Protothecal enteritis as a cause of protein-losing enteropathy in a bull

Prototheca spp are achlorophyllic saprophytic algae found in wastewater, sewage, agricultural waste, and possibly elsewhere in the environment. Infections with these organisms have been reported in cattle, humans, and dogs; affected cattle commonly develop mastitis. A 5-year-old Brahman-cross bull was evaluated because of a history of diarrhea and weight loss. The history and physical examination and clinicopathologic findings were similar to those associated with granulomatous enteritis caused by Mycobacterium avium subsp paratuberculosis (Johne's disease), which is the most common protein-losing enteropathy of cattle. However, diagnostic tests for paratuberculosis yielded negative results. Biopsy specimens from the ileum, jejunum, and ileocecal lymph node were collected for histologic examination and preparation of tissue impression smears; Prototheca-like organisms were identified. Because of the poor prognosis associated with this infection and the lack of safe and economical therapeutic agents for cattle, the owner decided to euthanatize the bull. Infection with Prototheca organisms was confirmed postmortem. As this case illustrates, protothecosis may be a cause of granulomatous enteritis in cattle.     

Prototheca zopfii mastitis in a herd of dairy cows

Mastitis caused by the colourless alga Prototheca zopfii was diagnosed in 17 of 120 cows in a dairy herd. Infection occurred in animals varying from 3-14 years old and was present in one to four quarters of each cow. Nine cases were associated with clinical mastitis characterised by the presence in milk of flakes or small clots. Somatic cell counts consistent with subclinical mastitis (>500 x 10(3) cells/ml) were recorded in five of the eight remaining cows. Histological examination of udder tissue showed the presence of granulomatous lesions associated with the presence of Prototheca. The problem was identified and controlled by repeated microbiological examination of milk samples from all lactating cows and immediate culling of infected animals. P. zopfii was also recovered from environmental water samples on this farm. It is suggested that infection may have occurred as a result of teat sores caused by trauma from a milking machine, and the tendency for cows to lay down on a race, the surface of which was sometimes flooded by drain water in which Prototheca were present.     

Disseminated protothecosis causing acute blindness and deafness in a dog

A 3-year-old Collie bitch was euthanatized two weeks after the onset of blindness and deafness. The hearing deficit had been localized by clinical signs, brain stem auditory evoked responses, and impedance audiometry. Protothecosis was diagnosed by cytologic and histologic examinations. The organism was identified as Prototheca zopfii . Organisms and granulomatous lesions were found in kidney, heart, liver, skeletal muscle, thyroid gland, colon, bronchial lymph node, brain, and cochlea.     

Canine protothecosis: review of the literature and report of an additional case

A review of protothecosis in dogs revealed that this malady usually begins in the gastrointestinal tract and progresses to systemic involvement. Clinical signs generally include bloody diarrhea or blood-stained feces as well as blindness, ataxia, and polyuria. Histologically, myriads of protothecal organisms in different stages of development are found in the granulomatous lesions. Two main species have been culturally identified: Prototheca zopfii and P wickerhamii. In the absence of cultural studies, species identification can be accomplished readily by immunofluorescence. The present case involved P zopfii infection in a 5-year-old female Cocker Spaniel that had bloody diarrhea, with a history of bloody diarrhea 6 months earlier.     

Occurrence of Prototheca zopfii, a mastitis pathogen, in milk

Prototheca zopfii was isolated from cow-composite milk and bulk tank milk by a new selective Prototheca enrichment method. Repeated testing of cow-composite milk from individual cows resulted in P. zopfii isolation data indicating a strong statistical correlation of P. zopfii with specific cows. Prototheca sp. were isolated from the milk of 31 of 79 cows in a single herd, and contamination was discounted as the source. Prototheca sp. were also recovered from 28 of 787 bulk tank milk samples from the Eastern U.S.A., and 22 of 69 temperature control milk samples from pooled dairy farm milk from delivery trucks.     

