Cow ’ s Mastitis produced by Unicellular Algae of the Genus Prototheca , an Emerging Disease : Treatment Evaluations ( Literature Review )

Mastitis induced by Prototheca have become emerging diseases over the last 10-15 years, evolving endemically in some farms, and leading to important economic losses.The diseased cows eliminate the algae by milk, with the risk of transmission to humans, which is why protothecosis is part of the zoonosis group.Many antibiotics and antifungals have been tried in the treatment of mastitis, but without result, even though the sensitivity of isolated strains was detected in vitro.In the present study we present the antibiotics and antifungals used by various authors in the treatment of mastitis with Prototheca spp, as well as the results obtained.Among antibiotics, good and consistent efficacy was obtained with: gentamicin, neomycin, kanamycin, colistin. In antifungals, good results include: amphotericin B, miconazole, mycostatin, ketoconazole, econazole, itraconazole, batrafen and clotrimazole.It has been concluded that mastitis produced by Prototheca species (especially P. zopfii) are incurable, and the slaughter of sick animals is recommended.In order to avoid spreading within the herd, monitoring will be carried out by applying a screening program and avoiding contaminated sources from the environment. Keywords—Mastitis, protothecosis, therapy, emerging


INTRODUCTION
Unicellular green algae (Chlorococcales) are described as lower plants, which contain chlorophyll and a cellulosic cell wall.Exceptions to this rule are algae of Prototheca genus, which have lost their ability to synthesize chlorophyll, becoming heterotrophs (Calley et Lloyd, 1964); (Lagneau, 1996).As a consequence, prototeca are considered to be unicellular, saprophytic, chlorophyllless, mutant algae of the Chlorella genus.The first description is made by Krüger who isolated them from the bark of the trees (Krüger, 1894).Prototheca is distinguished from bacteria and fungi by: size, shape, reproduction (formation of endospores), and the fact that the cell wall does not contain glucosamine and muramic acid (Milanov et Suvajdzic a , 2006).Taxonomic framework of the Prototheca: Domain: Eukaryota, Kingdom: Viridiplantae, Phylum: Chlorophyta, Class: Trebouxiophyceae, Order: Chlorellales, Family: Chlorellaceae, Genus:Prototheca.Although 12 species have been described over time, according to current taxonomic data, only the following are now recognized: P. zopfii, P. wickerhamii, P. stagnora, P. ulmea, P. moriformis and P. blaschkeae (Lass-Flürl etMayr, 2007); (Marques Sara et al., 2008) but recently, other two species were also included:P.cutis (Satoh et al., 2010) și P. miyajii (Masuda et al., 2016).P. zopfii is more frequently involved in animal diseases and P. wickerhamii in human disease, without a clear differentiation from this point of view.The first case of mastitis in cows was reported in 1952 in Germany (Lerche, 1952).Subsequently, the disease was described in many other farms in Europe, Asia, America, Australia and Africa.

II.
EPIDEMIOLOGY Prototheca are ubiquitous in nature, being isolated from a wide variety of habitats, such as vegetation, river water, and even marine water (Tyler et al., 1980); (Gonzalez, 1996); (Thompson et al., 2009).Almost all authors report their isolation from mud deposited on tree bark, wet soil around trees, vegetation, floodplains, acidic water, sewage, clogged ponds (Pore et al., 1983); (Huerre et al., 1993).Isolation has also been reported from green fodder, shelters, drains, milk, milking machines, bulk milk tanks, shelter floors, barns (Gonzalez, 1996); (Moubamba a et al.,  1997).Prototheca can be found in the faecesof adult cattle (Enders et Weber, 1993), but also in the faeces of other animal species (horses, pigs, rats).Various sources mention ahigh frequency isolation of P. zopfii (94%), and more rarely of P. wickerhamii (6%) (Anderson et Walker, 1988).Moisture (even the presence of wetlands) is a factor  (Ricchi et al., 2013).Within the species P. zopfii, three biotypes were described, which were reclassified into 2 genotypes (1 and 2) and a new species renamed as P. blaschkeae (Roesler et al., 2006).Genotype 2 is thought to be pathogenic, has greater adhesion capacity to mammary epithelium and is considered to be the main cause of mastitis in cows (Moller et al., 2007); (Pieper et al., 2012); (Ahrholdt et al., 2012); (Shahid a et al., 2017).Some data mention the isolation from milk samples taken from the same farm, both P. zopfii genotype 2 (predominantly) and genotype 1 (uncommon) and P. blaschkeae (Bozzo et al., 2014).The ability of P. zopfii to produce mastitis in cow was also demonstrated by experimental infections (Dion, 1982); (McDonald a et al., 1984); (Bergmann b , 1993).Epidemiological study of clinical mastitis in dairy cattle reveal the endemic character of these infections in the case of favoring factors, recalling in particular: repeated intramammary infusions, antibiotic pretreatment, antibiotic abusive treatments and the action of various immunosuppressive factors (often difficult to identify), associated with inadequate hygiene conditions, both at farm and milking levels.All these factors increase the risk of mastitis with Prototheca (Tenhagen et al., 1999); (Corbellini et al., 2001); (Pieper et al., 2012) (Kirk et Mellenberger, 2011).Some data indicate that mastitis caused by Prototheca have a peak in the 2nd lactation month (Tenhagen et al., 1999).Cows with and without mastitis can shelter and eliminate Prototheca species in a permanent or intermittent manner (Gonzalez, 1996).Algae can persist in the mammary gland for 45 to 100 days, or even more, and as a consequence they pose a real danger to other cows in the flock.Algal persistence is mentioned for up to 13 months in the dry period (Spalton, 1985)and serum antibodies are absent 6 months after lactation but will reappear following parturition (Dion, 1982).

