Chapter 13

MYCOTIC MASTITIS

(Yeasts, moulds, actinomycetes, colourless algae)

Mycotic mastitis has been associated with over 26 species of fungi in probably both primary and secondary roles and has generally followed antibiotic treatment of bacterial mastitis. Clinical symptoms are very variable and the detection and isolation of the fungus are necessary for diagnosis. Recovery is generally spontaneous but may take up to five months. No treatment has proved really effective. Infection by Cryptococcus neoformans is the most serious form of disease.

Disease name: Mycotic mastitis

Pathogens:

 

Other isolates of uncertain status from mastitis cases are listed below, the figures given after each species indicating the number of individual cases on record:-

Hansenula anomala (Hansen) H. & P. Sydow, 1; H. angusta Wickerman, 1;Pichia farinosa (Lindner) Hansen, 22; Sporobolomyces roseus Kluyver & v. Niel., 1; Candida albicans (Robin) Berkh., 3; C. catenulata Diddens & Lodder, 2; C:guilliermondlii Cast.) Langeron, 5; C. krusei (Cast.) B.erkh., 9; C. mesenterica (Cieiger) Diddens & Lodder, 1; C. mycoderma (Reess.) Lodder & Kreger van Rij, 1; C. pulcherrima (Lindner) Windisch, 1; C. rugosa (Anderson) Diddens & Lodder, 2;C. solani Lodder & Kreger van Rij 2; C. zeylanoides ( Cast.). Langeron & Guerra; 3; Torulopsis candida (Saito) Lodder,5; T. famata (Harrison) Lodder & Kreger van Rij, Geotrichum candidum Link, 3; Protothecii portoricensis Cif., Ashf. & DaImiiu, 1; various identified moulds were mentioned by Ainsworth & Austwick (1955a), and many unidentified yeasts and actinomycetes have been found by other authors.

References from which this list was compiled and which are not included above or in the text are:-

Benito-Trujillo et al., 1955; Blank,1957b; Kauker,1955; Munch-Petersen, 1954; Seele & Lauer , 1955; Smith, 1934.

Hosts. Cattle; goat.

Geographical distribution. Europe and North America.

Although bacteria are the predominant cause of bovine mastitis, single cases and occasional outbreaks of the disease have been attributed to infection by fungi. Only a few of these have been substantiated by the experimental reproduction of the disease with the suspected causal organism, but it is most likely that a number of the species of fungi regularly isolated from mastitis milk samples and at present considered to be without pathogenic significance, will eventually be shown to infect the bovine udder under certain conditions.

Apart from the almost prophetic suggestion by Thom (1928) that in certain cases the fungi found in milk might have come from udders with mycotic disease, the first reference to fungi as the cause of mastitis was by Rolle (1934), who isolated yeasts from five cows being fed on brewery by-products. There is little doubt that mycotic mastitis existed before the advent of antibacterial antibiotics, but since then there has been an increasing number of cases reported almost invariably associated with prior antibiotic treatment of diagnosed or suspected bacterial mastitis.

From a study of the literature it has not always been easy to relate the fungi which have appeared in cultures of mastitis milk to their actual presence in the affected udder and it is probable that a number of isolates were contaminants derived from the cowshed air, the teat surfaces, or the collecting vessel. Possibly the copious growth in milk of Geotrichum candidum (Syn. Oidium lactis) reported by early workers, the presence of Saccharomyces exiguus found in normal milk by Zürn (1877) and the 17 "species of filamentous fungi" listed by Ainsworth & Austwick (1954), were all contaminants. It is the yeasts and actinomycetes which seem to be best adapted to growth in the ducts and acini of the udder, by virtue of their ability to break up into small vegetatively-reproducing units, thus closely resembling the bacterial method of growth. From the point of view of rapid spread and penetration filamentous fungi are at a disadvantage when growing in a liquid medium if compared with the yeasts and the only certain case of mastitis due to a mould, is that briefly reported by Ainsworth & Austwick (1955a) in which a fungus, thought to be Aspergillus fumigatus, was present in a large mycetoma in the udder of a goat.

