 7, 8, 32–35, 41. Of the 74 human cases identified to species level, 70 involved P wickerhamii and only four were P. zopfii infections 1. P. wickerhamii is the only species isolated from feline lesions 10–12. P. zopfii has been incriminated in most cases of disseminated protothecosis in dogs 7, 8, 31, 33 and all cases of protothecal mastitis in cattle 7, 35.

A tentative diagnosis of protothecosis can be made by cytologic or histologic examination of representative tissue or fluid specimens, while definitive diagnosis is by culture. In previously reported cases, Prototheca organisms have been identified cytologically in urine sediment, vitreous humor, rectal scrapings, skin nodule aspirates and impression smears from infected tissues. Positive cultures have been obtained from vitreous humor, urine, blood, CSF, cutaneous lesions, and other affected tissues 7, 8, 38.

Prototheca can be cultured using blood agar and common mycologic media. Prototheca spp. grow rapidly on Sabouraud's dextrose agar (without cycloheximide) at 25–37oC, forming smooth, white or creamy yeast-like colonies, typically within 48 h 1, 6. Characteristic organism morphology, in all stages of development, can be recognised by staining a smear from colonial material using Gram's iodine solution 8. Lactophenol cotton blue slide preparations of these colonies reveal characteristic sporangia, containing sporangiospores 5. Identification to the species level relies on sugar and alcohol assimilation tests, susceptibility to clotrimazole, or by immunohistochemistry 5, 7, 35. Protothecosis can also be diagnosed by histologic evaluation of biopsy specimens. Organisms are variably visible in haematoxylin and eosin (H&E) stained sections, but their cell walls stain vividly with periodic acid Schiff (PAS), Gomori's methenamine silver and Giemsa 7, 8. Prototheca organisms range from 3–30 µm in diameter. P. wickerhamii cells tend to be round, forming sporangia (7–13 µm) containing up to 50 spherical sporangiospores. In contrast, P. zopfii cells are typically oval or cylindrical in shape, producing sporangia (14–25 µm) containing up to 20 sporangiospores 5.

Little has been established about the pathogenesis of Prototheca infections. It is thought that cutaneous infection in humans and cats result from traumatic inoculation of organisms from contaminated environmental sources 7. In people, concurrent therapy with corticosteroids appears to facilitate the establishment of disease 5. In dogs, it is likely that the colon is the primary site of infection with subsequent dissemination to other tissues after a variable time.

It has been suggested that immune dysfunction may account for the sporadic occurrence of canine protothecosis 36, 42, with an inherited immune deficiency suggested to explain the apparent susceptibility of Collies 22, 43. There is discrepant information in the human literature about the role of immune compromise in disease pathogenesis. In a review of 63 human cases, Wirth et al. 2 found olecranon bursitis and localized cutaneous infection developed more commonly in immunocompetent patients, whereas disseminated cutaneous lesions and visceral involvement mainly affected immunodeficient patients. In contrast, Leimann 5 considered disseminated disease to occur in both immunocompetent and immunocompromised patients. Disease in immunosuppressed patients is thought to be more florid 40.

As might be expected for a rare, sporadic disease, optimal therapy for protothecosis is not well established 5, 6, 43. Available information demonstrates major discrepancies between in vitro susceptibility data and the response to antimicrobials in vivo. Most localized human cases have been successfully treated by surgical excision of lesions combined with therapy with a variety of therapeutic agents 1, 40. There has, however, been little consistency in the clinical response to a given antimicrobial, with treatment failures necessitating use of alternate drug(s). Furthermore, long-term therapy (several months to years) is required in many instances. Agents with reported efficacy include amphotericin B (AMB; topically or systemically), itraconazole and fluconazole 1, 5, 40, 43, 44. Amikacin and tetracycline have been utilized also in some recent case studies 45. There is no information concerning the use of newer azoles or caspofungin. To date, intravenous (IV) AMB (in one form or another) is considered the most effective drug for treating protothecosis 6. The use of a wide range of antimicrobial agents, either singly or in combination, has been reported in dogs with systemic protothecosis, although none would appear to have convincing efficacy in the face of disseminated or CNS disease.

This multi-institutional study records 13 new cases of systemic canine protothecosis from Australia diagnosed over a 17-year period. To make this analysis more complete, findings from four previously reported Australian cases 37–40 are also included. To the best of our knowledge, these represent all confirmed canine cases in Australia during this period. This cohort of Australian patients was compared with the 27 canine protothecosis cases reported from outside Australia.

Methods

This was a retrospective study, with the index case being a patient treated by the first two authors. Twelve additional ‘new’ cases were obtained by contacting veterinary internists, ophthalmologists, neurologists, pathologists and dermatologists in private or institutional referral practice throughout Australia and New Zealand, and by appealing for cases at the 2005 ‘Science Week’ of the Australian College of Veterinary Scientists. Four previously published Australian cases 37–40 and 27 published overseas 13–38 were obtained by searching electronic databases (Medline, CAB) using the key words Prototheca, protothecosis, canine and dog(s). Cases were included only if sufficient clinical and laboratory data was provided to ensure that the diagnosis was correct and that comparisons with other reported cases was useful. Conference abstracts that were impossible to locate using the aforementioned electronic databases were not included.

Findings for all cases were collated. The age, breed, gender and place of domicile of affected animals were recorded. The first detected clinical sign(s) are listed separately to clinical signs that developed subsequently. Clinical pathology and diagnostic imaging abnormalities are listed for the Australian cases. The duration of illness, defined as the time between the first observation of clinical sign(s) and death or euthanasia, was calculated, as was outcome. Laboratory methods used to confirm the diagnosis, either ante- or post-mortem, were recorded. The anatomic sites of infection, based on clinical signs, laboratory and imaging data, and necropsy findings were tabulated. Where possible, it was noted whether a full or incomplete necropsy was performed. Where determined, the Prototheca species was listed.

Results

The clinical and laboratory findings from 17 Australian cases of canine protothecosis are summarized in Table 1. Following presentation of the overall data set, two representative cases are described in detail to illustrate specific aspects of this disease germane to its diagnosis. Canine protothecosis has been divided into three clinical forms: (i) cutaneous, (ii) systemic – where dogs showed involvement of a single body system, apart from skin and (iii) disseminated – where cases had evidence of haematogenous and/or lymphatic dissemination from the primary site of infection to one or more other tissues. In many cases, however, there was insufficient data to distinguish whether cases were in class (ii) or (iii).

Table 1

The clinical and laboratory findings from 17 Australian cases of canine protothecosis

