Original Article Equine Mammary Gland Disease with a Focus on Botryomycosis: a Review and Case Study E

EQUINE VETERINARY EDUCATION / AE / JULY 2012 357

Original Article Equine mammary gland disease with a focus on botryomycosis: A review and case study E. Smiet*, G. C. M. Grinwis†, J. G. B. van den Top and M. M. Sloet van Oldruitenborgh-Oosterbaan Department of Equine Sciences and †Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Keywords: horse; equine udder; mastitis; botryomycosis; bacterial granulomatous disease; mastectomy; Staphylococcus aureus

Summary stained with Gram’s stain – is indicated for the diagnosis of acute inflammation and tentative identification of Mammary gland problems occur incidentally in horses and bacterial organisms prior to culture and antibiotic one of the rarer conditions is botryomycosis (bacterial susceptibility results being available (McCue and Wilson pseudomycosis, bacterial granuloma, staphylococcal 1989). Ultrasonographic examination of the mammary pseudomycetoma). This article includes a short review gland is a useful adjunctive diagnostic modality (Spadari of equine mammary gland problems inappropriate et al. 2008) that provides excellent visualisation of the lactation, mastitis and neoplasia and botryomycosis, and glandular parenchyma and allows efficient, noninvasive additionally 2 clinical cases of botryomycosis of the udder evaluation of mammary enlargement (Brendemuehl 2008). resulting from Staphyloccocus aureus infection will be Cell counts in excess of 100 x 106 cells/l during lactation discussed. Both cases involved nonpregnant, nonlactating and 400 x 106/l during involution are regarded as mares referred for chronic mammary inflammation with pathological (Böhm et al. 2009). draining abcessation. In both mares, botryomycosis In chronic problems, histological examination of a caused by S. aureus was confirmed by histopathology and biopsy of abnormal tissue is required to establish a definitive a bacterial culture. Both mares recovered fully after diagnosis. Fine needle aspiration biopsies are often surgical hemimastectomy under general anaesthesia. unrewarding and thus a wedge biopsy or a hollow needle (Trucut) biopsy is required (Brito et al. 2008; Shank 2009). Introduction Botryomycosis, also called bacterial granuloma or staphylococcal pseudomycetoma, is most commonly In horses, diseases of the mammary glands are uncommon encountered in the scrotal region after castration when compared to dairy cattle and other ruminants (Knottenbelt 2009) or after skin lacerations on the limbs or (McGladdery 1998). Galactorrhoea, mastitis, neoplasia the chin and lip (Knottenbelt 2003, 2009). Occasionally and chronic infections occur occasionally (Knottenbelt botryomycosis occurs in the tissues and skin of the udder 2003; Bader and Busch 2006). Trauma may also be a cause (Weber 2006). for udder enlargement but usually resolves quickly with This article gives a brief review of the literature on the hydrotherapy alone (Knottenbelt 2003). most common equine mammary gland disorders and on Examination of a mare with a unilateral or bilateral botryomycosis in general. Two clinical cases of enlargement of the udder should include a thorough history botryomycosis in the equine mammary gland are and a careful general clinical examination (Brendemuehl described in depth, including diagnosis, treatment and 2008). Thereafter the mammary gland should be observed, outcome. palpated and a sample of the secretion should be Review expressed from the teat (Brendemuehl 2008). Aseptic collection of milk or mammary exudate is necessary for Inappropriate lactation cytological examination and bacterial culture (McCue and Wilson 1989). The microscopic evaluation of a thin smear – Inappropriate lactation, also known as galactorrhoea and *Corresponding author email: [email protected] witch’s milk, occurs rarely and is seldom reported. Although

