Mycopathologia140: 89–97, 1998.© 1998Kluwer Academic Publishers. Printed in the Netherlands.

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Experimental candidal mastitis in goats: Clinical, haematological,biochemical and sequential pathological studies

Pritpal Singh, N. Sood, P.P. Gupta, S.K Jand & H.S. BangaDepartment of Veterinary Pathology, Punjab Agricultural University, Ludhiana, India

Received: 20 February 1997; accepted in final form 16 February 1998

Abstract

The present study, first of its kind, was conducted with the objectives to understand hitherto little known aspectsof candidal mastitis, like its sequential pathology, pathogenesis and clinico-biochemical changes. For this purpose,unilateral intramammary inoculation of 10 goats withCandida albicans(1.2× 107 yeast cells) resulted in thedevelopment of mastitis, with gross and microscopic lesions being restricted to the infected udder halves only andwithout dissemination of infection to the opposite uninfected udder halves as well as other organs of the body.The experiment was continued for 40 days and after infection, there was sharp fall in milk yield andCandidaalbicanswas directly demonstrated in the milk and re-isolated from the milk and udder tissues up to 30th dayafter inoculation. An increase in total immunoglobulins in the milk and plasma along with increase in total plasmaproteins were also observed. Haematology revealed leukocytosis and neutrophilia. Microscopically, there wasacute purulent mastitis, which later became chronic, nonpurulent and interstitial with formation of granulomas. Itwas concluded thatCandida albicanswas highly pathogenic to the lactating goat mammary gland even withoutimmunosuppression or antibiotic treatment, resulting in severe irreversible tissue damage and nearly completeagalactia.

Key words: Candida albicans, goats, mastitis, pathology, experimental, pathogenesis, haematology, biochemicalchanges.

Introduction

Yeasts are ubiquitous in nature and have been fre-quently implicated in causation of subclinical andclinical mastitis in dairy animals [1] and man [2]. Amajority of yeast isolates from mastitic milk belongedto genusCandida[3–11]. However, the relationshipbetween lesions and candida is imprecisely understood[12] and pathogenesis of candidal mastitis in goats ispoorly documented. The present study was, therefore,undertaken to assess pathogenicity, sequential clinico-pathological and pathogenetic alterations induced byC. albicansin the lactating goat udder.

Materials and methods

Experimental animals:The present experiment wasconducted on 15 lactating female goats aged 2–4 years

which were given food and waterad libitum.The goatswere kept under observation for a fortnight in a thor-oughly cleaned premises before the start of the experi-ment and were adjudged to be healthy. All the animalswere free from sub-clinical mastitis and no bacteriaor fungi were isolated from the pre-inoculation milksamples.

Preparation of experimental inoculum:The strain ofC. albicansused in this study was isolated from anatural case of caprine mastitis and the isolate wasgrown on Sabouraud’s dextrose agar (SDA) contain-ing 0.03% chloramphenicol. After incubation for 5–6days at 37◦C, the growth was harvested by flush-ing with sterile phosphate buffer saline (PBS, pH 7.4)containing 0.05% Tween-20. The suspension was ho-mogenized with the help of a magnetic stirrer andthe blastoconidia concentration was adjusted to 60×106/ml using a haemocytometer.

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Figure 1. Markedly swollen and enlarged right udder half (R) of agoat 24 hrs after infection withCandida albicans.

Experimental design:The animals were randomly di-vided into 2 groups of 10 infected and 5 control goats.The right udder halves of each goat in the infectedgroup was inoculated intramammarily with 2 ml oftheCandidainoculum, whereas the left udder halvesof each infected goats and both of the udder halvesof 5 control goats were similarly infected with 2 mlof sterile PBS, pH 7.4 containing 0.05% Tween-20.The animals in two groups were kept in separatelyand distinctly located rooms. The control goats werealways attended, milked, fed and watered before han-dling the infected goats or contaminated material. Theanimals in both the groups were closely observed dailyfor gross changes in their udders and milk secretions,Milk samples collected before and after inoculationwere subjected to mastitis tests like bromothymol blue(BTB), modified Whiteside and California mastitistests and somatic cell counts (SCC). Blood and milksamples drawn at 0, 5, 10, 15, 20, 25, 30, 35 and

Figure 2. Markedly enlarged (about 2 times) right (R) infected ud-der half; teat and right supra mammary lymph nodes, 5 days afterinfection withC. albicans.