No. Breed Age (yrs) & gender Initial clinical signs Subsequent clinical signs Clinical pathology & diagnostic imaging abnormalities Duration of illness (months) Out come Diagnosis Anatomic sites of infection Necropsy Prototheca specieRegion 
Boxer 1.5 FN Colitis Hindlimb lameness Proliferative periosteal lesions in diaphysis of both femurs Bone lesion aspirate cytology & culture Colon, Bone, Heart, Kidney, CNS, Liver, Pancreas Full (eyes not examined) P. zopfii Brisbane, South East QLD 
Boxer 2 FE Colitis Weight loss N/A 12 Colonic histopathology, faecal culture, rectal scrapes negative Colon, Mesenteric lymph nodes, Kidneys Full P. wickerhamii Brisbane, South East QLD 
Boxer 2.5 ME Colitis Weight loss N/A Colonic histopathology Colon N/A N/A Townsville, Far North QLD 
Boxer Cross 3 FE Polypdipsia, polyuria, incontinence Vision loss, neurologic signs N/A Vitreous cytology & culture; urine culture Eye, CNS, Kidneys N/A P. zopfii Toowoomba, South East QLD 
Boxer 4 FN Colitis, urinary incontinence, polydipsia, polyuria Vision loss, deafness, lameness, neurologic signs Multiple areas of periosteal new bone on diaphyses of both femurs and left humerus Urine sediment cytology & culture; Rectal scraping cytology; Colon histopathology Colon, Bone, Eye, Heart, Kidney, CNS, Liver, Intestine, Lymph nodes, Skeletal muscle Full P. zopfii Cessnock, NSW 
Boxer 4.5 FN Neurologic signs Deafness, vision loss Polyclonal gammopathy, Ultrasonography – detached retinas, Aqueous-centesis – granulomatous inflammation, CSF –eosinophilic pleocytosis CSF culture CNS, Eye N/A P. zopfii Gold Coast, South East QLD 
Boxer (Blogg & Sykes, 95) 5 FN Vision loss Melaena, sudden death N/A 1 week Died Cardiac & ocular histopathology Eye, Heart, Intestine Limited N/A Cairns, Far North QLD 
English Cocker Spaniel 4 MN Pyrexia, large abdominal lymph nodes Neurologic signs Hyperglobulinaemia Died Histopathology of tissues collected at necropsy Liver, Mesenteric and renal lymph nodes, Pancreas, Stomach N/A P. zopfii (based on morphology) Brisbane, South East QLD 
English Cocker Spaniel (Meehan, 96) 8 FN Colitis Vision loss, deafness N/A Faecal culture; urine cytology & culture; colon histopathology Colon, Eye, Heart, Kidney, CNS, Liver Full P. zopfii Townsville, Far North QLD 
10 Staffordshire Bull Terrier 2 FS Vision loss Colitis Low gamma globulins Died Urine cytology and culture; rectal scraping; vitreous cytology and culture Eye, Colon N/A N/A South West WA 
11 Collie (Thomas 1990) 3.5 FE Colitis Neurologic signs, vision loss N/A Rectal scraping cytology Colon, Eye, Heart, Kidney, CNS, Liver, Caecum, Proximal ileum, Caeco-colic lymph nodes Full N/A Perth, WA 
12 German Shepherd Dog 3 FN Colitis Vision loss N/A Rectal scraping cytology; ocular histopathology Colon, Eyes N/A N/A Sydney, NSW 
13 Corgi 10.5 MN Colitis Vision loss N/A Colonic biopsy impression smear cytology & histopathology; rectal scrape negative Colon, Eye N/A N/A N/A Newcastle, NSW 
14 Maltese 4 MN Colitis Weight loss Hypoalbuminaemia, hypoproteinaemia, hypocholesterolaemia; thickening of the colonic wall and loss of layering ultrasonographically Colonic histopathology; negative urine cytology; negative faecal culture Colon N/A N/A Geelong, Victoria 
15 Australian Cattle Dog Wilkinson, 1988) 8 FN Colitis Cutaneous lesions, vision loss, rhinitis, nasal planum ulceration, footpad ulceration N/A 17 Skin & peripheral lymph node histopathology and culture; nasal & footpad swab culture Eyes?, Skin, Footpads, Peripheral lymph nodes Limited – not seen in SI or pancreas; no other histology done P. wickerhamii Townsville, Far North Qld 
16 Beagle cross 4 MN Skin lesions, polydipsia, ocular signs Neurologic signs, colitis  Skin biopsy; Skin and lymph node culture Skin, Lymph nodes N/A N/A Brisbane, South East Qld 
17 Kelpie cross 6 FN Colitis Ocular signs, neurologic signs  12 months at time of writing Alive Ocular histology Colon, Eye N/A Brisbane, South East Qld 
No. Breed Age (yrs) & gender Initial clinical signs Subsequent clinical signs Clinical pathology & diagnostic imaging abnormalities Duration of illness (months) Out come Diagnosis Anatomic sites of infection Necropsy Prototheca specieRegion 
Boxer 1.5 FN Colitis Hindlimb lameness Proliferative periosteal lesions in diaphysis of both femurs Bone lesion aspirate cytology & culture Colon, Bone, Heart, Kidney, CNS, Liver, Pancreas Full (eyes not examined) P. zopfii Brisbane, South East QLD 
Boxer 2 FE Colitis Weight loss N/A 12 Colonic histopathology, faecal culture, rectal scrapes negative Colon, Mesenteric lymph nodes, Kidneys Full P. wickerhamii Brisbane, South East QLD 
Boxer 2.5 ME Colitis Weight loss N/A Colonic histopathology Colon N/A N/A Townsville, Far North QLD 
Boxer Cross 3 FE Polypdipsia, polyuria, incontinence Vision loss, neurologic signs N/A Vitreous cytology & culture; urine culture Eye, CNS, Kidneys N/A P. zopfii Toowoomba, South East QLD 
Boxer 4 FN Colitis, urinary incontinence, polydipsia, polyuria Vision loss, deafness, lameness, neurologic signs Multiple areas of periosteal new bone on diaphyses of both femurs and left humerus Urine sediment cytology & culture; Rectal scraping cytology; Colon histopathology Colon, Bone, Eye, Heart, Kidney, CNS, Liver, Intestine, Lymph nodes, Skeletal muscle Full P. zopfii Cessnock, NSW 
Boxer 4.5 FN Neurologic signs Deafness, vision loss Polyclonal gammopathy, Ultrasonography – detached retinas, Aqueous-centesis – granulomatous inflammation, CSF –eosinophilic pleocytosis CSF culture CNS, Eye N/A P. zopfii Gold Coast, South East QLD 
Boxer (Blogg & Sykes, 95) 5 FN Vision loss Melaena, sudden death N/A 1 week Died Cardiac & ocular histopathology Eye, Heart, Intestine Limited N/A Cairns, Far North QLD 
English Cocker Spaniel 4 MN Pyrexia, large abdominal lymph nodes Neurologic signs Hyperglobulinaemia Died Histopathology of tissues collected at necropsy Liver, Mesenteric and renal lymph nodes, Pancreas, Stomach N/A P. zopfii (based on morphology) Brisbane, South East QLD 
English Cocker Spaniel (Meehan, 96) 8 FN Colitis Vision loss, deafness N/A Faecal culture; urine cytology & culture; colon histopathology Colon, Eye, Heart, Kidney, CNS, Liver Full P. zopfii Townsville, Far North QLD 
10 Staffordshire Bull Terrier 2 FS Vision loss Colitis Low gamma globulins Died Urine cytology and culture; rectal scraping; vitreous cytology and culture Eye, Colon N/A N/A South West WA 
11 Collie (Thomas 1990) 3.5 FE Colitis Neurologic signs, vision loss N/A Rectal scraping cytology Colon, Eye, Heart, Kidney, CNS, Liver, Caecum, Proximal ileum, Caeco-colic lymph nodes Full N/A Perth, WA 
12 German Shepherd Dog 3 FN Colitis Vision loss N/A Rectal scraping cytology; ocular histopathology Colon, Eyes N/A N/A Sydney, NSW 
13 Corgi 10.5 MN Colitis Vision loss N/A Colonic biopsy impression smear cytology & histopathology; rectal scrape negative Colon, Eye N/A N/A N/A Newcastle, NSW 
14 Maltese 4 MN Colitis Weight loss Hypoalbuminaemia, hypoproteinaemia, hypocholesterolaemia; thickening of the colonic wall and loss of layering ultrasonographically Colonic histopathology; negative urine cytology; negative faecal culture Colon N/A N/A Geelong, Victoria 
15 Australian Cattle Dog Wilkinson, 1988) 8 FN Colitis Cutaneous lesions, vision loss, rhinitis, nasal planum ulceration, footpad ulceration N/A 17 Skin & peripheral lymph node histopathology and culture; nasal & footpad swab culture Eyes?, Skin, Footpads, Peripheral lymph nodes Limited – not seen in SI or pancreas; no other histology done P. wickerhamii Townsville, Far North Qld 
16 Beagle cross 4 MN Skin lesions, polydipsia, ocular signs Neurologic signs, colitis  Skin biopsy; Skin and lymph node culture Skin, Lymph nodes N/A N/A Brisbane, South East Qld 
17 Kelpie cross 6 FN Colitis Ocular signs, neurologic signs  12 months at time of writing Alive Ocular histology Colon, Eye N/A Brisbane, South East Qld 

E, Euthanased.