several different clinical entities are included in this broad Most often, equine mastitis is caused by Streptococcus term (Knottenbelt 2003), variable mammary swelling with spp. Böhm et al. (2009) and McCue and Wilson (1989) inappropriate lactation is the most common clinical finding reported involvement of coagulase-negative in horses presented at the University Equine Clinic in Utrecht Streptococcus equi ssp. zooepidemicus. Bostedt et al. for investigation of udder problems (van der Kolk 2000). (1988) found that 51% of the cases were associated with Inappropriate lactation may occur in mature mares Streptococcus spp. with 70% of the cases being for several reasons. In pregnant mares it is usually a sign beta-haemolytic Streptococcus spp. In The Netherlands of impending abortion, fetal death, early placental the most common organisms found in equine mastitis are separation or placentitis (Knottenbelt 2003; Bader and Streptococcus and Staphylococcus spp. (van der Burg Busch 2006). In nonpregnant mares inappropriate lactation et al. 2011). Other agents found are Corynebacterium spp. and swelling of the udder may be associated with pituitary and Gram-negative bacteria including Escherichia coli, pars intermedia dysfunction (PPID/Equine Cushing’s Pseudomonas spp. and Klebsiella spp. (Bostedt et al. 1988; disease) (Knottenbelt 2003). If this condition is diagnosed McCue and Wilson 1989; Knottenbelt 2003; Bader and and (temporarily) treated, the udder swelling will often Busch 2006). Anaerobic mammary infections are resolve and lactation will cease (Knottenbelt 2003). uncommon (McGladdery 1998). Sometimes unilateral or bilateral udder swelling with It is important, however, to realise that examination of lactation is present without any clear underlying cause and milk samples of healthy mares during the first 7 days after this is called ‘witch’s milk’. In human medicine, witch’s milk delivery showed that over 40% of the mares and over 20% is fairly common in normal newborn infants and is of the milk samples were bacterially contaminated characterised by breast nodules and lactation up to age 2 (Bostedt et al. 1988). months. The condition is usually attributed to maternal The diagnosis of mastitis should be established using prepartum circulating hormones (Madlon-Kay 1986). A clinical signs and macroscopic examination of any very similar state may occur in newborn foals. abnormal mammary secretion. Cell counts and bacterial In a nonpregnant mature mare, the intake of cultures with antibiotic sensitivity, are especially important phyto-oestrogens may be a cause of the inappropriate for appropriate adjustment of therapy (Knottenbelt 2003; lactation but often no underlying cause is found Bader and Busch 2006). (Knottenbelt 2003). The majority of cases of mammary Therapy consists of frequent hand milking (‘stripping swelling with inappropriate lactation of unknown origin out’), cold water hydrotherapy of the oedematous udder, resolve spontaneously within several weeks to months. nonsteroidal anti-inflammatory drugs and systemic broad-spectrum antibiotics (McCue and Wilson 1989; Mastitis Knottenbelt 2003; Bader and Busch 2006). Trimethoprim-sulphonamide (TMPS) or a combination of The incidence of mastitis in the mare is low compared to penicillin and aminoglycoside are rational choices for initial that in cows (McCue and Wilson 1989; Knottenbelt 2003; antibiotic therapy until the results of bacterial culture are Bader and Busch 2006). However, in breeding mares the known. Antibiotic treatment based on the culture and incidence has been estimated to be as high as 5% in sensitivity results should be continued for at least 5–7 days normal breeding farms and 10% in problem herds (Böhm (McCue and Wilson 1989; McGladdery 1998). et al. 2009). Data from a group of 33 mares with mastitis Since the teats of horses have 2 or even 3 canals, each showed that 27.3% of cases occurred before parturition, of which is associated with separate compartments of 24.2% immediately after delivery, 12.1% during the middle glandular tissue, the use of intramammary antibiotics is and late stages of lactation, 9.1% immediately after controversial. Additionally, many teat injectors are too weaning of the foal and additionally 27.3% of cases large and can cause damage to the teat canal occurred outside the lactation period in nonpregnant (Knottenbelt 2003; van der Burg et al. 2011). In cases of mares (Bostedt et al. 1988). In the majority of cases both mastitis unresponsive to the treatment, neoplastic disease halves of the udder were affected (Bostedt et al. 1988). should be considered as part of the differential diagnosis Clinical signs of mastitis include a painful, swollen, firm (Knottenbelt 2003). and warm udder, with or without purulent discharge (Bader and Busch 2006). Oedema of the mammary gland and the Neoplasia of the mammary glands ventral abdomen is often present (Knottenbelt 2003; van der Burg et al. 2011). The udder may be so painful that The incidence of mammary neoplasia in horses is very low physical examination requires sedation (McGladdery (Hirayama et al. 2003; Knottenbelt 2003; Reesink et al. 1998). Mastitis can be accompanied by signs of systemic 2009). Quite often tumours are not diagnosed at first and disease such as fever, anorexia and even lameness will usually have been treated as mastitis (Reppas et al. (McCue and Wilson 1989). Some cases show no systemic 1996; Brito et al. 2008; Shank 2009). Affected mammary disease in spite of severe localised inflammation glands may be painful on palpation and warm. Often (Knottenbelt 2003). Unilateral atrophy or abcessation may there is a distinctive (a)symmetric swelling with or without also occur (McGladdery 1998). cutaneous ulceration. Purulent or haemorrhagic

