Etiology of Protothecosis in Animals
The genus Prototheca (family Chlorellaceae) are unicellular, colorless, achlorophyllous intracellular yeast-like microalgae, closely related to green algae of the genus Chlorella. A current molecular marker–based approach using the mitochondrial cytb gene proposed a new taxonomic classification of algae with 14 Prototheca species. Three are typically associated with dairy cattle: P ciferrii (formerly biovar I, or P zopfii genotype 1), P bovis (formerly biovar II, or P zopfii genotype 2), and P blaschkeae (formerly biovar III). Another three are associated with humans: P wickerhamii, P cutis, and P miyajii. The remaining species are P moriformis (P ulmea), P stagnora, P tumulicola, P zopfii, P cookei, P pringsheimii, P xanthoriae, and P cerasi, the last four of which were recently named (this new nomenclature is adopted here). Among the typical dairy cattle–associated species, P ciferrii has been found in the manure of cattle and the environment of dairy farms, P blaschkeae has been identified in the breeding environment of swine and as a sporadic agent of bovine mastitis, and P bovis has been described as a major primary cause of clinical protothecal mastitis in cattle Mastitis in Cattle With few exceptions, mastitis occurs when microbes enter the teat via the teat canal. Almost any microbe can opportunistically invade the teat canal and cause mastitis. However, most infections... read more herds globally.
Prototheca spp reproduce asexually. Sporangia (mother cells) form cytoplasmic sporangiospores (endospores or daughter cells), with spherical to oblong (oval) or wedge-shaped forms, ranging from about 3 to 30 mcm in diameter, depending on the species. Prototheca wickerhamii (3–10 mcm) has wedge-shaped sporangiospores radially arranged; P bovis (7–30 mcm) has oval or spherical sporangiospores usually larger than those of P wickerhamii.
Epidemiology of Protothecosis in Animals
Saprophytic algae from the genus Prototheca have been found in a wide variety of environmental niches and surfaces worldwide, including sewage, tree slime flux, plants, soil, manure, water (drinking cattle, river, lake, sea, swimming pools), sand, mud, and sewage, as well as transiently in the intestinal tract of animals. The algae are highly resistant to environmental conditions, particularly high humidity and organic matter.
Cattle, dogs, and cats are the main domestic animals in which Prototheca species have been isolated, although sporadic cases have been reported in goats, horses, and nondomesticated animals. Traumatic percutaneous lesions, contamination of mucous membranes, and invasion of the teat canal are likely the main routes of transmission of algae to domestic animals. Ingestion of milk from cows with protothecal bovine mastitis may be another route to infect calves.
Contamination of wounds and traumatic cutaneous-subcutaneous lesions apparently are main routes of protothecal infections among companion animals, in addition to oral transmission of algae to dogs by ingestion of contaminated water or food. Systemic or disseminated clinical signs of canine and feline protothecosis probably are related to debilitating conditions or immunosuppressive coinfections with viral agents. In retrospective case-series studies of dogs with cutaneous or systemic protothecosis, female Collies (US and Europe) and Boxers (Australia)>2 years old have shown a relative predominance, although this putative risk factor requires further investigation.
A substantial increase in the occurrence of clinical mammary protothecosis has been reported worldwide, particularly on farms with adequate control of other infectious bovine mastitis agents, such as staphylococci (Staphylococcus aureus), corynebacteria, and Streptococcus agalactiae. Protothecal bovine mastitis has been reported globally, with outbreaks recorded in South America (Brazil), North America (US, Canada, and Mexico), Asia (Japan and China), Oceania (New Zealand), and some countries of Europe (Denmark, England, and Italy).
Economic losses to endemic herds are related to veterinary care expenses for treatment and services, reduced milk production, and premature culling of affected animals.