N/A, not available.

MN, male neutered.

QLD, Queensland.

NSW, New South Wales.

All 17 dogs in the Australian cohort had systemic or disseminated disease. Clinical and/or necropsy evidence for dissemination was evident in 15 dogs. Large bowel involvement alone was present in two dogs (cases 3,14), while in others (e.g., case 2) it was likely from clinical data and disease chronology that the infection was restricted to the colon early in the clinical course. One dog survived. Of the 16 non-survivors, 13 were euthanased, while three died. The duration of illness ranged from 7 days to 17 months, with a median of four months.

The majority of cases were pedigree dogs, and all but two were medium- to large-breeds. Six of 17 cases were Boxer dogs, and one was a Boxer-cross. There were two English Cocker Spaniels, and one each of the Collie, German shepherd, Staffordshire bull terrier, Australian cattle dog, Maltese and Corgi breeds. In addition, there was a Beagle-cross and a Kelpie-cross. Twelve of 17 dogs were females (three entire females, nine spayed females), while the remaining five were male (one entire, four neutered). The median age at the time of diagnosis was four years (range 18 months to 11 years).

Clinical signs and physical findings were most often referable to gastrointestinal, ocular and neurologic disease. Eleven dogs showed large bowel signs as their principal initial abnormality, and three more developed overt signs of colitis subsequent to presentation. This was manifested typically as episodic large bowel diarrhoea with fresh blood, evident for four months (on average) before diagnosis or before signs of dissemination developed. Thus, 14/17 cases showed signs of large bowel involvement at some stage. Other than weight loss and colitis, three dogs (cases 2,3,14) showed no other outward sign of infection. Three dogs showed polyuria, polydipsia and urinary incontinence from the outset (one with [case 4] and two without [cases 5,16] colitis) and three showed ocular signs from the outset (cases 7,10,16). Further signs that developed as the disease progressed were most often ocular and/or neurologic in nature, including deafness. These additional signs usually developed close to the time of death or euthanasia. Twelve dogs showed ocular involvement some time in the clinical course.

Four of the 17 dogs (cases 4,6,7,8) did not show overt signs of large bowel involvement, however the colon was not closely evaluated ante- or post-mortem in these patients, and subclinical colitis cannot be excluded. The first of these (case 8), followed an atypical clinical course: during evaluation for pyrexia and enlarged abdominal lymph nodes, the dog convulsed and died immediately after an exploratory laparotomy. Interestingly, ‘yeast-like’ organisms, reported as contaminants, had been detected in urine sediment by the laboratory four weeks earlier. The second dog (case 6), developed ataxia followed by deafness and vision loss, while the third (case 4) showed polyuria, polydipsia and urinary incontinence followed by loss of vision and ataxia. The remaining dog (case 7) showed vision loss followed rapidly by melaena and sudden death.

Two dogs (cases 15,16) had disseminated disease with cutaneous lesions, with Case 15 also showing ocular signs. Two dogs (cases 1, 5) developed symptoms due to haematogenous osteomyelitis during disease progression This was manifest as overt lameness of multiple limbs, with pain localizing to the mid-shaft of proximal long bones. Lesions were evident radiographically in both cases as diaphyseal periosteal new bone production (Figure 1 a,b), more suggestive of mycotic osteomyelitis than neoplasia. Although osteolysis was not prominent in routine radiographs, microradiography of sections of affected bone obtained at necropsy from one patient (case 5) displayed lytic change in addition to prominent new bone formation (Fig. 1c).

Fig. 1

(a) Hematogenous algal osteomyelitis affecting the femoral diaphysis in a dog (case 1) with disseminated protothecosis. Note the irregular periosteal new bone formation. (b) A similar lesion is present in the femur of a different patient (case 5), although the periosteal new bone is much smoother in appearance. (c) Microradiography of the femur (ex vivo) obtained from case 5 at necropsy. Note that the higher resolution of this imaging reveals there is lysis as well as periosteal new bone formation in the osteomyelitis lesion in the diaphysis

Fig. 1

(a) Hematogenous algal osteomyelitis affecting the femoral diaphysis in a dog (case 1) with disseminated protothecosis. Note the irregular periosteal new bone formation. (b) A similar lesion is present in the femur of a different patient (case 5), although the periosteal new bone is much smoother in appearance. (c) Microradiography of the femur (ex vivo) obtained from case 5 at necropsy. Note that the higher resolution of this imaging reveals there is lysis as well as periosteal new bone formation in the osteomyelitis lesion in the diaphysis

Protothecosis was diagnosed in a variety of ways. Positive identification of organisms was most often based on cytology of rectal scrapings (4/6 cases attempted; Fig. 2), endoscopic or full thickness colon biopsy histology (6/6 cases attempted; Figs. 3 and 4), urine sediment examination (5/6 cases attempted; Figs. 5 and 6) and urine culture (4/4 cases attempted) and histopathology of multiple tissue specimens obtained at necropsy or laparotomy. The diagnosis was also achieved using oculocentesis cytology and culture (3/3 cases attempted), ocular histopathology (1 case), cytology and culture of bone aspirates (1 case), colon biopsy impression smear cytology (1 case), CSF culture (1 case), faecal culture (2 cases) and skin lesion and lymph node histology and/or culture (2 cases).

Fig. 2

DiffQuik stained smear of a rectal scraping from a dog (case 5) with protothecal proctitis. Note the Prototheca organisms against a background of faecal bacteria. Again, the morphology is consistent with Prototheca zopfii.

Fig. 2

DiffQuik stained smear of a rectal scraping from a dog (case 5) with protothecal proctitis. Note the Prototheca organisms against a background of faecal bacteria. Again, the morphology is consistent with Prototheca zopfii.

Fig. 3

Morphological appearance of Prototheca organisms in sections of the colonic wall. The sections show the mild mixed inflammatory infiltrate in the lamina propria consisting of plasma cells, neutrophils and macrophages. Interspersed amongst the inflammation are moderate numbers of large oval organisms. Although organisms are easily discernible in H&E stained sections (a), they are better visualised in PAS (b) and methenamine silver stained (c) sections. The organism morphology in these photomicrographs is consistent with a Prototheca zopfii infection.

Fig. 3

Morphological appearance of Prototheca organisms in sections of the colonic wall. The sections show the mild mixed inflammatory infiltrate in the lamina propria consisting of plasma cells, neutrophils and macrophages. Interspersed amongst the inflammation are moderate numbers of large oval organisms. Although organisms are easily discernible in H&E stained sections (a), they are better visualised in PAS (b) and methenamine silver stained (c) sections. The organism morphology in these photomicrographs is consistent with a Prototheca zopfii infection.

Fig. 4

Granulomatous colitis and arteritis due to Prototheca zopfii. A thrombus is evident in the lumen of an artery on the serosal portion of the the colonic wall (a). Organisms can be seen invading the artery (b), although they are easier to visualize in (c) because of the higher magnification and the periodic acid Schiff (PAS) stain.

Fig. 4

Granulomatous colitis and arteritis due to Prototheca zopfii. A thrombus is evident in the lumen of an artery on the serosal portion of the the colonic wall (a). Organisms can be seen invading the artery (b), although they are easier to visualize in (c) because of the higher magnification and the periodic acid Schiff (PAS) stain.

Fig. 5

Appearance of Prototheca zopfii organisms in a wet preparation of urine (from case 5) at high power (Original magnification×660).

Fig. 5

Appearance of Prototheca zopfii organisms in a wet preparation of urine (from case 5) at high power (Original magnification×660).