Sources of Prototheca spp in a dairy herd environment

A source of Prototheca spp causing mastitis in a herd of 263 milking cows in North Carolina was investigated. Of 38 samples from the dairy environment, 18 (47%) contained Prototheca spp. Isolation sites included cattle drinking water; a feed trough; mud, dirt, and excreted feces from a dirt lounging area; water, sludge, mud, and vegetation from a creek in the lounging area; and the floor of a freestall barn. Samples were collected from 5 additional dairies, including one dairy with and 4 dairies without a history of protothecal mastitis. Prototheca spp were isolated from 48 (25%) of 190 samples from various sites on all 6 dairies. Isolates were P zopfii (45; 94%) and P wickerhamii (3; 6%). Isolation frequency ranged from 4 to 47% of samples/dairy. There was no apparent difference in the isolation frequency of Prototheca spp from samples from dairies with or without a history of protothecal mastitis. Sites characterized by wetness and the presence of organic matter most commonly yielded Prototheca spp. Because Prototheca spp appear to be common in the dairy environment, factors in addition to presence in the environment may be important in development of protothecal mastitis.     

Urinary tract manifestations of protothecosis in dogs

Records of 13 dogs with systemic infection with Prototheca sp. from 3 veterinary teaching hospitals were reviewed. Acute renal failure secondary to disseminated infection with Prototheca zopfii was diagnosed in 2 dogs. In 1 dog, acute renal failure developed during administration of immunosuppressive drugs for treatment of anterior uveitis. During diagnostic evaluation of this dog, Prototheca sp. organisms were noted in urine sediment and renal biopsy specimens. In the 2nd dog, acute renal failure was diagnosed after treatment for bacterial cystitis. After diagnosis of protothecosis, organisms were successfully isolated by aerobic urine culture. Both dogs with acute renal failure did not respond to conventional medical therapy. In total, Prototheca sp. was noted in urine sediment in 4 of 8 dogs and successfully cultured from urine in 5 of 7 dogs. Four of 5 dogs had organisms noted in the kidneys on histopathologic examination. In all dogs, the species identified was P zopfii. Sensitivity testing of 3 isolates revealed wide differences in in vitro drug resistance. Examination and culture of urine is recommended as a practical method for diagnosis of systemic infection with Prototheca sp.     

A new staining solution for the morphological studies of fungi and Prototheca.

A new staining solution named "PHOL" is described for studying the morphology of clinical and environmental isolates of fungi and Prototheca. In total 69 isolates were used for assessing the efficacy of this newly formulated stain. The solution has shown good ability to stain all 62 isolates of fungi and 7 of Prototheca, and thus helped in the identification of the organisms. The new solution has the potential to stain the young as well as old isolates of fungi and Prototheca. It is emphasized that a wider application of this newly introduced inexpensive and safe staining solution will help the mycologists to study the morphological characteristics of yeast, molds, and Prototheca obtained from man, animals and environment.     

Development of cell content and shedding of Prototheca spp. in milk from infected udder quarters of cows

It was the objective of this study to analyse shedding patterns and somatic cell counts in cows and quarters infected with Prototheca spp. and to evaluate two approaches to identify infected animals by somatic cell count (SCC) or by bacteriological analysis of pooled milk samples. Five lactating dairy cows, chronically infected with Prototheca spp. in at least one quarter were studied over 11 weeks to 13 months. Quarter milk samples and a pooled milk sample from 4 quarters were collected aseptically from all quarters of the cows on a weekly basis. Culture results of quarter milk and pooled samples were compared using cross tabulation. SCC of quarter milk samples and of pooled samples were related to the probability of detection in the infected quarters and cows, respectively. Shedding of Prototheca spp. was continuous in 2 of 8 quarters. In the other quarters negative samples were obtained sporadically or over a longer period (1 quarter). Overall, Prototheca spp. were isolated from 83.6% of quarter milk samples and 77.0% of pooled milk samples of infected quarters and cows. Somatic cell counts were higher in those samples from infected quarters that contained the algae than in negative samples (p < 0.0001). The same applied for composite samples from infected cows. Positive samples had higher SCC than negative samples. However, Prototheca spp. were also isolated from quarter milk and pooled samples with physiological SCC (i.e. < 10(5)/ml). Infected quarters that were dried off did not develop acute mastitis. However, drying off had no effect on the infection, i.e. samples collected at calving or 8 weeks after dry off still contained Prototheca spp. Results indicate that pre-selection of cows to be sampled for Prototheca spp. by SCC and the use of composite samples are probably inadequate in attempts to eradicate the disease. However, due to intermittent shedding of the algae in some cows, single herd sampling using quarter milk samples probably also fails to detect all infected cases. Therefore, continuous monitoring of problem cows with clinical mastitis or increased SCC in herds during eradication programs is recommended.     