40 days post inoculation were also subjected to esti-mation of total proteins and total immunoglobulins.Haemoglobin (Hb), total leukocyte count (TLC) anddifferential leukocyte counts (DLC) of blood were alsodetermined at these intervals. Two randomly selectedanimals from the infected group and one animal fromthe control group were killed on days 5, 10, 20, 30and 40 after infection and were subjected to detailedpost-mortem. Gross changes, if any, were recordedand tissues from both the udder halves, supramam-mary lymph nodes, lungs, heart, kidneys, liver, spleen,brain, abomasum, intestine, ovaries and uterus werecollected in 10% neutral buffered formol saline. Paraf-fin sections, 5µm thick, were cut and stained withhaematoxylin and eosin (H&E) [13].

Re-isolation and demonstration of Candida:Re-isolation of the fungus was attempted from milk andudder lesions, pieces of which (collected in sterilized

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containers) were cultured on SDA slants at 37◦C for2-7 days. Demonstration of the fungus in impressionsmears of milk and from udder tissues and paraffintissue sections was done with the help of Grocott’smethanamine silver (GMS), Periodic acid schiff (PAS)and combined GMS-H&E [14]. Statistical analysiswas carried out by using one way analysis of variance[15].

Results

Clinical signs and changes in milk:The infectedgoats did not show any systemic response and theirappetite and body temperature remained unaffectedduring the experiment. However, within 24 hrs, theirright infected udder halves developed clinical masti-tis characterized by pain, hot to touch, tenderness andtheir enlargement as compared to contralateral left ud-der halves (Figure 1). The right udder halves remainedlarger till the end of the experiment. On palpation, theright supramammary lymph nodes of all the infectedgoats were found to be swollen and enlarged as com-pared to their contralateral lymph nodes. The averagemilk yield from the infected udder halves decreasedsharply and progressively from day 1 after infectionand was reduced from 180.31 ml/day before infectionto 12.38 ml/day on day 40. The mastitic mammarysecretions from all the infected goats were thick, co-agulated, creamy and sometimes blood tinged duringfirst 6 days and later the secretions became thin andflocculent by 20 DPI and the secretions were almostwatery by 40 DPI. There was no change in colour,consistency or amount of milk from the uninocu-lated halves of udders of the experimental as well asthe control animals. The average SCC of mammarysecretions from infected udder halves increased sig-nificantly from the pre-inoculation basal value of 0.35× 106/ml to 52.25× 106/ml on day 20. Thereafter,it decreased gradually till day 40, although remainingmuch higher than basal value as yet (10.21× 106/ml).Total proteins in mammary secretion of infected udderhalves showed a progressive and significant decreasefrom pre-infection basal value of 3.6 gms% to 2.88gms % on day 40, while, total immunoglobulins re-vealed a progressive and significant increase frombasal value of 67.21 mg% to 142.8 mg% by day 25.

Haematological examination:Total plasma proteinswere progressively increased from basal value of 6.25gm/dl till day 25, while total immunoglobulins in

plasma were progressively and significantly increasedfrom basal value of 1.58 gms/dl to 2.02 gms/dl on day35. Haemoglobin of all the infected and control goatsremained almost unaltered, whereas TLC showed amarked increase from pre-infection basal value of 8.56× 103/cumm to a peak value of 12.67× 103/cummon day 20. In addition, there was a significant neu-trophilia that peaked on day 10 (60.37%).