A large-scale study of the molecular characterization of 342 Prototheca isolates obtained from bovine mammary infections in different countries revealed a major frequency of P bovis (90.6%), followed by minor frequencies of P blaschkeae (8.8%) and P ciferrii (0.6%), confirming the predominance of P bovis as a primary cause of cattle mastitis. Globally, Prototheca spp have been isolated in < 10% of mastitis cases and bulk tank milk samples subjected to microbiological culture. In a study conducted on 50 dairy farms in the Republic of Korea from 2015 to 2017, P bovis was isolated from 187 of 2,508 (7.5%) quarter milk samples. Likewise, an overall incidence of bovine mastitis by P bovis of 4.6% has been reported in dairy farms located in southeastern Poland, sampled from 2016 to 2017. A large-scale investigation of clinical bovine mastitis etiology among 10 Brazilian dairy herds from 2017 to 2019 identified Prototheca species as the primary agent in 113 of 4,275 (2.6%) clinical cases. Nonetheless, outbreaks of protothecal mastitis may affect ~30% of cattle herds and have been associated with contamination of intramammary therapy procedures, and with poor milking and environmental hygiene conditions.
On endemic farms, the organism has been recovered from different environmental sources, particularly with high humid or wet habitats that contain abundant organic matter in the soil, where animals are driven or resting. These farm sources include streams or stagnant ponds, mud and manure surround milking areas (outdoor runs, paths, resting areas), feed, cattle drinking water, and feces of cattle and calves, as well as milking machine surfaces, the floor of the milking area, the material of the compost barn, and the cow’s bed. Improper milking hygiene, deficiencies in premilking measures, and excessive amounts of organic material in the milking environment increase the risk of protothecal mammary infections; protothecal mammary infections can also be transmitted from infected to healthy cows during milking. Furthermore, flies are potential vectors of algae in the farm environment. Poor teat hygiene practices and improper hygiene in intramammary infusions are strong herd-level risk factors for protothecal infections; especially risky are procedures such as the use of dry cow teat sealant and infusions with nonintramammary formulations.
Prototheca bovis may persistently infect the epithelia of mammary glands during lactation and persist through the dry period, acting as a source of infection in the next lactation to other cows. A 2018 study identified P bovis among fecal samples of cattle and calves from the same herds with a history of mammary protothecosis, suggesting infection of calves by milk, and a connection between the occurrence of protothecal mastitis and the fecal cycle of algae between the environment, cattle, and calves.
Pathogenesis of Protothecosis in Animals
Because they lack chlorophyll, Prototheca species do not perform photosynthesis; they require external sources of carbon and nitrogen (ie, they are heterotrophic), resulting in a saprophytic lifestyle and opportunistic pathogen behavior in animal and human infections. Prototheca algae are intracellular pathogens that reproduce asexually. During cell maturation (growth), the cytoplasm undergoes cleavage or internal septation, leading to multiple and irregular divisions of the cell, forming 2–20 sporangiospores (endospores). The pressure of the enlarging sporangiospores breaks the cell wall, and sporangiospores are released to begin a new reproduction cycle. In adequate nutrient, temperature, pH, and humidity conditions, this process repeats every 5–6 hours. The enlargement of internal sporangiospores promotes the destruction of infected cells and tissues. Prototheca-induced infections cause pyogranulomatous inflammation of the tissues. Any cell or tissue may be infected by these algae; reported infections of mammary glands, brain, skin, eyes, intestine, skeletal muscles, liver, lymph nodes, and kidney tissues, in addition to phagocytes, demonstrate the high infective potential of Prototheca.
The pathogenicity of Prototheca-induced infections remains not fully clear. The rigid cell wall structure, suppression of cell-mediated immune response, evasion of phagocytic mechanisms of macrophages and neutrophils, and biofilm-producing ability of the algae probably contribute to the establishment, multiplication, and persistence of the organism in inflammatory foci. Virulent strains possess intracellular persistence that, in turn, induces a pyogranulomatous reaction, destroying different cells and tissues, including mammary parenchyma, and leading to limited immune response and tissue resolution. Among isolates from cattle with mammary infections, biofilm production has been shown to be species dependent, with P bovis being a strong biofilm producer.