One of the more unsatisfactory aspects of the disease is the lack of specific determination of the isolated fungi, for only 10 of the 20 accounts of mycotic udder infection give the name of the fungus involved. Few data on the relative importance of the different species are available, but reference to the list of species and the numbers of cases reported for each, shows that some species occur much more frequently than others. A start on the experimental production of the disease with known fungi has been made by Redaelli (1957a,b) and these papers should provide very useful information for pathogenicity studies.

With so few background data, mycotic mastitis is not a commonly recognized disease but there is some evidence that it might be more frequently reported if mycological examination of mastitis milk was undertaken instead of relying on normal bacteriological cultural and staining techniques. The species of fungi so far isolated from mastitis milk show a wide range of types with a predominance of yeasts. So far there are on record some 430 individual cases of mycotic mastitis of which over half occurred in 13 outbreaks, one involving 106 cows. One of the few estimates of incidence in a herd has been made by Burkey et al. (1951), who recorded 64 cases during 1945-50 with the yearly incidence in a herd varying from none to 8% of all cases of mastitis.

Symptomatology.

At present there is no evidence that mycotic mastitis is clinically distinguishable from the bacterial disease, but further study may reveal criteria which might be used for a tentative diagnosis. Cryptococcal mastitis (Cryptococcus neoformans infection) is by far the most serious type and it has already been mentioned under cryptococcosis. Pounden et al. (1952) found that the symptoms were most variable, but a number of cows showed a characteristic gradual development over a period of several days of severe swelling in more than one quarter which persisted for a long time. In the outbreak described by Simon et al. (1953) loss of appetite and a rise in temperature were noted, but the absence of toxaemia was a feature. Galli (1954), on the other hand, reported that swelling occurred in all four quarters within 12 hours of the administration of penicillin for the treatment of streptococcal mastitis, and was accompanied by cessation of milk production, subcutaneous oedema of the udder, perineum and sternum, and swelling of the supramammary and internal iliac lymph nodes, but no rise in temperature. In all cases the drop in milk yield was dramatic and in many cows, permanent.

In the cases associated with other yeasts, the symptoms appear to have been much milder (Galli, 1954), with the acute phase of the disease characteristically occurring 2-3 days after antibiotic treatment of bacterial mastitis and being followed by a gradual recovery over a comparatively long period of up to five months (Hulse, 1952), during which time yeast cells may still be detected in the milk. The nature of the secretion varies in the different stages of the disease but greyish or yellowish floccules have been reported (Andersen & Jorgensen, 1949) and the cryptococcal infection may produce a mucilaginous secretion.

Pathology.

Yeast cells and actinomycete filaments can be detected in wet preparations of the udder secretion, but are not always easy to distinguish from the other bodies in milk. Smears stained with the periodic acid-Schiff technique are probably the most satisfactory for distinguishing yeast cells but actinomycete filaments require Gram staining or an acid-fast technique for differentiation.

It is most probable that a characteristic tissue response to fungal infection occurs with a well-defined cellular reaction with polymorpho-nuclear cells, eosinophiles and histiocytes predominating together with epithelial cells. The histopathology of the udder lesions has been investigated only in cases of cryptococcal mastitis. In these Innes et al. (1952) described the changes from the acute to the chronic stage which ended in the destruction of all except the fibrous framework of the gland. Galli (1954) found granulomatous foci up to the size of a pea in the udder tissue.

Mycology

The isolation of yeasts and filamentous fungi from milk is normally accomplished without difficulty by using malt or Sabouraud's glucose agar preferably with added antibacterial antibiotics. Incubation should normally be carried out at 37° and 25°C. Aerobic actinomycetes maybe isolated on blood or nutrient agar at 37°C. but will not grow in the presence of the antibiotics.

The filamentous fungi, e. g. Aspergillus fumigatus, may be recognized by their morphology on direct examination but almost all yeast species require extensive study of their morphological and physiological characters before they can be identified. Reference to this problem has already been made under moniliasis, and it must be stressed here that the identity of the species involved in the case or outbreak is of great importance. The monograph of Ladder & Kreger van Rij (1952) has greatly facilitated the identification of yeasts, but at present these fungi are still a specialized field of mycology .