Protothecal granulomatous meningoencephalitis in a dog

A case of central nervous system protothecosis in a dog is reported. A three-year-old male Maremma sheepdog was referred with a two month history of diarrhoea associated with progressive tetraparesis, depression and right circling. Stupor, severe proprioceptive deficits, bilateral decreased thoracic limb flexor reflexes and bilateral deficit of the menace reaction were detected on neurological examination and a multi-focal neurological localisation was suspected. Histopathological evaluation revealed multi-focal granulomatous foci in the thalamus, hippocampus and caudal brainstem containing numerous oval-rounded organisms with a thick, periodic acid-Schiff-positive and Gomori's methenamine silver-positive cell wall, a basophilic cytoplasm and one nucleus. Scattered sporangia containing two to four endospores were also observed. Morphological features were consistent with Prototheca species. Ultrastructurally, numerous degenerated algae were present within macrophages mainly lacking nuclei and cytoplasmic organelles. Generally, protothecosis in dogs is characterised by systemic signs because of a multi-organ involvement, and haemorrhagic colitis or ophthalmologic signs are the most frequent presenting signs. However, protothecosis should be added, also in Europe, to the list of the differential diagnoses in adult dogs with a multi-focal neurological localisation even in absence of other clinical signs.     

Cytologic,histologic, microbiologic,and electron microscopic characterization of a canine Prototheca wickerhamii infection

An adult dog was presented for chronic cough and a recent development of ulcerated, erythematous nares with nasal discharge. Cytology of enlarged peripheral lymph nodes revealed many intracellular and extracellular organisms. These round or rarely oval organisms measured approximately 5-9 µm in diameter and frequently contained several globular structures, ranging from deeply basophilic to magenta. A thin, clear halo was present. Smaller 1-2 µm, magenta forms were also observed. Fungal culture yielded small, wet, raised, irregularly shaped, white to pale tan colonies. Microbiologic staining of cultured material revealed features suggestive of algae. Histopathology of the lymph nodes revealed marked granulomatous inflammation with intralesional algal organisms suggestive of Prototheca. Electron microscopic findings were also consistent with protothecosis. Polymerase chain reaction, followed by direct DNA sequencing, identified the organism as Prototheca wickerhamii. A brief literature review discussing protothecosis in veterinary medicine is included.     

An isolate of Prototheca wickerhamii from systemic canine protothecosis

The isolate from a canine disseminated protothecosis was identified to be Prototheca wickerhamii by its morphological and biochemical characteristics. The isolate was grouped into a cluster identical to the type strain of P. wickerhamii, ATCC 16529(T) in phylogenetic trees based on the small subunit (SSU) and the 5' end of the large subunit (LSU) ribosomal DNA (rDNA); the cluster was close to that including the other Prototheca species. However, the strains of P. wickerhamii, SAG 263-11 and Pore 1283 belonged to a cluster different from the other isolates of Prototheca species and closely related to those of Auxenochorella species. Therefore, P. wickerhamii could be divided into two distinct genetic groups, one group close to the other Prototheca spp. including a standard strain of P. wickerhamii, and another group consisting of isolates previously reported to be close to the Auxenochorella species.     