Gross lesions:Gross lesions were noticed only inthe infected right halves of the udders. On day 5,post inoculation, these halves and their supramam-mary lymph nodes were enlarged about twice the sizeof their left counterparts (Figure 2). On cutting thesetissues, congestion and necrotic foci (0.5 to 1 cm indiameter) were visible. Varying sized nodules (pinto pea) were observed on the external and cut sur-faces of the infected right udder halves on 10th DPI,and became more conspicuous on subsequent inter-vals. Similarly, the right supramammary lymph nodesremained enlarged as compared to their left counter-parts throughout the experiment. No gross patholog-ical changes were noticed in the uninoculated udderhalves of experimental and both the udder halves ofcontrol animals.

Histopathology:The infected right udder halves ofthe goats killed on 5 DPI showed acute diffuse pu-rulent mastitis, characterized by marked neutrophilinfiltration in the acini (Figure 3). PAS, GMS andGMS-HE stained sections revealed pseudohyphae andbudding forms ofC. albicans(Figs. 4 and 5). Onlyclumps of distorted yeast like structures but no pseudo-hyphae, were visible in macrophages and giant cellsat 20 DPI (Figure 6). There was also presenceof phagocytosed blastoconidia/chlamydosporeswithinthe macrophages in a few places (Figure 7). By 10thDPI the neutrophils were degenerated and their num-bers in the acini had decreased but infiltration oflymphocytes, plasma cells and macrophages in theinterstitial tissue was evident. In places, giant cellgranulomas were seen and small pseudohyphae andbuddingCandidaorganisms were visible inside themacrophages and foreign body giant cells. A chronicgalactophoritis first observed on day 10 was consis-tently observed till 40th day of the experiment char-acterized by marked hyperplasia of lining epitheliumduring initial days and was seen as stratified squamousmetaplasitic epithelium by 20 through 40 DPI, besidesthere was also organization of exudate into fibroustissue leading to complete occlusion of their lumens.

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Figure 3. Udder (5th DPI). Acute diffuse purulent mastitis characterized by marked neutrophilic infiltration in the lumina of acini. H&E× 300.

Figure 4. Udder (5th DPI). Pseudohyphae and budding yeasts ofC. albicansmixed with purulent exudate in the lumina of acini. GMS-H&E× 300.

By 20th DPI, mastitis became chronic with markedfibroplasia in the interlobar septa and interacinar in-terstitial tissue replacing the acini and also leadingto pseudolobule formation by day 30 (Figure 8), andleaving only small islands of secretory tissue in fi-brous tissue pool by 40th DPI. In places, lymphoid

cell infiltration/aggregates were found replacing theudder parenchyma on 30 and 40 DPI (Figure 9).Smears of mastitic mammary secretions from infectedright udder halves showed the presence of elongatedpseudohyphae and blastoconidia/chlamydospores on 5DPI (Figure 10).

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Figure 5. Udder (5th DPI). Same as in Figure 4 showing branched pseudohyphae more clearly. GMS× 300.

Figure 6. Udder (20th DPI). Clumps of distortedCandidaorganisms in the macrophages and giant cells. Note the absence of pseudohyphae.GMS× 300.

The right supramammary lymph nodes of allthe infected goats showed reactive lymphadenitis.The lymph nodes also revealed degeneratedCan-dida organisms mostly within macrophages in theirmedullary region at 40 DPI.

The uninoculated udder halves, their ipsilaterallymph nodes and other organs of the animals ofboth the infected and control groups did neither re-veal any significant pathological change norCandidaorganisms.

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Figure 7. Udder (20th DPI). Large number of blastoconidial chlamydospores mostly within phagocytes. PAS× 300.

Figure 8. Udder (30th DPI). Extensive fibrosis in the interlobular septa dividing the lobules into pseudolobules. H&E× 70.

Discussion

In the present study, severe clinical mastitis developedwithin 24 hrs of Candida inoculation tallying with sim-ilar observations of several other workers [16–19]. Theintramammary inoculum containing 1.2× 107 Can-dida cells produced marked clinical mastitis in the

right udder halves of all the infected goats, whichsupported the viewpoint thatCandidaalone was capa-ble of causing mastitis, if employed in sufficient dose[1, 20–25]. These findings were, however, contraryto the popular belief that previous antibiotic treat-ment [10, 26–33] or immunosuppression [34, 35] werenecessary for setting up of clinical candidal infections.