III. PATHOGENESIS
Prototheca is considered as germ with moderate pathogenicity and virulence (Huerre et al., 1993); (Răpuntean et al., 2004).However, once they enter the body, they produce local granulomatous lesions, but they can spread lymphatically, causing a systemic or even generalized infection, with fatal evolution.Such evolutionary forms have been described in cows (Taniyama et al., 1994),dogs (Tyler et al., 1980); (Gaunt et al., 1984); (Imes et al., 1977); (Souza et al., 2009), and in humans after immunosuppressive therapy (Wolfe, 1976) or immunocompromised hosts (Wirth et al., 1999).In the case of cows, penetration into the mammary gland occurs at the level of the streak canal, frequently after milking, when the teat meatus remains partially open and the nipples come into contact with a heavily contaminated environment (Costa a et al., 2006); (Gonzalez, 1996); (Langoni et al.,3013).Spreading during milking is not significant, but may be important for poor milking hygiene (Kirk et Mellenberger, 2011).Protothecahas a tendency to invade the connective tissue between the nipple and the abdominal region, as well as the lymph nodes.It is noted that, often,the algae remain restrictedin the mammary gland and regional lymph nodes, causing inflammatory granulomas (Jensen et al., 1998).The microorganisms are phagocytosed, by macrophages and neutrophils, where they can proliferate, resisting the host's defense mechanisms, generating an infection that cannot be defeated by therapy.Mastitis caused by Prototheca differs from those produced by other microorganisms by the fact that the inflammatory reaction is poorly expressed or does not occur at all.There are cases where neither congestion nor edema occurs, and the decrease in milk production is due to the destruction of the secretory tissue (Moubamba b , 1997).

IV.
CLINICAL AND PATHOLOGICAL ASPECTS Mastitis with Prototheca may develop as clinical and subclinical forms (Langoni et al., 1995); (Schlenstedt et al., 1997).Most often, it evolves chronic, subclinical, with decreased milk production.Milk secretion becomes watery, sometimes with the presence of gray flocks (Wawron a et al., 2013).The mastitis may also develop acutely (frequently after parturition), comprising one or more quarters, with severe hardness, accompanied by fever, inappetence, drastic reduction of milk production, also affecting retro-mammary lymph nodes, which are hypertrophic (Furuoka et al., 1989); (Schelenstedt et al., 1997); (Spalton, 1985); (Wawron a et al., 2013).In the majority of cases, infected cows stays undetected, until the affected quarter is almost dry [115].Many infected cows have only a decrease in milk production, in which, along with Prototheca cells, there is a high number of somatic cells (Tenhagen et al., 1999) (Hodges et al., 1985).