Cryptococcus neoformans is undoubtedly the most important yeast causing mastitis, and its early detection is essential if a severe outbreak involving the slaughter of affected animals is to be prevented. Fortunately it is rare but the outbreaks already referred to are evidence of its virulence. It may be detected in the udder secretion by its large cells, 5-20 micra in diameter, each surrounded by a wide, transparent mucilaginous capsule. Colonies on malt agar are cream or buff coloured and usually extremely slimy, characters shared by some of the saprophytic Cryptococcus spp., so that final identification must rest on the physiological tests and intracerebral inoculation of mice.

Of the other commonly isolated yeasts, Pichia farinosa is one of the more frequent (Lernau et al., 1947). It forms a white, powdery-surfaced colony on malt agar, and its elongated cells fuse (conjugate) in unequal pairs (anisogamously) to form 4-spored asci. Saccharomyces fragilis forms a smooth, greyish-brown, matt, creeping colony on malt agar, has narrow elongated cells, bean-shaped ascospores, and closely resembles Candida krusei which is considered to be its imperfect (non-ascosporic) state. In the genus Trichosporon, the hyphae split up into arthrospores as well as forming normal budding yeast cells. It may be noted that Candida albicans, so often involved in human and avian infections, has been found associated with mastitis on three occasions only, and has in fact rarely been isolated from cattle, van Uden & do Carmo Sousa (1957) having failed to find it among 131 yeast isolates obtained from 252 bovine caeca. These authors considered that it was replaced by C. tropicalis as a common alimentary commensal and it is not surprising that the latter species figures as a moderately frequent cause of mastitis (see Sipka, 1957).

The characters of Nocardia asteroides have already been described under nocardiosis , and descriptions of several of the filamentous fungi isolated from milk will be found under aspergillosis and mucormycosis. The colourless pathogenic alga Prototheca greatly resembles a yeast both in its cultural appearance and its morphology , but instead of budding it forms four or more vegetative spores inside each of the large oblong cells (6 -16 x 5 -11 micra) and these are freed by the rupture of the parent cell wall into two equal parts. It is not clear at present whether the two species isolated from mastitis cases, P. zopfii and P. portoricensis , are identical but the pathogenicity of the former for the bovine udder has been shown by Lerche (1952).

Epidemiology

The use of antibiotics as the standard treatment for bovine mastitis makes the evaluation of fungi as a primary cause of the disease extremely difficult. Whenever details are available it appears that fungal infection has almost always followed antibiotic therapy, occasionally without any evidence that bacteria were primarily involved. Among the preceding bacterial infections reported have been those caused by Corynebacterium pyogenes, Escherichia coli, Serratia indica, Staphylococcus aureus, Streptococcus agalactiae, Str. faecalis, and Str. uberis, and it is therefore probable that in many cases the fungi are secondary invaders colonizing the necrotic material left after the elimination of the bacteria. This may be the reason why Redaelli (1957a, b) found that he could set up primary mastitis in cows and goats with only four species of yeast; possibly five other species frequently reported from mastitis cases can establish infection only in already damaged tissue.

The source of the fungi, particularly of the yeasts associated with mastitis is virtually unknown, for knowledge of their natural occurrence is very scanty. The normal yeast floras of plant and animal substrates and that in the air are just becoming known and the only material linked with the occurrence of mycotic mastitis has been "brewers grains" - the soaked remains of the malted barley after the wort has been drained off. These "grains" have a high feeding value and although initially without a yeast flora, they appear to be an excellent substrate for yeast growth and most probably become quickly contaminated. Because only the wort is inoculated with brewers' yeast, "brewers' grains" never come in contact with this species (Saccharomyces cerevisiae) and in fact it has never been reported from an udder infection. At least six outbreaks have been associated with the feeding of brewers' grains, but unfortunately in no case was the causal organism identified. Airborne yeasts are predominantly non-fermentative ( di Menna, 1955b) and rarely seem to be involved, but many of the filamentous fungi are known to be abundant as spores in the air of cowsheds. Sometimes the infecting fungus is in therapeutic material or its diluent, for Andersen & Jorgensen (1949) found that the distilled water used to dilute the stock penicillin solution contained a similar yeast to that isolated from the mastitis cases.