Fig. 6

DiffQuik stain of urine sediment showing more detailed morphology of Prototheca organisms from case 5. The oval shape of organisms is characteristic of Protothecazopfii. Original magnification×330.

Fig. 6

DiffQuik stain of urine sediment showing more detailed morphology of Prototheca organisms from case 5. The oval shape of organisms is characteristic of Protothecazopfii. Original magnification×330.

A Prototheca spp. was identified by culture in 7/17 cases, with P. zopfii isolated from five dogs and P. wickerhamii in two others. In one additional dog (case 8), the organism was thought to be P. zopfii based on organism morphology in tissue sections. P. zopfii infections were associated with more aggressive disease, with earlier and more widespread dissemination than the two P. wickerhamii cases.

Specific treatment was attempted in only six dogs. Two patients, both with granulomatous colitis due to P. wickerhamii (cases 2, 15), were treated with AMB protocols (one intravenous [IV], one subcutaneous [SC]). One (case 2) was given itraconazole in addition to AMB. Both patients showed a partial response, surviving for 12 and 17 months, respectively and without developing overt signs of dissemination. The one surviving patient (case 17) was treated using both amphotericin B and itraconazole, and at the time of writing, is receiving on-going therapy with itraconazole. A poor response was observed in two dogs treated with ketoconazole, and one case was euthanased before the response could be properly assessed.

Five dogs were subjected to full necropsy (cases 1,2,5,7,9). Four of these cases had Prototheca granulomas in multiple tissues including colon, kidney, eyes, heart, liver, brain, pancreas, lymph nodes and skeletal muscle. One of these dogs (case 2) had disease caused by a P. wickerhamii infection of 12 months duration; although its clinical signs were referable to colitis only, lesions at necropsy were found in mesenteric lymph nodes and kidneys in addition to the colon. In cases studied histologically, disease was most severe in the mucosal tunic of the colon, suggesting the infection extended from the mucosa to the deeper layers of the colonic wall. There was also evidence of Prototheca invading blood vessels of the colonic wall, with thrombi containing organisms within the lumen of these vessels (Fig. 4).

Two dogs had limited necropsies only. One dog (case 15) with cutaneous and ocular disease due to P. wickerhamii and a clinical course of 17 months had no lesions in the small intestine or pancreas. However, the large bowel was unfortunately not examined. Another patient (case 7), who survived for only 8 days, had only myocardial and ocular tissues examined, both of which contained protothecal organisms.

Eleven cases were from Queensland, three from New South Wales, two from WA and one from Victoria (Fig. 7). A preponderance of cases from Queensland is clearly evident as five of the six Boxer dogs and the Boxer cross dog came from this region.

Fig. 7

Geographical distribution of 17 cases of systemic canine protothecosis from Australia.

Fig. 7

Geographical distribution of 17 cases of systemic canine protothecosis from Australia.

Case studies

The spectrum of clinical presentations of canine protothecosis is illustrated by two representative cases provided below.

Case report 1

A four-year-old, spayed Boxer bitch (Table 1, Case 5) was referred with a four-month history of polydipsia, polyuria, urinary incontinence and chronic intermittent large bowel diarrhoea. Incontinence had not responded to oestrogen therapy and diarrhoea persisted despite a diet change. In the two weeks prior to presentation the dog showed progressively worsening vision, became deaf, and developed a left forelimb and right hind limb lameness. The dog was deaf, blind (no menace and no dazzle response) and in lean body condition. There was mild enlargement of the left mandibular and superficial cervical lymph nodes. Pain was evident on palpation of tissues near the mid-shaft of the left humerus and right femur. Neurologic deficits were not evident in any limb. Ophthalmic examination revealed bilateral exudative retinal detachments and anterior uveitis. Frank blood was evident on digital rectal examination. Based on these findings, a disseminated infectious, immune-mediated, or neoplastic disease was suspected. Soon after admission, the dog developed an acute vestibular ataxia, with disorientation, positional horizontal nystagmus, left head tilt and circling to the left.

Routine haematology and serum biochemistry were unremarkable. CSF cytology and culture, peripheral lymph node aspirates, thoracic radiographs and a bone marrow aspirate revealed no abnormal findings. Colonoscopy revealed a thickened, irregular, ulcerated, friable mucosa. Colonic mucosal pinch biopsies were submitted for histologic evaluation.

Analysis of both a concentrated wet preparation and a cytospin preparation (Fig. 5) of urine collected by cystocentesis revealed numerous oval, variably-sized organisms both free and within macrophages. In cytospin preparations, these organisms stained densely with DiffQuik (Fig. 6). Protothecosis was tentatively diagnosed. Cytology of rectal scrapings showed numerous organisms of identical morphology (Fig. 2). Radiographs of the left humerus and both femurs showed multiple, smooth, solid, regular areas of periosteal new bone surrounding the diaphysis (Fig. 1). Urine culture on blood agar at 37oC resulted after overnight incubation in a heavy growth of dull, creamy colonies (1–3 mm diameter) but growth on Sabouraud's dextrose agar at 25oC was slower. Diff Quik® and Gram stained smears from colonial material demonstrated organism morphology consistent with a Prototheca spp. Identification at the National Reference Laboratory in Medical Mycology (Royal North Shore Hospital) established the isolate was P. zopfii. Histologic examination of the colon biopsies demonstrated severe ulcerative colitis, dominated by a neutrophilic infiltrate with numerous Prototheca organisms throughout the sections.

A diagnosis of disseminated protothecosis was made, and a grave prognosis given. The dog was euthanased and a full necropsy performed. Prototheca granulomas were evident in colon, small intestine, kidneys, myocardium, eyes, liver, brain, lymph nodes and skeletal muscle.

Case report 2

A two-year-old, entire Boxer bitch (Table 1, Case 2) was presented with a three-month history of episodic large bowel diarrhoea, frequently associated with haematechezia. Over this period, the dog showed mild weight loss and lethargy, and had failed to respond to dietary trials, anthelmintic and empiric antimicrobial therapy.

Physical examination was normal apart from a mild generalised peripheral lymphadenomegaly, and fresh blood and mucus on digital rectal examination. Lymph node aspirates were unrewarding. Colonoscopy (Fig. 8) demonstrated patchy hyperaemic regions in the mucosa of the rectum and descending colon. Chronic ulcerative colitis was diagnosed by histologic examination of H&E-stained rectal pinch biopsies, and Prototheca organisms were subsequently identified in PAS-stained sections. Culture of rectal scrapings for Prototheca spp. was unsuccessful.

Amphotericin B was administered twice weekly as a subcutaneous infusion using a protocol developed to treat canine cryptococcosis54. In addition, the dog was given itraconazole (10 mg/kg per os [PO] once daily). The dog's stools gradually became more formed, but did not return to normal. Sulphasalazine (250 mg PO every 6 h [QID]) was added to the treatment regimen. After 10 AMB treatments over 5 weeks (cumulative AMB dose 5 mg/kg), colonoscopy was repeated. The rectum and colon appeared normal grossly and histologically, with no Prototheca forms evident in PAS-stained sections. AMB was discontinued, while itraconazole and sulphasalazine were continued for a further two weeks.

The dog remained clinically well for an additional three months, then relapsed. Colonoscopy demonstrated rectal and colonic inflammation, and colon biopsies demonstrated eosinophilic inflammation. Prototheca forms were not identified. Prednisolone (1 mg/kg PO every 12 h) was administered, but diarrhoea persisted and recurrent protothecosis was strongly suspected. The owners declined further therapy. The dog became increasingly debilitated and was euthanased three months later, i.e., nine months after initial diagnosis and 12 months after the first detection of clinical signs. At necropsy, Prototheca lesions were found in the colon, mesenteric lymph nodes and kidneys. Prototheca granulomas were not identified in the small intestine, liver, stomach, lung, thymus, bladder, adrenals, thyroids, parathyroids or brain. P. wickerhamii was isolated from colonic swabs collected at necropsy. In summary, the clinical course in this case was more indolent than case 1, with limited dissemination, even after corticosteroid administration.