V.
DATA ON THERAPY Diseases caused by unicellular algae of the genus Prototheca generally respond poorly or not at all to treatments.Numerous antibiotics and antifungals were used in the treatment, but the results were often contradictory, depending on how the disease evolved (localized or systemic infection), the species of affected animal and the age of the disease.Protothecais considered resistant to most antimicrobials (Baumgartner, 1997).In the case of Prototheca mastitis in cows, although many therapeutic trials have been made, the results were, in most cases, unsatisfactory.Although treatments have often been established based on the antibiogram results, which demonstrate in vitro susceptibility of Prototheca, this has not been correlated with in vivo efficacy.For this reason, many authors consider that the treatment of mastitis caused by Prototheca is difficult, inefficient and totally non-economic (Almeraya, 1994); (Moubamba b , 1997); (Kirk, 1999).(Lagneau, 1996); (Filippsen et al., 1999); (Wawron b et al., 2013).Among the antifungal agents, in vitro inhibitory action, and some more or less satisfactory results in clinical cases, have been demonstrated to amphotericin B (0.15 μg/ml) on both human and animal isolates.It is a polyclonal macrolide antibiotic, recommended in invasive fungal infections, with diverse etiology, with fungistatic or fungicidal effect.In vitro testing by broth dilution of 5 Prototheca spp.Strains, against amphotericin B or a combination with flucytosine (5-FC) and rifampicin revealed that amphotericin B combined with rifampicin produced the best effect at the most decreased CMI, and flucytosine combined with amphotericin B demonstrated the lowest effect (Srimuang et al., 2000).Following the exposure of the P. zopfii and P. wickerhamii to sub-inhibitory concentrations of amphotericin B, it was found that ultrastructural changes of the treated cells occurred, including mitochondrial swelling, cell organ degradation, accumulation of lipid droplets and starch granules in the cytoplasm, as well as charges of the inner layer of the cell walls (Segal et Socher, 1981).The study on the correlation of the lipid composition in P. wickerhamii (16529 ATCC) and sensitivity to some antimicrobial agents: polyenes, polymyxins and imidazoles, showed that the presence of ergosterol in the neutral lipid fraction of the membrane is probably responsible for increased susceptibility to amphotericin B; and the presence of a large amount of free fatty acids in the membranes, creates susceptibility to imidazoles.The membrane determinants of susceptibility to polymyxin B were less defined (Sud etFeingole,1979).P. wickerhamii and P. zofii strains were resistant to fluconazole and itraconazole, demonstrated by E-test and cultivation on the casitone yeast extract agar(Blaschke-Hellmessen, 1996).Treatment with nistatin has also been shown to be ineffective, although the Prototheca has been shown to be susceptible in vitro (Goudswaard, 1977).Casal et Gutierrez a (1981) have investigated, in vitro, the action of several antibiotics against P. wickerhamii strains, and found inhibitory effect on amikacin, colistin, dibekacin, framichetin, gentamicin, kanamycin, lividomycin, neomycin, polymyxin, paromomycin, ribostamycin, sisomicin and tobramycin, being recommended for treating protothecosis in humans with P. wickerhamii infection.Casal et Gutierrez c (1983) by examining the in vitro susceptibility of Prototheca strains to ribostamycin, specified that concentrations of 4 mcg/ml were required to inhibit 100% of P. zopfii strains; 16 μg/ml to inhibit 100% of P. stagnora strains and 95% of P. wickerhamii strains.The determined values were inferior to plasma ones, obtained after the ribostamycin injection, and this antibiotic was recommended for the treatment of human protothecosis in case ofP.wickerhamii infection.The same authors have found that susceptibility to clotrimazole (50 μg/disc) allows the differentiation of P. zopfii (which is resistant) from P. wickerhamii (which is susceptible).Casal et Gutierrez e (1995), recommends the testing of the susceptibility to neomycin (30 μg /disc) which allows the differentiation of P. wickerhamii and P. zopfii from P.  1998) by testing a number of 16 strains of P. zopfii isolated from cows with mastitis, showed that they all proved to be resistant to clotrimazole.Răpuntean Gh.,et al.,(2004) have tested the in vitro susceptibility of 16 strains of Prototheca zopfii, isolated from cattle mastitis (agar gel diffusion technique) against 26 antibiotics and 10 antifungals.Of the antibiotics, full sensitivity (100%) wasrecorded at: lincocin-forte (16-32 mm), neomycin (24-30 mm), gentamicin (14-20 mm), kanamycin (14-30 mm) and colistin (22-28 mm).All strains were resistant to lincomycin-spectinomycin, tylosin, novobiocin, spiramycin, penicillin, ampicillin, tetracycline, clindamycin, ofloxacin, ciprofloxacin, cephalothin, ceftazidime, amoxicillin, erythromycin, flumequin, trimethoprim, and chloramphenicol.Of the antifungals, total sensitivity (100%) was recorded to mycostatin (16-22 mm), econazole (18-24 mm), batrafen (cyclopiroxolamine) (20-30 mm), amphotericin B (10-12 mm).In some antifungals, resistance was also recorded: 2 strains showed resistance to ketoconazole and clotrimazole, and to itraconazole 3 strains showed resistance.Linares et al., (2005),following testing of 104 strains of P. wickerhamii and 2 control strains, all strains were shown to be susceptible to voriconazole, the inhibition being produced at ≤ 0.5 μg/ml, indicating that this substance may be an option for treating infections with Prototheca spp.Marques et al., (2006) mentions the susceptibility of P. zopfii and P. wickerhamii strains isolated from mastitis cows to amphotericin B and nystatin, the latter being more active.Milanov b et al., (2006) have pointed out that Prototheca isolates from mastitis cows were susceptible in vitro to nystatin and amphotericin B, intermittently sensitive to polymyxin B, gentamicin and neomycin and resistant to kanamycin, enrofloxacin, ceftriaxsone, streptomycin, amoxicillin, tetracycline, penicillin, lincomycin and novobiocin.Lassa et Malinowski (2007) by testing the in vitro susceptibility of 168 strains of algae isolated from cow's mastitis versus 10 antifungals and 10 antibiotics (disc diffusion method), showed only sensitivity to aminoglycosides and gentamicin (75.5%), kanamycin (71.7%) and neomycin (30.6%), and from antifungal only to nistatin, amphotericin B and pimaricin (natamycin).Buzzini et al., (2008) conducted a screening on the sensitivity of 105 strains of P. zopfii isolated from different locations of dairy cows farms versus a panel of polyene antibiotics, find a good in vitro efficacy of amphotericin B and pimaricin, followed by nystatin and filipin, mentioning that two strains were resistant to all 4 tested polyenes.Bouari et al., (2011) finds, by the E-test, an in vitro inhibitory effect on ketoconazole and amphotericin B against P. zopfii strains and oneP.wickerhamii reference strain.Sobukawa et al., (2012) shows by E-test that P. zopfii genotype 1 strains have been shown to be more susceptible to amphotericin, gentamicin and kanamycin than the genotype 2 and resistant to itraconazole.Wawron b et al., (2013) by examining 27 strains of P. zopfii isolated from cow's milk, mentions the 100% resistance of strains to several antifungals and antibiotics: clotrimazole, fluconazole, econazole, flucytosine, cefoperazone, cephalexin, enrofloxacin, lincomycin, oxytetracycline and 92.6% to miconazole; nistatin, ketoconazole and amphotericin B shows the best inhibitory activity; sensitivity was also recorded to gentamicin (96.3%), kanamycin (59.3%) and polymyxin (59.3%).In addition to antibiotics and antifungals, the sensitivity of Prototheca to other chemical substances was also tested.Bodenhoff et Madsen (1978) have tried to treat Prototheca mastitis, with ethidium bromide, that was found to be active in vitro, but unsuccessfully in vivo.It is shown that the number of excreted algae decreases, but complete healing is not obtained.In the histological sections made from the udder, after the slaughter of the animal, microorganisms were present both intracellularly and extracellularly.Bergmann a (1993)reports the use of tetramisole and levamisole hydrochloride by intramammary administration in mastitis cows produced by P. zopfii using 40 ml/quarter (4 mg/kg mcg) in 6 administrations simultaneously with milking.Algae elimination is suppressed for at least 6 hours.Administration of 200 ml/quarter, results in suppression of elimination for 36 hours.The same author mentions that oral administration of nilverm (overdose) does not lead to the suppression of the elimination of microorganisms through milk.[115]; [116].In all cases, treatments have been ineffective, and algae persist in tissues, even during dry periods.No spontaneous healing of Prototheca mastitis has been reported.(Lassa et Malinowski, 2007).