奶牛中型无绿藻性乳房炎

目前已有12个国家报道无绿藻性奶牛乳房炎,造成严重的经济损失并对公共安全存在潜在的危害.2010年春季,北京地区某奶牛场暴发中型无绿藻引起的临床型乳房炎.从患有临床型乳房炎的10头奶牛的11个乳区分离到8株酵母样微生物,鉴定为中型无绿藻.用16种抗生素药敏纸片进行药敏试验,结果显示中型无绿藻仅对庆大霉素、链霉素和阿米卡星等氨基糖苷类抗生素敏感.同化试验证明该分离株能发酵葡萄糖和丙三醇.本试验在我国首次报道了中型无绿藻能引起奶牛临床型乳房炎.     

Eradication of Prototheca zopfii infection in a dairy cattle herd

Protothecosis is a severe form of mastitis in dairy cows caused by colorless algae of the genus Prototheca. Since P. zopfii is highly resistant to all known chemotherapeutics, infected cows must be removed from the herd. Eradication measures are difficult since many chronically infected cows may become intermittent shedders. Therefore, cultural methods are insufficient for control measures. In order to eradicate Prototheca zopfii-mastitis in dairy cattle herds, two isotype specific indirect ELISA for detection of IgA and IgG1 in whey were used in a dairy herd highly affected with protothecal mastitis. All cows (n = 313) were tested four times in intervals of six months. Milk specimens were examined in parallel by cultivation and serologically using two indirect ELISA systems for specific IgA and IgG1 in whey. Cows tested Prototheca positive were consequently separated from the herd and slaughtered. At the first examination, 15.6% of the animals were found positive by culture, and 23.3% were positive in at least one of the ELISA systems. Within two years, protothecal prevalence and incidence decreased to zero indicating that the eradication strategy used was successful. In summary, serological identification of P. zopfii-infected lactating cows is an useful tool to eradicate protothecal bovine mastitis in infected herds.     

Paraparesis as initial manifestation of a Prototheca zopfii infection in a dog

A case of protothecosis causing non‐ambulatory paraparesis in a dog without clinical evidence of disseminated infection is described. A five‐year‐old female Labrador retriever was referred with a 10‐day history of progressive non‐ambulatory paraparesis and lumbar pain as the only physical and neurological abnormalities. Lumbar myelography revealed severe extradural spinal cord compression extending from L4 to L7 vertebrae, and a right hemilaminectomy was performed. Surgical findings included an adherent whitish hard ill‐defined mass. Cytology and biopsy results disclosed the presence of algae enclosed in a matrix of chronic inflammatory infiltrate. Culture confirmed the presence of Prototheca species. Neurological improvement occurred within a month, and the dog received antifungal treatment without evidence of clinical disseminated disease for 6 months, but died after a generalised tonic–clonic seizure. Post‐mortem examination revealed multiple foci of inflammatory granulomatous infiltrate and algae‐like structures in the brain, lumbar intumescence and cauda equina. Prototheca zopfii was identified using molecular biology methods.     

Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan

This study is the first investigation on Japanese isolates of Prototheca zopfii from bovine mastitis and the cow-barn surroundings by molecular characterization to clarify routes of infection for bovine protothecal mastitis. We performed isolation of Prototheca from cow-barn surroundings (drinking water, sewage and feces) and milk samples from cases of bovine mastitis. Genotypes of the 32 isolates of P. zopfii from cow-barn surroundings and 67 isolates from mastitis were analyzed by genotype-specific PCR assays and restriction fragment length polymorphism (RFLP) assays. All mastitis isolates were identified as P. zopfii genotype 2. Conversely, 29 isolates from cow-barn surroundings were identified as P. zopfii genotypes 1 and 3 isolates as genotype 2, respectively. Given these results, both genotypes of P. zopfii could exist in cow-barn surroundings, but no sites were identified as frequent sources of P. zopfii genotype 2. P. zopfii isolates should thus be further explored with regard to genotype to clarify the reservoir of etiological agents in bovine Prototheca mastitis.     