Retrospective analysis of cases reported from overseas

The 27 non-Australian cases of canine protothecosis reported previously are presented in Table 2. Two dogs with cutaneous disease were successfully treated (cases 19,20). Interestingly, Prototheca cells were identified in the urine of one of these survivors (case 20), indicating likely dissemination to the kidneys.

Table 2

The 27 non-Australian cases of canine protothecosis

Case No. & Reference Breed Age (years) & gender Initial clinical signs Subsequent clinical signs Duration of illness (months) Outcome Diagnosis Anatomic sites of infection Prototheca species Country 
 1 Povey, 1969 Springer Spaniel 3.5 ? Colitis, posterior paresis Polydipsia, haematuria 3 days Died Necropsy histopathology Rectum, Heart, Kidney N/A England 
 2 VanKruiningen, 1969 Boxer 9 F Chronic ocular pathology, polyuria, polydipsia Vision loss, colitis 10 Euthanased Urine cytology; necropsy histopathology Intestine, Eye, Heart, Kidney, Brain, Liver N/A USA 
 3 Van Kruiningen, 1970 Springer Spaniel 3 M Colitis Cachexia Died Necropsy diagnosis Colon, Small intestine, Lymph nodes N/A USA 
 4 Carlton, 1973 Collie 5.5 M Vision loss Diarrhoea Euthanased Ocular histopathology Eye N/A USA 
 5 Sudman, 1973 Miniature Schnauzer 2 F Chronic otitis externa & nasal discharge, cutaneous lesions  Euthanased Multiple tissue histopathology; aural & nasal exudate culture Skin, Internal nares, Cervical lymph node, Lung? P. wickerhamii USA 
 6 Buyukmihci, 1975 Collie 8.5 FN Colitis Vision loss Euthanased Subretinal fluid cytology; multiple tissue histopathology at necropsy Intestine, Eye, Heart, Kidney, Liver, Lymph node, Spleen, Lung N/A USA 
 7 Saunders, 1975 7 M Ocular pathology  Vitreous centesis & culture Eye P. zopfii 
 8 Holscher, 1976 Collie 1.5 F Colitis Unable to stand Euthanased Multiple tissue histopathology Colon, Heart, Kidney, Liver, Lymph node P. wickerhamii USA 
 9 Imes, 1977 Doberman Pinscher 6 FN Ocular pathology Hindlimb lameness, deafness, vision loss, loss of smell, posterior paresis 6 weeks Euthanased Multiple tissue histopathology Eye, Kidney, Brain, Liver, Spleen, Lung P. zopfii South Africa 
10 Tyler, 1980 Cross-breed 4 FN Ataxia Paresis, head tilt, depressed mentation ocular pathology 2.5 Euthanased CSF & kidney culture; multiple tissue histopathology Eye, Heart, Kidney, Brain, Spinal Cord, Liver P. zopfii & P. wickerhamii USA 
11 Migaki, 1982 Cocker Spaniel 5 FN Colitis Ataxia, seizures Died Kidney histology; histology at necrospy Colon, Kidney, Liver, Lymph node P. zopfii North & South America 
12 Migaki, 1982 Pers com Holscher Cross-breed 3 F Colitis Neurologic signs including circling, deafness Kidney & colon histopathology Colon, Kidney, CNS P. zopfii USA 
13 Cook, 1984 Collie 3 FE Colitis Vision loss, deafness Euthanased Kidney impression smear cytology; histopathology Colon, Eye, Heart, Kidney, Brain, Cochlea, Liver, Skeletal muscle, Lymph node, Thyroid P. zopfii USA 
14 Font, 1984 Hungarian Vizsla 2 FE Colitis Vision loss Euthanased Colon & ocular histopathology Colon, Eye P. wickerhamii USA 
15 Gaunt, 1984 Siberian Husky 4 FS Lameness Vision loss, colitis, sudden death Died Multiple tissue histopathology; liver culture Colon, Eye, Heart,Kidney, Brain, Liver, Skeletal muscle, Thyroid, Spleen P. zopfii USA 
16 Meredith, 1984 Dalmatian 10 FN Vision loss Lymphadenopathy, seizures, sudden death 3 weeks Died Vitreous centesis cytology & culture; popliteal lymph node impression smear cytology; histology at necropsy Eye, Lung, Heart,Diaphragm, Thyroid, Kidney, Liver, Pancreas, Brain P. zopfii USA 
17 Rakich, 1984 Collie 3 ? Colitis Depressed mentation, ataxia, vision loss, sudden death 1.5 Died Urine sediment & rectal scraping cytology; multiple tissue histopathology Colon, Eye, Heart, Kidney, Brain, Spinal Cord, Liver, Pancreas, Skeletal muscle, Lymph node, Thyroids, Aorta, Spleen P.zopfii USA 
18 Moore, 1985 Cross-breed 7 ? Vision loss Polydipsia, polyuria, sudden death Died Vitreous cytology & culture; blood culture; histology at necropsy Eyes, Heart, Kidney, Brain, Aorta P. zopfii USA 
19 Macartney, 1988 Greyhound 10 ME Multiple cutaneous masses  Dog in remission when article written Skin nodule aspirate culture & histopathology Skin Spp. unable to be determined Great Britain 
20 Ginel, 1997 Collie 4 ME Cutaneous lesions on scrotum, footpads, trunk & limbs; rhinitis, nasal depigmentation  24 Dog in remission when article written Skin nodule aspirate cytology & culture; urine sediment cytology; skin histopathology Skin, Urinary tract P. wickerhamii Spain 
21 Schultze, 1998 Cross-breed 5 FS Vision loss Colitis, depression, circling 10 Euthanased Vitreous humor cytology & culture; ocular histopathology; multiple tissue histopathology Colon, Eye, Optic nerve, Heart, Kidney, Brain, Liver, Pancreas P. zopfii USA 
22 Schultze, 1998 Cross-breed 8 FN Vision loss Colitis, deafness, depressed mentation, disorientation Euthanased Vitreous humor cytology; multiple tissue histopathology Colon, Eye, Optic nerve, Heart, Kidney, Brain N/A USA 
23 Rallis, 2002 German Shepherd Dog 3.5 ME Colitis Severe weight loss Euthanased Colon & rectal histopathology Colon N/A Greece 
24 Hosaka, 2004 Cross-breed 10 FS Colitis Vision loss, seizures Died Colon & colonic lymph node histology; vitreous cytology; colonic mucosal, blood & vitreous culture Colon, Eye, Heart Kidney, Stomach, Abdominal lymph nodes P. zopfii Japan 
25 Strunck, 2004 Cocker Spaniel 2 MN Colitis  12 Died without evidence of dissemination-cause of death unreported Rectal scraping cytology; colon histopathology Colon P. zopfii USA 
26 Pressler et al., 2005 Cross-breed 8 FN Uveitis Central vestibular signs, colitis, acute renal failure 3 weeks Euthanased Urine sediment examination, urine culture, rectal scrapings, subretinal fluid, CSF Variety of organs including kidneys P. zopfii USA 
27 Pressler et al., 2005 Samoyed 10 FN Polydipsia/polyuria Chorioretinitis, hyphaema, LTFU-presumed died Vitreal fluid, urine culture (but not sediment examination) NA P. zopfii USA 
Case No. & Reference Breed Age (years) & gender Initial clinical signs Subsequent clinical signs Duration of illness (months) Outcome Diagnosis Anatomic sites of infection Prototheca species Country 
 1 Povey, 1969 Springer Spaniel 3.5 ? Colitis, posterior paresis Polydipsia, haematuria 3 days Died Necropsy histopathology Rectum, Heart, Kidney N/A England 
 2 VanKruiningen, 1969 Boxer 9 F Chronic ocular pathology, polyuria, polydipsia Vision loss, colitis 10 Euthanased Urine cytology; necropsy histopathology Intestine, Eye, Heart, Kidney, Brain, Liver N/A USA 
 3 Van Kruiningen, 1970 Springer Spaniel 3 M Colitis Cachexia Died Necropsy diagnosis Colon, Small intestine, Lymph nodes N/A USA 
 4 Carlton, 1973 Collie 5.5 M Vision loss Diarrhoea Euthanased Ocular histopathology Eye N/A USA 
 5 Sudman, 1973 Miniature Schnauzer 2 F Chronic otitis externa & nasal discharge, cutaneous lesions  Euthanased Multiple tissue histopathology; aural & nasal exudate culture Skin, Internal nares, Cervical lymph node, Lung? P. wickerhamii USA 
 6 Buyukmihci, 1975 Collie 8.5 FN Colitis Vision loss Euthanased Subretinal fluid cytology; multiple tissue histopathology at necropsy Intestine, Eye, Heart, Kidney, Liver, Lymph node, Spleen, Lung N/A USA 
 7 Saunders, 1975 7 M Ocular pathology  Vitreous centesis & culture Eye P. zopfii 
 8 Holscher, 1976 Collie 1.5 F Colitis Unable to stand Euthanased Multiple tissue histopathology Colon, Heart, Kidney, Liver, Lymph node P. wickerhamii USA 
 9 Imes, 1977 Doberman Pinscher 6 FN Ocular pathology Hindlimb lameness, deafness, vision loss, loss of smell, posterior paresis 6 weeks Euthanased Multiple tissue histopathology Eye, Kidney, Brain, Liver, Spleen, Lung P. zopfii South Africa 
10 Tyler, 1980 Cross-breed 4 FN Ataxia Paresis, head tilt, depressed mentation ocular pathology 2.5 Euthanased CSF & kidney culture; multiple tissue histopathology Eye, Heart, Kidney, Brain, Spinal Cord, Liver P. zopfii & P. wickerhamii USA 
11 Migaki, 1982 Cocker Spaniel 5 FN Colitis Ataxia, seizures Died Kidney histology; histology at necrospy Colon, Kidney, Liver, Lymph node P. zopfii North & South America 
12 Migaki, 1982 Pers com Holscher Cross-breed 3 F Colitis Neurologic signs including circling, deafness Kidney & colon histopathology Colon, Kidney, CNS P. zopfii USA 
13 Cook, 1984 Collie 3 FE Colitis Vision loss, deafness Euthanased Kidney impression smear cytology; histopathology Colon, Eye, Heart, Kidney, Brain, Cochlea, Liver, Skeletal muscle, Lymph node, Thyroid P. zopfii USA 
14 Font, 1984 Hungarian Vizsla 2 FE Colitis Vision loss Euthanased Colon & ocular histopathology Colon, Eye P. wickerhamii USA 
15 Gaunt, 1984 Siberian Husky 4 FS Lameness Vision loss, colitis, sudden death Died Multiple tissue histopathology; liver culture Colon, Eye, Heart,Kidney, Brain, Liver, Skeletal muscle, Thyroid, Spleen P. zopfii USA 
16 Meredith, 1984 Dalmatian 10 FN Vision loss Lymphadenopathy, seizures, sudden death 3 weeks Died Vitreous centesis cytology & culture; popliteal lymph node impression smear cytology; histology at necropsy Eye, Lung, Heart,Diaphragm, Thyroid, Kidney, Liver, Pancreas, Brain P. zopfii USA 
17 Rakich, 1984 Collie 3 ? Colitis Depressed mentation, ataxia, vision loss, sudden death 1.5 Died Urine sediment & rectal scraping cytology; multiple tissue histopathology Colon, Eye, Heart, Kidney, Brain, Spinal Cord, Liver, Pancreas, Skeletal muscle, Lymph node, Thyroids, Aorta, Spleen P.zopfii USA 
18 Moore, 1985 Cross-breed 7 ? Vision loss Polydipsia, polyuria, sudden death Died Vitreous cytology & culture; blood culture; histology at necropsy Eyes, Heart, Kidney, Brain, Aorta P. zopfii USA 
19 Macartney, 1988 Greyhound 10 ME Multiple cutaneous masses  Dog in remission when article written Skin nodule aspirate culture & histopathology Skin Spp. unable to be determined Great Britain 
20 Ginel, 1997 Collie 4 ME Cutaneous lesions on scrotum, footpads, trunk & limbs; rhinitis, nasal depigmentation  24 Dog in remission when article written Skin nodule aspirate cytology & culture; urine sediment cytology; skin histopathology Skin, Urinary tract P. wickerhamii Spain 
21 Schultze, 1998 Cross-breed 5 FS Vision loss Colitis, depression, circling 10 Euthanased Vitreous humor cytology & culture; ocular histopathology; multiple tissue histopathology Colon, Eye, Optic nerve, Heart, Kidney, Brain, Liver, Pancreas P. zopfii USA 
22 Schultze, 1998 Cross-breed 8 FN Vision loss Colitis, deafness, depressed mentation, disorientation Euthanased Vitreous humor cytology; multiple tissue histopathology Colon, Eye, Optic nerve, Heart, Kidney, Brain N/A USA 
23 Rallis, 2002 German Shepherd Dog 3.5 ME Colitis Severe weight loss Euthanased Colon & rectal histopathology Colon N/A Greece 
24 Hosaka, 2004 Cross-breed 10 FS Colitis Vision loss, seizures Died Colon & colonic lymph node histology; vitreous cytology; colonic mucosal, blood & vitreous culture Colon, Eye, Heart Kidney, Stomach, Abdominal lymph nodes P. zopfii Japan 
25 Strunck, 2004 Cocker Spaniel 2 MN Colitis  12 Died without evidence of dissemination-cause of death unreported Rectal scraping cytology; colon histopathology Colon P. zopfii USA 
26 Pressler et al., 2005 Cross-breed 8 FN Uveitis Central vestibular signs, colitis, acute renal failure 3 weeks Euthanased Urine sediment examination, urine culture, rectal scrapings, subretinal fluid, CSF Variety of organs including kidneys P. zopfii USA 
27 Pressler et al., 2005 Samoyed 10 FN Polydipsia/polyuria Chorioretinitis, hyphaema, LTFU-presumed died Vitreal fluid, urine culture (but not sediment examination) NA P. zopfii USA 