VI. PREVENTION AND CONTROL
Infections of the mammary gland with Prototheca have grown in importance, being more and more frequently reported on some farms, with an emerging character (Lagneau, 1995); (Bozzo et al., 2014); (Milanov b  et al., 2016); (Alves et al., 2017).It is concluded that the treatment of mastitis caused by Prototheca is totally unprofitable, and the slaughter of the infected animals is recommended, thus eliminating the polluting sources (Baumgartner, 1997); (Costa b et al., 1996); (Schlenstedt et al., 1997).(Migaki, 1988); (Kirk et Mellenberger., 2011): [115]; [116].The management and conditions in the shelter where the animals are kept, greatly influence the occurrence of mastitis with Prototheca species, the outer environment being considered the main epidemic source.Other factors influencing the occurrence of algal mammary infections are poor hygiene of milking equipment, milk collection and storage containers, and poor sanitary hygiene in shelters.It is advisable to avoid leaving the cows to adopt the lying position immediately after milking because the sphincter of the teat canal is relaxed, it does not close instantly, allowing easy access of various microorganisms that can penetrate to the level of glandular acins (Costa a et al., 1996); (Jánosi et al., 2001); (Langoni et al., 2013).Avoid contact of nipples with drainage water, which can be highly contaminated.The effort of a strictest sanitation should be followed and achieved throughout the milking process to avoid any contamination.(Costa b et al.,  1996).Intensive and prolonged antibiotic treatments should be avoided, which often lead to the increase and spread of antibiotic resistance and therapeutic inefficiency (Lagneau,1996).
In the cow mastitis control programs, in several countries, unicellular algae of the genus Prototheca are also included among potential pathogens (Schlenstedt et al., 1997); (Kirk, 1999); (Kirk et Mellenberger, 2011).Detection of infected cows could be done by bacteriological (cultivation) and serological (ELISAi) exams for the detection of IgA and IgG1 (Roesler et Hensel, 2003), but also through other techniques (fluorescence, PCR, MALDI-TOF mass spectrometry etc).Control measures are preferable (with all the stress the animals are subjected to) to identify the eliminators, and epidemiological investigations should be systematically applied even if the disease is sporadic.Due to the spontaneous emergence and lack of efficacy of the medication used, the slaughter of infected cows seems to be the only method to be used to eradicate the infection (Schlenstedt et al., 1997).At the same time, it is necessary to improve farm management and milking hygiene(Costa a et al., 2006); (Baumgartner, 1997).