discharges from the teat canal(s) may be present (Shank abscessation, mostly caused by Staphylococcus aureus 2009). In some cases mastitis treatment can reduce the and occasionally by other bacteria such as Pseudomonas severity of the signs; this might initially support the diagnosis aeruginosa, Proteus spp. or E. coli (Winslow 1959; Bader of mastitis; however, the signs usually do not resolve and Busch 2006). completely and reappear when treatment is discontinued Botryomycosis was first described in the horse in 1870 as (Shank 2009). Evidence of pain is not useful in distinguishing a granulomatous lesion in the lung (Bollinger 1870), mastitis from mammary tumours (Shank 2009). Prendergast although the relationship to bacterial infection was not et al. (1999) reported pain as a clinical sign in 3 cases recognised. The term botryomycosis was used to describe diagnosed with mammary carcinoma, while McCue and the grape-like appearance of the granules (botryo) and Wilson (1989) reported pain in 15 of 28 mastitis cases. an erroneously presumed fungal aetiology (mycosis) Ultrasonographic examination of the udder is a (Winslow 1959; Bonifaz and Carrasco 1996). The bacterial useful diagnostic modality in the diagnosis of mammary aetiology was recognised in the early 20th century neoplasia, and the diagnosis can be supported by the (Magrou 1919). The condition is also referred to as rectal detection of iliac lymphadenopathy (Knottenbelt ‘staphylococcic actinophytosis’, ‘granular bacteriosis’, 2003). ‘bacterial pseudomycosis’ and even ‘actinobacillosis’ Histopathological examination of the abnormal tissue is (Bonifaz and Carrasco 1996). the definitive means of diagnosis of mammary neoplasia. As there is controversy about the terminology of Fine needle aspiration biopsies are often unrewarding as botryomycosis and other similar clinical conditions with cytology may be misleading. Wedge biopsy or a different aetiologies, Bonifaz and Carrasco (1996) hollow-needle (Trucut) biopsy provides a much more proposed the following definitions: reliable histopathological diagnosis (Brito et al. 2008; Shank 2009). • Mycetoma: this is conveniently divided into eumycotic Equine sarcoid is the most common tumour found on the mycetoma (caused by true fungi), actinomycotic mammary gland (Knottenbelt 2003), although strictly it is a mycetoma (caused by aerobic actinomycetes) and skin tumour rather than a mammary tumour. Sarcoids may mixed mycetoma (caused by 2 or more fungal significantly affect the teat and the teat canal. Melanoma microorganisms) occurs in the udder with some frequency and these are • Actinomycosis: this is caused by anaerobic often embedded within the glandular tissue; most are actinomyces that are usually part of the normal/ benign and well demarcated but occasionally they can be commensal microflora malignant, large and invasive (Knottenbelt 2003; Bader and • Botryomycosis: this is caused by nonfilamentous Busch 2006). Further, histiocytomas and lymphomas have aerobic and anaerobic bacteria, such as S. aureus, been reported (Reesink et al. 2009; Shank 2009; Mendes Pseudomonas spp., and Bacteroides spp. et al. 2011). However, neither melanoma, histiocytoma nor lymphoma are genuine mammary tumours. The most Botryomycosis can be distinguished from actinomycosis common ‘true’ neoplasm of the equine udder is the very and mycetoma by histopathological examination of a malignant (adeno)carcinoma (Munson 1987; Reppas et al. biopsy (wedge biopsy or Trucut biopsy). 1996; Prendergast et al. 1999; Hirayama et al. 2003; Lous The shape and staining characteristics of the granules et al. 2009). One benign mammary adenoma has been serve to differentiate it from actinomycosis and mycetoma described (Spadari et al. 2008). (Bonifaz and Carrasco 1996). Unlike the granules of Treatment of mammary neoplasia requires complete mycetoma and actinomycosis, botryomycosis granules surgical resection of the affected tissue and as far as are nonfilamentous and do not stain readily with the possible, the regional lymph nodes (McGladdery 1998). Grocott-Gomori methenamine-silver nitrate stain (Bonifaz In general, the prognosis for a mammary tumour is poor and Carrasco 1996). Botryomycosis can be divided into because many cases are presented very late and often cutaneous botryomycosis and visceral botryomycosis after micrometastasis has already occurred (Foreman (Bonifaz and Carrasco 1996). et al. 1990; Bader and Busch 2006). Hormone- Histopathologically, the causative organism, which is dependence, such as occurs in man and cats, is not a generally S. aureus, appears to be encapsulated in recognised feature of equine cases, at least not in the granules, and this process is thought to protect it from the malignant cases, and thus hormonal treatment is not effects of antibiotics. This morphological pattern is called generally useful (Reppas et al. 1996; Spadari et al. 2008). the Splendore-Hoeppli phenomenon and is characterised The recently described equine mammary adenoma may by in vivo formation of intensely eosinophilic material be an exception to this (Spadari et al. 2008). (radiate, star-like, asteroid or club-shaped configurations) around microorganisms (bacteria, fungi and parasites) Botryomycosis or biologically inert material (Hussein 2008). This characteristic formation of the peribacterial (or Botryomycosis is a rare, chronic, granulomatous perifungal) Splendore-Hoeppli material probably prevents suppurative disorder with the formation of micro- phagocytosis and intracellular killing of the insulting agent