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Figure 9. Udder (30th DPI). Lymphoid aggregates replacing the acini. H&E× 150.

Figure 10. Milk smear from the infected udder half showingCandidaorganisms in the form of long pseudohyphae and budding yeasts on 5thDPI. PAS× 300.

Marked agalactia as observed by us, was alsorecorded in mycotic mastitis earlier [37–39]. Simi-larly, qualitative changes as well as rise in SCC inmastitic mammary secretions from mycotic mastitishad been documented previously [3, 20, 25, 38–44].

The decrease in total proteins in milk as observedin this study and also reported in cryptococcal mastitis

[38], might have been either due to their increasedutilization/degradation or decreased secretion by thedamaged acinar epithelium [35, 38]. The increase intotal immunoglobulins in milk from 20 DPI onwardsindicated mounting of local humoral immune response[45, 46], in whichCandidaspecific 1gG, 1gM and1gA antibodies may be secreted by B-cells [36].

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The increase in total Igs along withα andβ glob-ulins might have contributed to the increase in totalplasma proteins [47–49]. These observations corre-lated well with the histopathological findings of lym-phoid aggregates in the udder tissue as well as reactivehyperplasia of ipsilateral supramammary lymph nodesin the chronic stages of the disease. The haematologicfindings of marked leukocytosis and neutrophilia cor-related well with histopathologic evidence of severepurulent mastitis, particularly during early stages ofthe infection.

No systemic disturbances were recorded in thepresent study, which mimicked the findings of Holf-man et al. [18] and Verma and Kalra [25]. However,others [20, 27, 44] reported systemic disturbances likefever and anorexia in candidal mastitis,

Microscopically, the presence of numerous neu-trophils in the acini and milk ducts at 5 DPI andlater, vindicated the prime role that neutrophils play indisposing of candidal infections [50–52]. Both blasto-conidia and pseudohyphae forms were observed in themilk acini and ducts at this stage. It is thought thatblastoconidia adhere to the epithelial/mucosal cellsand cause irritation, resulting in congestion, oedemaand exudation of neutrophils, while, pseudohyphaeare more invasive and may penetrate the epithe-lial/mucosal barriers to enter the interstitial tissue [34,36]. The formation of well defined granulomas com-posed of macrophages, giant cells, lymphocytes andplasma cells at 10 DPI plays a pivotal role [36, 53, 54].Moreover, human milk macrophages have been shownto effectively phagocytize and killCandida albicansin vitro [55, 56]. The same was corroborated by thehistochemical evidence of degenerated/disintegratedcandidal bodies within macrophages in milk smearsat that stage. With the progression of days, masti-tis became more and more chronic with replacementof secretory tissue with extensive fibrosis along withinfiltration of chronic inflammatory cells includinglymphocytes, macrophages and plasma cells in the in-terstitial tissue. These findings refuted those of Kuo[44], who observed little cellular infiltration in the in-terstitial connective tissue in candidal mastitis, whichmay be due to the simple reason that his experi-ment was only of 5 days duration. In addition, inthe chronic stages, blastoconidia/chlamydiospores ofthe Candidawere seen mostly inside macrophages,which further augmented our stand that pseudohyphaemight have penetrated the epithelial barrier and werelater overcome and localized by host’s phagocyticand immune trap. The extensive parenchymal fibrosis

and fibrotic occlusion of lactiferous ducts amply justi-fied the nearly complete cessation of milk yield fromaffected udder halves during chronic stages.

Von-Veen and Kremer [57] also reported that insome cases of mycotic mastitis in cows the fungus wascleaned spontaneously. The infection in the presentstudy also appeared to be self resolving with the pas-sage of time, although it resulted in severe irreversibleudder damage.

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Address for correspondence:Dr. P.P. Gupta, Department of Veteri-nary Pathology, Punjab Agricultural University, Ludhiana-141004,India.Phone: 91-161-401960-75, Ext. 275 & 324; Fax: 91-161-400945