VII.
CONCLUSIONS Prototheca mastitis have become emerging, and leading to important economic losses.Treatment of mastitis with Prototheca in cattle, often established on the basis of antibiogram results, which demonstrate in vitro susceptibility to various medicinal products, has been shown to be ineffective in vivo.For this reason, mastitis produced by Prototheca species are believed to be incurable, treatment is doomed to failure and totally non-economic.It is advisable to remove diseased or carrier animals from the stock, thereby eliminating the sources of contamination.

Journal of Forest, Animal and Fisheries Research (IJFAF) [Vol-1, Issue-4, Nov-Dec, 2017] https://dx.doi.org/10.22161/ijfaf.1.4.3 ISSN: 2456-8791
Through numerous in vitro research, these algae have been shown to be sensitive to some antibiotics, such as "polyene and aminoglycoside", as well as some "azole" antifungals.The percentage of susceptible strains is higher for antifungals, which is why they were more commonly used in therapeutic trials..A strain of P. zopfii isolated from mastitis cows was found to be sensitive to natamycin and nystatin but they only have local action (Moubamba b et al., 1997).In vitro resistance to various antimicrobial agents of Prototheca isolates, from mastitis cases, has been reported by other authors(Costa b et al., 1996);

-1, Issue-4, Nov-Dec, 2017] https://dx.doi.org/10.22161/ijfaf.1.4.3 ISSN: 2456-8791 stagnora
Lagneau (1996)hand, the sensitivity to ribostamycin (60 mcg/disc) is a criterion for differentiation between Candida (resistant) and Prototheca (inhibited) (Casal et Gutierrez d , 1986).Costa b et al.,(1996)as a result of the treatments carried out in cows with subclinical mastitis caused by Prototheca spp, conclude that these algae do not respond to antimicrobial treatment, requiring the elimination of the affected animals as the best way of controlling the disease.AlsoLagneau (1996),states that the treatment of protothecosis is a real problem caused by the lack of response to common anti-mastitis medications and that treated cows are not recovered.Linareset al., (1998) following the examination of 11 Prototheca strains by E-test, against 5 antifungal agents (flucytosine, ketoconazole, itraconazole, and amphotericin B), showed only susceptibility to amphotericin B.It is stated that the E test is easy to perform and provides MIC values similar to those obtained using other techniques.Aalbaek et al., (