leading to chronicity of the infection (Hussein 2008). The Historically botryomycosis in horses was mainly eosinophilic deposits are thought to represent antigen- recognised as a post castration complication (Knottenbelt antibody complexes and debris from host inflammatory 2009), but the botryomycotic lesions may disseminate cells, but may also represent glycoproteins, lipids and through skin, bone, tendons and other organs (van Dorssen calcium derived from host leucocytes. The exact 1960). Cutaneous botryomycosis usually requires skin molecular pathway involved in the development of the damage (e.g. pruritus from insect bite hypersensitivity or Splendore-Hoeppli phenomenon is uncertain (Hussein wounds) (Knottenbelt 2009). Clinically, the infection is 2008) and it is not always present (Scott and Miller 2003). characterised by one or more chronic, purulent The pathogenesis of botryomycosis is unclear but it discharging infected skin ulcers, with infiltration of the probably involves a delicate balance between the underlying tissues, forming nodules and/or suppurative virulence of the infectious agent, the bacterial strain, plaques. The condition is seldom painful or pruritic inoculum size, presence of foreign bodies and the host (Knottenbelt 2009). immune response (Kimmelstiel and Easley Jr 1940; Saadat Once a bacterial strain has been isolated, it should be et al. 2007). identified and an antibiotic sensitivity profile performed Botryomycosis occurs in man and animals worldwide (Bonifaz and Carrasco 1996). In horses, long courses of (Bonifaz and Carrasco 1996). Cutaneous and visceral antibiotics are often unrewarding and thus surgical forms of human botryomycosis have been described excision, if feasible, is the therapy of choice (Knottenbelt (Winslow 1959; Saadat et al. 2007). Cutaneous 2009). Repeated surgical interventions may be required botryomycosis may occur anywhere on the body (Saadat along with concurrent prolonged culture-guided antibiotic et al. 2007) but traumatic injuries are a recognised treatments (Knottenbelt 2009). predisposing factor. Visceral botryomycosis is rare and can mimic carcinoma macroscopically. General malaise, fever Cases and leucocytosis can be present. Both immunocompetent and immunosuppressed Case A patients, for instance due to acquired immuno-deficiency syndrome, alcoholism, diabetes mellitus, leukaemia, post A 3-year-old maiden Friesian mare was admitted to the transplantation immunosuppressive medication or the Equine University Clinic in Utrecht with a 3 month history of long-term use of steroids, can be affected by draining abcessation of the left mammary gland. Before botryomycosis (Schlossberg et al. 1998; Saadat et al. 2007). referral, the mare had been treated by the referring Treatment consists of surgical removal of the affected veterinarian with TMPS at a dose of 30 mg/kg bwt q. 12 h tissue. In human patients, long-term treatment with for 14 days (Cubarmix)1, phenylbutazone 2 mg/kg bwt q. broad-spectrum antibiotics only is often unrewarding 12 h per os (Pro-Dynam)2 and thorough cleaning of the (Findlay and Vismer 1990; Bonifaz and Carrasco 1996), draining tracts. She was referred as result of the failure of although in individual cases it has been successful treatment and the development of a fever. (Machado et al. 2005; Coulibaly et al. 2008; Biemans et al. At admission the mare was bright, alert and responsive. 2010). As described in one case, laser vaporisation could Her clinical parameters were unremarkable. She was in be an additional therapy (Leffell et al. 1989). good body condition, and sound at walk and trot. In animals, cutaneous botryomycosis has been The only abnormalities identified were multiple firm, described in guinea pigs (Bostrom et al. 1969), dogs nodular, ulcerative lesions of different diameters within (Scott 2007; Casamián-Sorrosal et al. 2008), cats the left mammary gland including the teat. A purulent (Sheikh-Omar and Abdullah 1985), a Siberian hamster discharge covered the skin of that area (Fig 1). Palpation (Eshar et al. 2010) and horses (van Dorssen 1960; revealed a firm, painful, warm asymmetric swelling of 20 ¥ Knottenbelt 2003, 2009). 10 ¥ 15 cm with nodular skin lesions. Rectal examination Visceral botryomycosis has also been reported in was unremarkable; there was no indication that the animals, with pulmonary involvement in a Scottish Highland process had extended to abdominal or inguinal lymph steer (Miller et al. 2001), the pericardium of a dog nodes. (Casamián-Sorrosal et al. 2008), the nasopharynx of a cow A complete blood cell count and biochemistry panel (Thompson et al. 2001) and in the uterus of mares showed values to be within normal limits (Table 1). (Ellenberger et al. 2006). Mammary involvement has only Ultrasonographic examination of the udder revealed been described very rarely in horses (Knottenbelt 2003; multiple fluid filled abscesses up to 1 cm in diameter. These Bader and Busch 2006; Weber 2006), goats (Sharma and were present up to a depth of 3–4 cm and were Iyer 1974) and once in an African elephant (Sleeman et al. surrounded by normal mammary tissue. Some abscesses 2003). invaded the teat canal, subcutis and surrounding tissues; Botryomycosis can be experimentally induced in mice no gas could be detected. (Akiyama et al. 1996), rabbits (Magrou 1919), guinea pigs A sample of the mammary secretion was obtained and (Kimmelstiel and Easley Jr 1940), sheep (Aynaud 1928), and cultured. Streptococcus equisimilis, coagulase negative S. horses (van Dorssen 1960). aureus and a Corynebacterium organism were identified.