E, Euthanased.

N/A, not available.

MN, male neutered.

FN, female neutered.

E, Euthanased.

N/A, not available.

MN, male neutered.

FN, female neutered.

USA, United States of America.

All 25 dogs that died or were euthanased were proven, ante or post-mortem, to have systemic or disseminated protothecosis. Collies (6 dogs) were overrepresented and there were seven crossbreds and one Boxer. Of the 27 cases, gender was described in 24, with 17 cases involving females and only seven in males. The median age at diagnosis was 5 years, patients ranging in age from 18 months to 10 years. The median duration of the terminal illness for the 25 systemic/disseminated cases was 4 months (range 3 days to 12 months).

Most dogs showed combinations of alimentary, ocular and neurologic signs. Many dogs showed chronic episodic colitis alone as their initial symptom, followed by the development of ocular and/or neurologic signs. Two dogs (cases 25, 23) showed only signs of colitis; necropsy was performed in one of these, with lesions found in colon but not in the eyes, liver, kidney or lung (case 23). Seven of 27 cases showed no clinical signs of large bowel disease (cases 5, 7,9,10,16,18,27), but it is not clear if the large bowel was specifically evaluated in these patients. One of these dogs (case 5) showed otitis externa, nasal discharge and cutaneous lesions referable to upper respiratory tract disease, presumably following aspiration of organisms into the nasal cavity. Mild to severe weight loss was common. Sudden death occurred in four cases (cases 15,17,18,19); in three of these, death was thought to be due to protothecal myocarditis.