Clinical Findings of Protothecosis in Animals
A variety of clinical signs of protothecosis, acute or chronic, have been described among some domestic species, particularly chronic mammary infections in cattle, hemorrhagic enteritis and systemic infections in dogs, and cutaneous lesions in cats.
Mammary infections appear to be the major clinical manifestation of protothecosis among domestic animals. Protothecal species are considered environmental agents in the etiology of bovine mastitis. Prototheca bovis is a major species involved in mammary infections of cattle; P blaschkeae and P wickerhamii are less frequent agents of infection. Mostly cows show clinical mammary manifestations, although some animals may have a silent course of infection (subclinical mastitis). Prototheca infections occur during lactation or dry periods. Watery, white to yellow milk, with pus or flakes, along with edema and indurative mastitis, have been observed in clinical forms. Mammary protothecosis substantially decreases milk production and increases somatic cell count. Supramammary lymph nodes may be enlarged. Occasionally, algae may spread from mammary glands and infect other organs. Algae may be eliminated intermittently in milk—a fact that may make microbiological diagnosis on herds difficult. Although some animals may resolve clinical signs spontaneously, protothecal mastitis tends lead to long-term infections that are commonly refractory to conventional intramammary or systemic therapy.
Canine protothecal infections are sporadic, although an increasing number of cases have been reported in companion animals. Dogs are infected predominantly by P bovis and P wickerhamii. Chronic, intermittent hemorrhagic diarrhea (presence of mucus, hematochezia), and progressive weight loss appear to be the main clinical signs, and they indicate a distal GI tract infection. The oral route is important to the establishment of canine disease. Other organs affected include the skin, eyes, CNS, liver, spleen, kidneys, and lymph nodes. A retrospective study of systemic protothecosis in 17 dogs in Australia from 1988 to 2005 revealed that hemorrhagic colitis represented the first referable clinical sign, with a history of chronic evolution. In addition to bloody diarrhea (64.7%), the dogs had various other manifestations, especially blindness (70.6%) and different neurologic signs (seizures, ataxia, and central vestibular disease) compatible with the multifocal disorder (47%). In these dogs, Prototheca spp, P zopfii, and P wickerhamii were identified, and P bovis showed earlier dissemination and more severe disease than P wickerhamii. The course of disease was fatal in 16 of the 17 (94%) dogs.
Other multisystemic clinical signs of canine protothecosis include fever, urinary incontinence, polyuria, polydipsia, osteomyelitis, myocarditis, lymphadenomegaly, and cutaneous forms (crusts on footpads, ulcerated nodules). A variety of ophthalmologic signs have been described, commonly bilateral, including progressive loss of vision, uveitis, retinitis, synechiae, and glaucoma. Molecular diagnosis enabled P bovis detection in a dog from Brazil with chronic hemorrhagic diarrhea and weight loss, along with a history of contact with the environment of a dairy herd, suggesting that cows or their environment were the sources of transmission to the dog.
Protothecal infections are rarer in cats than in dogs. Prototheca wickerhamii appears to be a predominant etiologic agent. Contamination of wounds or traumatic percutaneous inoculation of algae resulting from cat fights are proposed as transmission routes. Firm, nonulcerated, nonpruriginous, single to multiple, cutaneous-subcutaneous papules, nodules, or masses, with scabs to an ulcerated aspect, are present predominantly on the forehead, nose, base of the tail, pinnae, and distal aspects of the limbs. Usually, regional lymphadenomegaly is not observed with skin lesions in cats.
Rare cases of P wickerhamii infection in a goat with respiratory distress and ulcerated nodules in the muzzle and pinna, as well as pyogranulomatous rhinitis in a mare caused by coinfection of Prototheca spp and Pithomyces chartarum, have been reported. Systemic protothecosis has also been recorded among nondomesticated animals, including fruit bats, deer, beavers, snakes, rats, and fish.