The cultured bacteria were suspected to be due to Either long-term antibiotic treatment with a very contamination during sample collection. Fluid from several guarded prognosis, or surgical excision of the affected of the pockets was aspirated with a fine needle. A thin gland with a fair prognosis were suggested to the owners, smear of this fluid stained with Wright-Giemsa (Diff-Quick) who opted for surgery. A hemimastectomy of the affected showed granulocytes and erythrocytes, but no intracellular gland was performed under general anaesthesia in dorsal bacteria were identified. A bacterial culture of the recumbency. A purse string suture was placed over the left aspirated fluid revealed S. aureus. teat canal and a wide elliptical incision was made around A single tissue sample obtained by punch biopsy (6 mm the teat. The affected gland was dissected free by a Biopsy Punch Standard Miltex)3 was submitted for combination of blunt and sharp dissection. Large vessels histopathological examination and a second for bacterial were ligated with polygalactin 0 (Vicryl)4. All the affected culture. Histopathological examination showed a chronic, tissue and approximately 90% of the left mammary gland nonspecific, perivascular dermatitis with granulation tissue were removed. After placement of a latex penrose drain and the bacterial culture again revealed S. aureus, which (Kendall)5 the wound was closed in 4 layers: fibrous was only resistant to ampicillin. connective tissue in 2 layers with a simple interrupted suture pattern (polygalactin 0), the subcutis simple continuously with polygalactin 2-0,and the skin was stapled (Tohamed)6. The resected tissue was submitted for pathological examination. This revealed a severe, chronic, multifocal to coalescing suppurative mastitis with lesions containing Splendori-Hoeppli material (Fig 2) and intralesional bacteria (Fig 3). The findings were consistent with botryomycosis. Histopathology of the associated inguinal lymph nodes showed only reactive hyperplasia. No neoplastic changes were identified. Following surgery, the wound was cleaned on a daily basis and the mare received i.v. ceftiofur q. 12 h 5 mg/kg bwt (Excenel)7 for 4 days, followed by TMPS 30 mg/kg bwt q. 12 h per os (Sulfatrim)1 for 7 days. Meloxicam at 0.6 mg/kg bwt q. 24 h was administered for 8 days (Metacam)8 with tramadol 4 mg/kg bwt q. 12 h per os (Tramagetic)9 for 6 days. Due to severe post operative pain 10 Fig 1: Case A showed multiple firm nodules of different diameter at an epidural catheter (19 gauge Perifix Standard) was the left side of the mammary gland, including the teat, with and placed for the first day to provide an optimal analgesic without ulceration and purulent discharge. regime for the mare; this consisted of morphine sulphate