In 19 cases, the species of Prototheca was determined, i.e., 14 were P. zopfii, four were P. wickerhamii, and in one case both species were isolated. In three additional dogs, for which limited clinical information was available 36, disseminated disease with renal involvement was associated with P. zopfii infections, while protothecosis was diagnosed without positive culture in a further two dogs. In all cases, P. zopfii infections were associated with aggressive disease, with earlier and more widespread dissemination than in P wickerhamii infections. One of the two dogs with cutaneous disease that was successfully treated had a P. wickerhamii infection; in the other survivor, the species was not identified.

Discussion

This study is the largest series of Prototheca cases recorded in the literature. A concerted attempt was made to contact veterinarians in Australia and New Zealand likely to encounter such cases, with a view to providing a consensus picture of the pathogenesis of this rare but devastating infection. We were successful in obtaining information on a representative cohort of canine patients, providing new insights into disease pathogenesis. Interestingly, no feline cases were encountered during our attempts to recruit cases retrospectively.

A striking difference between this Australian series and reported cases from overseas is the preponderance of pedigree dogs, especially Boxers, in our survey. Whilst only one of 27 overseas reports involved a Boxer dog (case 17), six of our 17 cases were Boxers, with an additional Boxer-cross. Whereas the majority of the overseas cases were in crossbred dogs, all but three of our cases were purebreds. It was of great interest that apart from one Maltese and one Corgi (in our cohort), and one miniature Schnauzer (in the overseas cohort), cases were generally in medium to large breeds likely to have an outdoors domicile. The preponderance of Boxer dogs suggests the possibility of a genetic defect predisposing to the development of this infection. This breed is at increased risk for developing a number of unusual infectious diseases including cryptococcosis 47, canine leproid granuloma syndrome 48, neosporosis 49 and canine histiocytic ulcerative colitis (an enrofloxacin-responsive disease of young Boxers proposed to have an infectious aetiology) 50, 51. Perhaps Boxer dogs have poor immunologic surveillance, or a specific immune defect rendering them less capable of successfully responding to infectious agents requiring effective cell mediated immunity to eradicate organisms from host tissues. Alternatively, a higher incidence of colitis (of whatever form) in this breed may facilitate progression from colonization, to penetration of the mucosa and lamina propria and hence invasion by an agent such as Prototheca, likely to be ingested from time to time in outdoor dogs.

In concordance with overseas reports, there was an over-representation of females in our study cohort. The reason for this is currently unclear, but is unlikely to be related to levels of female hormones per se, as most of the affected female patients were neutered. Although it is clear that dogs of any age can be affected, there was a tendency for young-adults to be affected in both cohorts. Although our overall case numbers were low, there was a trend for protothecosis to be more common in south east Queensland, a warm sub-tropical region. Interestingly, no cases could be identified from Tasmania or New Zealand (Grant Guilford, pers comm), two regions with a cooler climate, during the study period. The coastal distribution of cases (Fig. 7) likely reflects the fact that over 90% of the Australian human and owned canine population lives in coast cities. The large cluster of cases from south east Queensland is likely to reflect a true epidemiologic phenomenon, as this area has a population of only some 2 million people, compared to 4 million each for Sydney and Melbourne. The preponderance of cases in dogs of the Boxer breed in south east Queensland is interesting, as it raises a further possibility – that there is a genetic susceptibility to protothecosis in certain lines of Boxer dogs that are more commonly found in this location than elsewhere. The most recent study of protothecosis from the USA briefly described 13 cases recruited by retrospective data analysis from three university teaching hospitals over a 10-year period 36. Based on a comparison of the total owned canine populations of California, North Carolina, Texas and Australia, it would seem likely that protothecosis is more prevalent in Australia than North America.

Within the Australian cohort, no case of localized cutaneous protothecosis was encountered in either dogs or cats, although two dogs had multifocal skin lesions referable to haematogenous dissemination. All 17 Australian protothecosis patients had severe disease and all but one eventually died or were euthanased. Fifteen of the 17 showed evidence of dissemination, either ante-mortem or at necropsy, and in the two remaining dogs that showed gastrointestinal signs only during the clinical course, dissemination could not be excluded because a complete necropsy had not been performed.

The majority of dogs in both cohorts showed haemorrhagic colitis as the first sign of disease. Colitis varied in severity, was often episodic, and was typically present for several months prior to the development of signs due to dissemination. Prototheca organisms appear to have a tropism for well-vascularized tissues including the eye, CNS, kidneys and myocardium. Dissemination manifested most often as ocular and neurologic dysfunction. The natural course of canine protothecosis would appear to start with granulomatous colitis without other signs, but with slow progression and periods of apparent remission in response to non-specific therapy. Once signs of dissemination became apparent, death rapidly followed.

In this study cohort, 12/17 cases had ocular signs. Likewise, 15/20 dogs in the overseas cohort presented with, or developed, ocular lesions. Both eyes were usually affected, although progression was often asymmetric. Ocular disease with concurrent signs of large bowel diarrhoea is thus suspicious of protothecosis, and prompt evaluation of rectal scrape and urine sediment cytology is indicated. Ocular involvement is a marker for haematogenous dissemination from a focus in the colon, the complex vascular anatomy of the choroid presumably offering a favorable environment for deposition of endospores released from the primary site of infection.

Neurologic signs due to CNS infection were common (8/17 cases), although sometimes it took many months for such signs to develop. Neurologic manifestations of protothecosis were variable, although cases were generally considered to have multifocal disease. Signs included depression, behavioral change, deafness, cervical pain, vestibular signs, circling, ataxia, paresis and seizures. CSF fluid analysis was often helpful in establishing the presence of inflammatory disease, however organisms were not evident even when cytocentrifugation was used. Sometimes the presence of eosinophilic pleocytosis confounded the issue by suggesting alternate diagnoses such as neural angiostrongylosis 51. Analysis of the data for patients in both cohorts suggests rectal cytology and urine sediment examination and culture are more likely to establish a cost effective diagnosis of disseminated protothecosis in cases with CNS disease, even when overt colitic signs are absent.

A variety of signs of multi-systemic disease, such as pyrexia, polyuria, polydipsia, urinary incontinence, multiple cutaneous lesions and sudden death, were present in a proportion of our cases. Fever is not a surprising finding in a patient with disseminated infection. Polydipsia and polyuria likely reflected kidney involvement, and organisms could be detected in urine sediment collected from these cases 36. Two of our Australian cohort had lameness and bone pain as a prominent feature, the first time protothecal osteomyelitis has been reported in dogs. Radiographic changes were similar to those seen in haematogenous mycotic osteomyelitis. Unlike German Shepherds with disseminated fungal infections 53, long bones rather than vertebral end plates were affected in both dogs. When present, bone lesions provide a convenient location from which to obtain material for diagnostic studies, especially when organisms cannot be seen in urine or rectal scrapings.