Diagnosis of Protothecosis in Animals
Hematologic imaging, and ocular examination
Serologic, electron microscopy, and molecular techniques
Routine identification of Prototheca has been based on microbiological culture and phenotypic aspects of colonies, cytologic and histologic examination, and micromorphology and biochemical activity (carbohydrate and alcohol assimilation) of the algae. Molecular methods have allowed the determination of species, genotyping, taxonomic reclassification, and novel species characterization of algae.
Milk, cutaneous lesions, liquor, urine, vitreous humor, feces, rectal scrapings, tracheobronchial washing, fragmenting of organs, milking-machine surfaces, and environment material from farms have been used to isolate the algae. Prototheca species may be isolated on conventional media, such as blood agar and Sabouraud agar, in aerobic conditions. The growth of algae is optimized between 25° and 37°C. Irregular to mucoid, wet to dry, white to gray or yellowish, nonhemolytic yeast-like colonies, 1–2 mm in diameter, are isolated between 2 and 5 days, in aerobic conditions, on sheep blood agar and Sabouraud agar, depending on the species of algae. Prototheca isolation media (PIMs) have enabled selective isolation of the algae, especially from contaminated specimens or environmental material. Gram and fungal staining techniques (eg, lactophenol cotton blue) enable microscopic visualization of the algae, based on isolated colonies. Spherical to oval or wedge-shaped gram-positive organisms (sporangia) are observed using Gram staining, along with pink structures that constitute broken cell walls of the algae. Staining with lactophenol cotton blue and Romanowsky variants enables the observation of sporangiospores. A panel of the assimilation of carbohydrates, alcohol, and other substrates (eg, glucose, galactose, n-propranolol, ethanol, glycerol, trehalose, sucrose, maltose, fructose, arginine, lactose) has been used to determine the species of Prototheca; however, molecular confirmation is recommended.
Hematologic and serum biochemical examinations often reveal neutrophilic leukocytosis (nonspecific inflammation) and hyperglobulinemia, along with typical renal and hepatic abnormalities when these organs are affected in disseminated canine protothecosis. Marked increases of leukocytes and protein concentrations may occur in dogs with CNS signs. A rigorous ophthalmologic examination should be considered in dogs with clinical signs compatible with disseminated protothecosis. Imaging examination commonly is nonspecific.
Indirect ELISA (identifying IgG and IgA isotypes) has been used to diagnose algae in serum and whey. Molecular methods such as conventional and multiplex PCR assay, PCR-restriction enzyme analysis (PCR-REA) assay (partial cytB gene), and sequencing (18S rRNA gene) have enabled species identification, genotyping, and reclassification of algae. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has enabled the identification of P bovis and P ciferrii recovered from domestic animals. Transmission and scanning electron microscopy are alternative methods used for confirmation of diagnosis, species, and identification of ultrastructures from Prototheca species, including organelles, sporangia, and sporangiospores.
Main gross lesions generally include white to yellow, granular or nodule-like plaques on the serosal surface of organs of domestic animals. Ulcers and cutaneous-subcutaneous nodules occur in canine and feline protothecosis. Canine enteric infections reveal erosions, nodules, mucus, and congestion of mucosa, along with mesenteric lymphadenomegaly and hemorrhagic fecal content. Enlargement of supramammary lymph nodes, congestion, nodules, and destruction of mammary parenchyma may occur in protothecal mastitis.