TABLE 1: Haematology and chemistry results of 2 mares (Cases A and B) with botryomycosis of the udder

Case A Case B Reference range

Haematology Packed cell volume (l/l) 0.32 0.38 0.32–0.42 Mean corpuscular volume (fl) 46.7 40.5 35.6–47.5 White blood cells (x109/l) 10.4 11.7 7.0–10.0 Segments (x109/l) 6.8 7.9 2.7–8.8 Bands (x109/l) 0 0 0.0–1.0 Lymphocytes (x109/l) 3.4 3.3 1.3–5.7 Monocytes (x109/l) 0.2 0.4 0.2–1.0 Eosinophils (x109/l) 0.1 0.1 0.0–0.6 Basophils (x109/l) 0 0.1 0.2–2.1 Chemistry Albumin (g/l) 34 39 26–37 Total protein (g/l) 66 75 52–79 a1-globulin (g/l) 1 1 1–7 a2-globulin (g/l) 9 10 3–13 b1-globulin (g/l) 6 7 4–16 b2-globulin (g/l) 7 6 3–9 g-globulin (g/l) 8 12 6–19 Urea (mmol/l) 3.8 - <8 Creatinin (mmol/l) 105 - 106–168 Calcium (mmol/l) 3.1 - 2.4–3.3 g-glutamyl transpeptidase (u/l) 12 - <20

Fig 2: Histology of Case A: pyogranulomatous inflammation with Fig 4: Case A about one week after surgery. centrally located accumulation of the causative agent showing the Splendori-Hoeppli phenomenon. continued to clean the wound at home with chlorhexidine 4% solution (Hibiscrub)14 twice a day for 10 days. In this case, because total surgical excision of the affected tissue was achieved, a full recovery was possible without the need for a long-term antibiotic treatment. At follow-up, 3 months post surgery, all clinical signs had resolved completely and the mare was performing well.

Case B

A 4-year-old Standardbred mare was presented to the Equine University Clinic in Utrecht with a 4 month history of a draining abscessation of the left mammary gland. Before referral she had been treated with an unknown oral preparation and anti-inflammatory drugs, which gave improvement of the condition, but the problem recurred after discontinuation of treatment. Fig 3: Histology of Case A: intralesional bacteria are found in the On admission she was bright, alert and responsive and centre of the pyogranulomatous inflammation (arrow). her clinical parameters were unremarkable. She was in good body condition and was sound at walk and trot. 0.2 mg/kg bwt q. 24 h (Morfine HCL)11, xylazine 0.1 mg/kg A painful, asymmetrical swelling of the left mammary bwt q. 8 h (Sedamun)12 and ketamine 0.25 mg/kg bwt q. 8 gland with multiple firm, nodular, ulcerative lesions of h (Narketan)13. The epidural catheter was removed 3 days different diameter were identified on the skin of the left side post surgery. The drain from the surgical wound was of the mammary gland including the teat. There was also a removed on Day 7 (Fig 4). serosanguinous-purulent discharge (Fig 5). On Day 12 both the wound and the right mammary A physical examination did not reveal any other gland became swollen and painful on palpation. Purulent abnormalities. General haematology and blood discharge from the gland was submitted for culture; biochemistry variables were within reference values multiple S. aureus spp. were identified and this also (Table 1). Mammary secretion was submitted for bacterial revealed sporadic meticillin-resistant S. aureus. The mare culture, revealing S. aureus. Fluid was aspirated from a was quarantined and treatment with marbofloxacin nodule with a fine needle after thorough cleaning. This fluid 6 mg/kg bwt q. 24 h i.m. (Marbocyl 2%)14 was instigated. also revealed S. aureus. Examination of a Gram-stained Additionally frequent hand milking, local hydrotherapy thin smear of the aspirate showed many leucocytes, and a and analgesics (meloxicam 0.6 mg/kg bwt q. 24 h, per os; moderate number of Gram-positive cocci. A Ziehl-Neelsen Metacam) were added to the treatment protocol. Over stain was negative. the next 10 days the wound healed and the mastitis Under a short general anaesthesia induced i.v. with resolved completely. The mare was discharged from the ketamine 2 mg/kg bwt (Narketan) and midazolam hospital 34 days after admission. The dedicated owner 0.06 mg/kg bwt (Midazolam) after premedication with