Clinical and necropsy data from the two cohorts provided strong circumstantial evidence for haematogenous and lymphatic spread of protothecosis from the primary infection site in the colon. Involvement of the eye and kidney reflects the tendency for infectious emboli to lodge in tortuous capillary beds, such as those of the uveal tract and glomerulus. Although neurotropism is harder to explain, the choroid plexus may be affected similarly, with secondary infection of contiguous neural structures. Similar phenomenology has been suggested to account for the distribution of lesion in disseminated Aspergillus terreus infections, where lesions tend to affect the uveal tract, kidney and bony vertebral endplates following dissemination from a primary pulmonary focus 53, 54. Likewise, disseminated cases of cryptococcosis sometimes involve the brain, eye, kidney and peripheral lymph nodes following dissemination from the primary infection in the sinonasal cavity 47. We speculate that haematogenous dissemination in all three diseases is related to the production of small infectious propagules within mammalian tissues, with secondary distribution of spores, or small daughter cells, through the circulation. Histologic support for this proposal stems from the finding of cases where Prototheca organisms could be visualised in thrombi within vessels of the serosal surface of the colon. Although not performed in our cases, blood culture may prove a useful non-invasive diagnostic test for detecting Prototheca spp in disseminated cases. Lymphatic spread is likely to occur also, accounting for involvement of abdominal and medial iliac lymph nodes in some patients. There was evidence for both haematogenous and lymphatic dissemination in two patients (cases 10,11).

A key observation from both cohorts was that colonic and rectal involvement was the most consistent feature of disease, even in the absence of overt signs of colitis. Although three dogs in our series did not show signs of alimentary disease, the gastrointestinal tract was not closely evaluated ante- or post-mortem and it is likely that owners were not astute enough to recognise colitic signs. No case subjected to a complete necropsy failed to have gross and histologic involvement of the large bowel. Respiratory involvement was recorded in only one of our cases (which also had colitis), lending support to the hypothesis that ingestion rather than inhalation or penetrating injury is the predominant mechanism by which this saprophytic organism initiates disease in dogs.

Exactly how and why Prototheca organisms initiate infection of the large bowel is unclear, although it seems most likely that the first step must be colonization of the mucus of the colon following ingestion of a large number of infectious propagules from an environmental source, such as dam water or stagnant pools. This would account for the overrepresentation of larger dogs with a predominantly outdoors domicile. Presumably, the next step involves a disruption of the normal epithelial barrier of the bowel wall, which permits organisms’ access to the submucosa where they can establish an infection. The permissive event may be concurrent colonic inflammation, such as non-specific colitis or canine granulomatous ulcerative colitis, or a process or agent that causes mechanical disruption of the epithelium, such as abrasion from impacted bones or damage associated with intestinal parasitism. Additionally, there is likely a genetic contribution to disease susceptibility and further investigation of cellular immune function in the Boxer breed is justified.

In some patients in the Australian cohort, colitic signs were present for many months prior to presentation. Infections with the longest ‘latent period’ prior to dissemination were attributable to P wickerhamii. We suspect that in these patients, disease initially localised to the colonic wall, spread to involve a greater length of the bowel over a protracted period, and eventually penetrated deeper into the colonic wall. It seems that haematogenous dissemination is only likely after extensive full thickness infection of the colonic wall has occurred. Two dogs showed gastro-intestinal signs only, and necropsy data available for one of these patients showed lesions confined to the colon, mesenteric lymph nodes and kidneys. Further work is required to describe the ultrasonographic features in such patients, to determine the nature and extent of changes in the colon and its draining lymph nodes, and whether organisms accumulate in the renal pelvis, as they do in disseminated aspergillosis 52.

Rigid or flexible colonoscopy provided excellent visualization of the primary lesions in patients so investigated. Abnormalities detected included markedly thickened and hyperaemic colonic mucosa, focal areas of necrosis and ulceration and oedematous, friable, mucosal folds with mucosal erosions that bled easily. Histologic examination of endoscopic biopsies confirmed the presence of colonic inflammation and in most cases demonstrated causal organisms. Taken together with histologic findings at necropsy, there was no doubt that organisms had a predilection for the colon and rectum, and a tendency to invade both longitudinally along the gut and circumferentially to involve the full thickness of the colonic wall through to the deep muscle layers and the serosa. The inflammatory infiltrate was mixed, with lymphoid cells, plasma cells, macrophages and some neutrophils. Numbers of organisms varied from case to case, as did the associated inflammatory response, with some cases having an apparent lack of an appropriate immune response. Organisms were sometimes seen invading blood or lymphatic vessels. Disconcertingly, organisms were sometime absent from representative pinch biopsies, and in one case, there was an eosinophilic response. At necropsy, Prototheca were often detected in colonic lymph nodes.

Insufficient canine cases of systemic protothecosis have been treated appropriately to make definitive comments concerning drug therapy. This situation is made even more difficult because in vitro susceptibility testing for algae are not well standardised. Thus, treatment guidelines have not been formulated, even for human patients. It is important to emphasise that the greatest chance of effecting a cure rests with early diagnosis, as it is easier to treat algal colitis than disseminated disease involving regions difficult to target with antimicrobials, such as the eye and brain. Thus, even though this is a rare disease, maintaining a high index of suspicion for its diagnosis in dogs (especially Boxers) with colitis that do not respond to standard dietary and drug therapy 50 is of the utmost importance. Agents likely to be most useful against Prototheca species include AMB, fluconazole, itraconazole, amikacin, tetracycline and possibly terbinafine. Cases with colitis are amenable to local therapy (e.g., AMB retention enemas) in addition to systemic therapy. Combination therapy with more than one agent is likely to be superior to monotherapy with an azole. There is sufficient evidence from cases in our series and others in the literature (Table 2) that use of AMB in the therapeutic regimen is mandatory, and a subcutaneous infusion protocol has been described 55 that is easy to follow and cost effective. Newer forms of delivering AMB with reduced nephrotoxicity such as liposomal and lipid complex preparations 56 may be even more effective, but are likely to be cost prohibitive for many owners for some time to come. The experimental use of novel biocides, such as polyhexamethylene bigaunide 57, orally and/or by enema is worthy of consideration also.

The prognosis for cutaneous canine protothecosis is clearly much better than for patients with systemic disease. In part, this is because disease is localised to the skin and subcutis in an otherwise immunocompetent host. Furthermore, surgical excision of severely affected tissues provides a strong basis upon which to add appropriate antimicrobial therapy. Furthermore, the tendency for these cases to be caused by P. wickerhamii further improves the prognosis, as this species is generally more susceptible to treatment than P. zopfii.

It is worth mentioning, briefly, that there are interesting conceptual similarities between protothecosis and pythiosis 58 in dogs. While Pythium insidiosum is an oomycte, rather than an algal organism, it occurs especially in tropical environments and manifests as either cutaneous or gastro-intestinal disease, with either upper and/or lower bowel involvement. Systemic dissemination has been described once in a two-year-old male German shepherd dog. Young, male, large breed dogs, particularly outdoor working breeds, are most often afflicted and affected dogs generally have a history of recurrent exposure to warm freshwater habitats.

In conclusion, protothecosis is a devastating sporadic disease of dogs. Based on theoretical considerations, early detection and aggressive treatment may result in improved outcomes for patients. Protothecosis should be included in differential diagnoses in any dog showing signs of multi-systemic disease, especially that involving the large bowel, CNS and eye. It is necessary to have a high index of suspicion for this disease in dogs with unusual or refractory colitis, especially in predisposed breeds such as the Boxer. In such cases, diagnostic investigations should include urine sediment examination and deep rectal scrapings for cytologic examination and urine culture. Finally, it may be prudent to recommend that, where possible, owners of dogs prevent their pets from drinking stagnant water, as it is likely that this represents the most likely route of infection.

Acknowledgements

The authors wish to thank John Mackie of VPS IDEXX laboratories for access to histology records, Annie Rose and Graeme Allan for radiographic interpretation, and Annette Litster and M Fischle for assistance with preparing the figures. The following veterinarians are thanked for referring cases and providing case material: L. Booth (Cessnock Veterinary Hospital), E. DillMacky (Animal Referral Hospital), S. Carter (Western Suburbs Veterinary Clinic), P. Noble (Herriot House Veterinary Surgery), R. Thomasson (Vetcall Ashmore) and L. Martinez (Stones Corner Veterinary Surgery), K. Mason (Animal Allergy and Dermatology Service) and S. Denley (Indooroopilly Veterinary Clinic).

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