Prototheca species may be diagnosed by cytologic and histologic examination of different tissue specimens (impression smears, biopsy or fine needle aspirates, rectal scrapings) staining by modified Gram stain, Romanowsky variants, Periodic Acid-Schiff (PAS), Grocott-Gomori methenamine Silver (GMS), and other staining methods. The algae exhibit typical sporangia (morulae or mother cells), with thick wall, basophilic cytoplasm, internal septations, and variable forms (oval, wedge-shaped), as well as many sporangiospores (ranging from 3 to 30 mcm in diameter), enabling the identification of species by micromorphology. Histologically, a chronic pyo- to granulomatous reaction is observed in different tissues, with the presence of sporangia containing sporangiospores surrounded by epithelioid macrophages, lymphocytes, neutrophils, plasma cells, occasionally multinucleated giant cells, and another variable contingent of cells, along with foci of necrosis. Cutaneous lesions exhibit hyperkeratosis, atrophy, ulceration, and follicular loss. Ophthalmic lesions include neuritis, inflammatory cell infiltration of the choroid and retina, with a variable number of intralesional algae.
Differential diagnosis of hemorrhagic enteritis by P bovis in dogs should include Salmonella spp, parvovirus, coronavirus, and intestinal parasites (genus Ancylostoma, Toxocara, Giardia, Isospora, and Cryptosporidium). Cutaneous-subcutaneous protothecosis of companion animals should be distinguished from infections caused by some bacteria (mycobacteria, Nocardia spp, Rhodococcus equi), parasites (Leishmania spp), and fungi and yeasts (Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces spp). A group of agents that cause chronic bovine mammary infections, refractory to conventional therapy (eg, Nocardia spp, Trueperella pyogenes, fungal organisms) may induce a similar clinical appearance of protothecal mastitis.
Treatment of Protothecosis in Animals
Prototheca spp have shown intrinsic resistance to a wide variety of pharmacological products with well-known antimicrobial action, including antibiotics, antifungals, algicides, antiseptics, and disinfectants—a fact that has stimulated in vitro and in vivo studies focused on the identification of an effective drug or product against the pathogen need for control and therapeutic approaches. Prognosis for bovine mammary infections and canine systemic (or disseminated) manifestations is poor, since response to medical therapy is usually poor.
In vitro susceptibility of Prototheca isolates from bovine milk and different clinical specimens of companion animals to some antimicrobials (gentamicin, amikacin, kanamycin, netilmicin, colistin sulfate, polymyxin B) and antifungals (amphotericin B, itraconazole, ketoconazole, nystatin, posaconazole) has been observed. Nevertheless, there are no standard guidelines to in vitro susceptibility tests for Prototheca spp, as well as a strong association of susceptible isolates with clinical response. In addition, in vitro algicidal effects of a panel of antiseptics, disinfectants, or sanitizing agents (sodium hypochlorite, iodine, hydrogen peroxide, chlorhexidine, thimerosal, copper sulfate, silver nitrate, peracetic acid, Tris-EDTA, ozone, dimethyl sulfoxide [DMSO], and guanidine) have been assessed, with variable results. Alternatively, DMSO (a small molecule that enhances permeabilization of cell membranes) and propolis (a natural product with antibacterial properties produced by bees) have been investigated for algicide effects. In vitro algicide effects have been described for dinitroanilines, a group of herbicides. Besides in vitro action against Prototheca, these diverse pharmacological products have promoted only temporary in vivo regression, and they do not substantially alter the outcome of clinical infections. Usually, clinical signs of protothecosis return, including bovine mastitis, as well as canine and feline clinical forms.
Various combinations of drugs have been proposed for a prolonged course of treatment for cutaneous and systemic (or disseminated) protothecosis among companion animals, including amphotericin B (0.25 mg/kg [cat] or 0.5–1 mg/kg [dog], IV, 3 times per week until cumulative dose of 8-12 mg/kg achieved) plus itraconazole (5–10 mg/k [dog and cat], PO, every 12 hours, for 4–6 weeks) or nystatin (100,000–500,000 IU [dog], PO, every 8 hours, for >90 days). Antifungal ketoconazole and fluconazole have been proposed for treatment as well. Nonetheless, the duration of therapy is speculative, and because long-term treatment is required, many animals may develop adverse reactions to drugs. Particularly in cases of protothecal mastitis, some antimicrobials, antifungals, and antiseptics or disinfectants that have shown in vitro algicidal effects are irritant or caustic to the mammary glands of cattle, and residues may cause adverse organic reactions in humans if ingested through milk or milk products.