Fig 5: In Case B the left part of the mammary gland was enlarged with firm nodules (with and without ulceration) and purulent discharge.

Fig 7: Case B during surgery: the botryomycotic process was large and extended under the skin cranial of the mammary gland.

(Aureomycin Salbe)19. After 10 days there was significant improvement, but 3 weeks after termination of the therapy, the left mammary gland was again swollen and painful. At this point the owner opted for radical surgical excision. Under general anaesthesia in dorsal recumbency a left sided mastectomy was performed. Prior to surgery all fistulae were flushed with a 2% povidone iodine solution (Betadine)20 and the site was prepared for aseptic surgery. During surgery (Fig 7) it became clear that the affected tissue extended cranially over the ventral body wall. This affected tissue and the overlying skin was removed also. Complete closure could not be Fig 6: Histological view of a deep biopsy of Case B: chronic established. After surgery the horse was treated with oral pyogranulomatous inflammation without a visible causative agent. doxycyclin (5 mg/kg bwt q. 12 h per os) (Doxymix) and the wound was cleaned daily with 2% povidone iodine detomidine 0.02 mg/kg bwt (Domosedan)15 2 sterile and treated with chlortetracycline ointment (Aureomycin hollow-needle biopsies (18 gauge Quick-core Biopsy Salbe). After 10 days the horse was discharged with Needle)16 were taken, one for histological examination advice to continue the oral doxycyclin for a further 7 and one for bacterial culture. The histological days. Hydrotherapy twice a day and turn out to pasture examination (Fig 6) revealed extensive fibrosis with a as much as possible were suggested. The wound healed large area of pyogranulomatous inflammation containing well by secondary intention and no post operative macrophages with lipofuscine pigment in the subcutis, complications occurred. Follow-up on this case with the with clusters of neutrophils indicating a chronic trainer 3 months later revealed that the mare was back in pyogranulomatous inflammation. Bacterial culture of the training and performed up to expectations. second biopsy revealed occasional S. aureus. Although the Splendori-Hoeppli phenomenon and intracellular Discussion bacteria were not visible, the macroscopic and microscopic findings combined with the bacterial culture Botryomycosis is a rare and incompletely understood resulted in the tentative diagnosis of botryomycosis. disease both in man and animals. The historically most The owner opted for a second opinion where common form of the condition in equids, namely after the diagnosis was confirmed. At the owner’s request, castration (the ‘champignon condition’), occurs very medical management was instigated with doxycyclin rarely in The Netherlands at present. This is probably due 5 mg/kg bwt q. 12 h per os (Doxymix 15%)17 for to improved castration techniques. Open castration of 14 days and phenylbutazon 2 mg/kg bwt q. 12 h per os standing horses is less used and most horses receive (Phenylbutariem)18 for 3 days. The udder was treated antibiotics pre- and post surgery. Van Dorssen (1960) locally with chlortetracycline ointment topically q. 12 h showed that a simple course of penicillin might prevent