Disseminated protothecosis diagnosed by evaluation of CSF in a dog

A 5-year-old female spayed Shetland Sheepdog Mix dog was evaluated for a history of recent seizure activity, progressive hind limb ataxia, polyuria, and polydipsia and no history of gastrointestinal signs. Physical examination findings included conscious proprioceptive deficits, ataxia, and anterior uveitis along with a hypermature cataract in the right eye. Results of a CBC, serum biochemical profile, urinalysis, and computed tomography scan of the brain were unremarkable. Cerebrospinal fluid (CSF) analysis revealed marked eosinophilic pleocytosis and rare organisms consistent with Prototheca spp within neutrophils and macrophages. On postmortem histologic examination, mononuclear inflammation and numerous intralesional algal organisms, similar to those seen on the cytologic preparation of CSF, were found in the brain, eyes, kidneys, and heart. Abnormalities were not detected on gross and histologic examination of the gastrointestinal tract. Cultures of CSF and subdural/olfactory bulb, but not intestinal tract, yielded growth of Prototheca spp, and PCR analysis and DNA sequencing confirmed the organism as Prototheca zopfii genotype 2. We have reported a rare case of disseminated protothecosis that was diagnosed by evaluation of CSF in a dog presented with neurologic signs and no overt enteric disease. Protothecosis should be considered as a rare cause of seizures, even in the absence of obvious enteric signs, and should be included in the differential diagnosis of eosinophilic pleocytosis.     

Prototheca zopfii mastitis in a herd of dairy cows

Mastitis caused by the colourless alga Prototheca zopfii was diagnosed in 17 of 120 cows in a dairy herd. Infection occurred in animals varying from 3–14 years old and was present in one to four quarters of each cow.

Nine cases were associated with clinical mastitis characterised by the presence in milk of flakes or small clots. Somatic cell counts consistent with subclinical mastitis (>500 × 10³ cells/ml) were recorded in five of the eight remaining cows.

Histological examination of udder tissue showed the presence of granulomatous lesions associated with the presence of Prototheca.

The problem was identified and controlled by repeated microbiological examination of milk samples from all lactating cows and immediate culling of infected animals.

P. zopfii was also recovered from environmental water samples on this farm. It is suggested that infection may have occurred as a result of teat sores caused by trauma from a milking machine, and the tendency for cows to lay down on a race, the surface of which was sometimes flooded by drain water in which Prototheca were present.     

Genotyping of bovine Prototheca mastitis isolates from Poland

Bovine mastitis due to unicellular, achlorophyllous algae of the genus Prototheca is a serious and complex ailment that accounts for high economic losses in the dairy industry. Bovine protothecal mastitis has been almost exclusively associated with only one species Prototheca zopfii. Recently, based on the 18S rDNA sequence analysis, P. zopfii has been divided into three genotypes (1-3), of which P. zopfii genotype 3 has been given the status of a new species Prototheca blaschkeae sp. nov. The aim of this study was to investigate the genotypic composition of the population of P. zopfii bovine mastitis isolates from Poland, according to that threefold classification. Of the 44 isolates tested, 43 (98%) were identified as P. zopfii genotype 2. Only one isolate was assigned to the newly established P. blaschkeae species. These findings clearly show the predominance of the P. zopfii genotype 2 in the etiology of bovine mammary protothecosis in Poland, but also underline the involvement of P. blaschkeae in the disease. The study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates, collected from different regions of Poland.     

Phenotypic and genotypic characterization of Prototheca zopfii in a dog with enteric signs

This is a case report of enteric protothecosis caused by Prototheca zopfii in an eight-year-old male mixed breed dog with a history of chronic bloody diarrhea, loss of appetite and weight loss. Algae were isolated from rectal scrapings in defibrinated sheep blood agar and dextrose Sabouraud agar. Cytological evaluation showed the presence of globular and cylindrical organisms with a defined capsule and variable number of endospores, characteristic of the genus Prototheca, in the rectum of the animal. Scanning electron microscopy of P. zopfii strains at different development stages confirmed the diagnosis of algal infection. Molecular identification using a conserved 18S rDNA gene sequence determined that the strain belonged to genotype 2. This report describes success on treatment of canine protothecosis, diagnosed based on clinical, cytological, microbiological, scanning electron microscopy and genotypical findings.