Wide surgical excision has been indicated for protothecal cutaneous lesions of dogs and cats, although a substantial number of animals subjected to excisional procedure or biopsy of solitary nodules developed systemic disease after the surgical approach.
Control and Prevention of Protothecosis in Animals
No specific measures are recommended to prevent or control protothecal infections in companion animals, except to avoid contact of wounds with possible environmental sources of the algae. Given the environment-borne nature of Prototheca species that cause bovine mastitis, prevention and control measures applied to environmental mastitis may be applied to mammary protothecosis.
Routine clinical and microbiological diagnosis of mastitis in cattle herds and cows recently acquired, adequate management and milking hygiene, the proper time for teat stimulation, gloves for milkers’ hands, individual towels for drying teats, and use of pre- (especially) and postmilking teat dip are general measures recommended to prevent environmental mastitis in dairy herds. Studies focused on in vitro action of iodine, sodium hypochlorite, and chlorhexidine against Prototheca spp isolated from milk have revealed algicide effects in low concentrations, indicating that these sanitizing agents may be used as teat dip solutions to prevent and control protothecal infections. In addition, offering food after milking, providing clean and dry housing and pre- and postmilking areas (with an emphasis on removing feces and organic material), regularly changing bedding, and chlorinating the water used in milking procedures are measures that should be considered for preventing environmental pathogens, including Prototheca spp.
On endemic dairy farms, early diagnosis, segregation of infected animals into a distinct group during milking, drying of the teat (only one teat), or culling of chronically infected animals or animals with multiple affected teats appear to be the main procedures for controlling protothecal mastitis. Besides an apparent higher virulence and severity of P bovis in mammary infections, prevention and control measures are similar regardless of algal species or genotypes.
Zoonotic Aspects of Protothecosis in Animals
Prototheca wickerhamii is the most common Prototheca species isolated from human cases of protothecosis. Protothecosis is considered an opportunistic, rare infection in humans, although an increase of more severe, systemic cases has been noted among patients with compromised host immunity, particularly those subjected to immunosuppressive therapy, organ transplant recipients, and HIV patients.
An occupational behavior of disease has been proposed. Infections in humans may occur through percutaneous inoculation of the algae and through contamination of wounds or mucous membranes with environmental reservoirs of the algae. Direct transmission through secondary contact of humans with diseased animals remains unclear.
Ingestion of contaminated bovine milk and milk products (cheese) has been suggested as a source of the transmission of algae from cattle to humans, and it poses a public health issue because Prototheca species may resist the temperatures applied to milk and milk derivatives.
Cutaneous, articular disorders (olecranon bursitis) and systemic or disseminated infections (peritonitis, hepatitis, encephalitis) are the main clinical signs of human protothecosis. Azoles and amphotericin B are the most common drugs administered to treat protothecosis in humans, although there is no consistency in the clinical response, particularly among patients with underlying conditions.
Prototheca spp are opportunistic microalgae associated with clinical infections in animals and humans.
The algae are ubiquitous in the environment, especially under conditions of high humidity and abundant organic matter.
Chronic mammary infections in cattle, hemorrhagic enteritis and systemic infections in dogs, and cutaneous lesions in cats are the main clinical signs in domestic species.
Besides in vitro susceptibility to some drugs with algicide action, the pathogen commonly is refractory to conventional therapy.
For More Information
Pressler BM. Chapter 67: protothecosis and chlorellosis. In: Greene CE, ed. Infectious Diseases of the Dog and Cat. 4th ed. Elsevier Saunders, 2012;696-701.