Perhaps the most significant finding in relation to the epidemiology of mycotic mastitis has been the discovery by Emmons (1955c) that a high proportion of the pigeon droppings he collected in barns contained Cryptococcus neoformans, although this fungus does not appear to cause any disease in the birds themselves. Dust from pigeon lofts, barn rafters or soil (Emmons, 1951) may thus have been the source of infection in the extensive outbreaks of cryptococcal mastitis already referred to. In each case the disease had followed antibiotic treatment.

Another factor concerning the epidemiology of the condition is the frequent occurrence of yeasts which form a pellicle in liquids. Surface films of these yeasts would tend to cling to dairy utensils and, under conditions of inadequate sterilization, could be transferred to the hands of the milkers and so to the udders of the cows. Contamination of dry-stored teat cups by a Candida sp. was reported recently by Simon & Hall (1955) during an investigation of an outbreak of mastitis apparently caused by the same fungus.

Treatment

With the exception of the cryptococcal infection, mycotic mastitis appears to be a self-limiting disease with an acute phase subsiding in most cases within a few days. A delay of two days in diagnosis is therefore often sufficient to allow for spontaneous recovery so that reports of successful treatment are very difficult to assess. Such treatments as have been used have been directed more towards the alleviation of the symptoms than to the control of the pathogen involved. Thus Galli (1954) treated nine cases of Candida pelliculosa infection by stripping out 3-4 times a day and found an improvement in a few days. Steele-Bodger (1953), dealing with 17 cases associated with unspecified yeasts, injected the infected quarters with an iodine and liquid paraffin preparation and claimed to have obtained improvement within 24 hours. Tucker (1954) tried cold applications and stimulants. Mastitis due to Nocardia asteroides (Sforza, 1946) and other aerobic actinomycetes would appear to respond to the normal therapy for bacterial mastitis as these organisms are mostly susceptible to the commonly used antibiotics.

Cryptococcal mastitis presents a much more serious problem in control because of the severe losses it may entail and the possible danger to man from contact with infective material, although there is no record of such transmission. In the outbreak described by Pounden et al. (1952), 23 compounds were tested in vitro against the fungus, but the few which proved active failed to show any real effect in vivo. With the same type of infection, Segretain et al, (1956) found that cycloheximide at a daily dosage of 24 mg. per quarter for nine days was well tolerated and apparently led to improvement in the condition.

Normal hygienic measures including the segregation of affected cows and the careful sterilization of dairy utensils appear to provide the best method of controlling outbreaks in the absence of any really effective udder treatment. The immediate cessation of antibacterial antibiotic treatment when this has proved ineffective, seems to be an important step, for enhancement of the pathogenicity of Candida albicans has been noted in mice and chick embryos treated with aureomycin and penicillin respectively (Seligmann, 1952; Foley & Winter, 1949), and it is possible that similar factors may operate in the bovine udder. A close watch should also be kept on the microflora of the secretion for the reappearance of pathogenic bacteria and for the gradual decrease in yeast cells.

Wilson (1957) considers that where bacterial mastitis has occurred there is no difference between the susceptibility of infected and uninfected quarters to subsequent yeast infection, and that recrudescence of mycotic infection in the same quarter is uncommon. Moreover once the initial phase is over the infection shows little tendency to spread to other cows.

Experimental inoculation

Rolle (1934) tried inoculating the udder of a normal cow with the yeast he isolated from a mastitis case and produced swelling within 24 hours which persisted for more than ten days, whilst yeast cells could be recovered from the milk for a month afterwards. Using Prototheca zopfii, Lerche (1952) found that a swelling occurred after eight days and that cells persisted in the milk for up to five months. In Hulse's (1952) experiments recovery took eight weeks after the inoculation of 300 million yeast cells into the teat canal, whilst Stuart (1951) reproduced the condition in two out of seven quarters only by infusion of secretion from infected udders, the incubation period being 16 hours in one and 3 days in the other. Up to 60 million cells of the isolate grown in pure culture failed to set up the disease .

In the most recent series of experimental inoculations, Redaelli (1957a, b) found that with Candida tropicalis, C. pelliculosa, and Saccharomyces fragilis reaction occurred in 1-6 days according to the number of cells injected and each species produced a different pathological response. With Cryptococcus neoformans symptoms did not appear until 16-36 days after inoculation. He failed to produce infection with Hansenula anomala, Pichia farinosa, Candida albicans, C. krusei and C. solani.

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