the development of botryomycosis, even if a S. aureus mammary gland tissue in Case B was not submitted for challenge is brought directly into the wound. This finding is histological examination. supported by the more recent research of Akiyama et al. Mastectomy is not a common procedure in equine (1996) who showed that, after experimental S. aureus practice. It is clearly an invasive procedure often infection of skin wounds in mice, the bacterial cells seem accompanied by a period of severe pain after surgery. to form a biofilm in a neutropenic surrounding as exists After surgery, Case A showed a severe pain response that shortly after inoculation. The formation of a biofilm by required strong analgesic measures to control it. Only few S. aureus seems to serve as a pseudocapsule, thereby studies describe the surgical techniques of mammary protecting the microorganism from the immune-system of resection and their outcome (Schiemann and Bartmann the host, leading to chronicity of the infection (Cheng 2004). It is, however, of utmost importance to remove all et al. 2009). It might well be that the formation of the diseased tissue if recurrence is to be avoided. biofilm triggers the formation of the Splennore-Hoeppli Both cases healed well by second intention and reaction as this phenomenon is thought to be a localised radical surgical excision gave a good result. The fact that immunological response to an antigen-antibody in Case B the surgical excision was performed after a precipitate related to fungi, parasites, bacteria or inert long course of antibiotics may explain why the duration materials. of the healing process seemed to be shortened In cutaneous cases, small wounds may be the entry compared to Case A. portal for commensal S. aureus. Although a decreased immune status is also considered a predisposing factor for Authors’ declaration of interests the development of human botriomycotic lesions (Brunken et al. 1983), little information is available in veterinary No conflicts of interest have been declared. species. Makama et al. (2006) mentioned that in human medicine, botryomycosis is associated with defects of cellular immunity, and in particular low lymphocyte counts. Acknowledgements Both equine cases in the present study had normal lymphocyte counts. In neither case in this study was there The authors would like to thank the colleagues Dr E. de any evidence of immunocompromise. Graaf-Roelfsema DVM, PhD, Spec. KNMvD Equine Internal The study of Kimmelstiel and Easley (1940) showed that Medicine, Dr R. Fleis-Keesler DVM, Dipl. ACVP and Dr D.J. a foreign body may also support the development of Riemersma, DVM PhD, Dipl ECVS for sharing their case data botryomycosis. No foreign body was found in the present and the student Femke Jacobs for her contribution to case cases. Little is known about the factors that might description. predispose certain bacteria to produce lesions of the botryomycotic type. Manufacturers’ addresses Effective treatment of botryomycosis depends on various factors including the nature of the infecting 1Dopharma, Raamsdonkveer, The Netherlands. organism, the site of the lesion and the host’s immune 2Leo Animal Health, Uldum, Denmark. status. Additionally, even if bacteria isolated from the 3Medipharchem Nederland B.V., Wormerveer, The Netherlands. 4 lesions are sensitive to the antibiotics used, the presence Ethicon, Bleu Ash, Ohio, USA. 5Covidien, Dublin, Ireland. of the Splendore-Hoeppli material as well as other tissue 6Skin Stapler, Goettingen, Germany. characteristics could prevent penetration of the drug. 7Pharmacia and Upjohn BV, Woerden, The Netherlands. Antimicrobial agents that have been used to successfully 8Boehringer Ingelheim, Alkmaar, The Netherlands. treat human botryomycosis include trimethoprim- 9Nycomed B.V., Hoofddorp, The Netherlands. sulphamethoxazole, minocycline, erythromycin and 10B. Braun AG, Melsungen, Germany. 11 cefazolin (Mehregan et al. 1991). In cases where Pharmachemie, Haarlem, The Netherlands. 12Eurovet Animal Health B.V., Bladel, The Netherlands. antibiotics are not effective, surgical excision is 13Vétoquinol B.V., ‘s Hertogenbosch, The Netherlands. recommended. In equine dermatology, it is known and 14Regent Medical Ltd, Manchester, UK. accepted that the best treatment for cutaneous 15Orion Corporation, Espoo, Finland. botryomycosis is surgical excision (Knottenbelt 2003) as 16Wilson-Cook Medical Inc., Winston-Salem, North Carolina, USA. even very long courses of antibiotics may not solve the 17Eurovet Animal Health B.V., Bladel, the Netherlands. 18 problem (Knottenbelt 2009). Riemser Arztneimittel AG, Germany. 19Alfarma, Luxembourg. In Case A the diagnosis was based on the history, 20Purdue Pharma LP, Stamford, Connecticut, USA. macroscopic appearance, histology and a positive culture. In Case B the history, macroscopic appearance and positive culture were the same, and the histological References examination revealed pyogranulomatous inflammation Akiyama, H., Kanzaki, H., Tada, J. and Arata, J. (1996) Staphylococcus but the Splendore-Hoeppli phenomenon was not aureus infection on cut wounds in the mouse skin: experimental identified. Unfortunately, the completely removed staphylococcal botryomycosis. J. Dermatol. Sci. 11, 234-238.

Aynaud, M. (1928) La botryomycose du mouton. Ann. Inst. Pasteur 42, Leffell, D.J., Brown, M.D. and Swanson, N.A. (1989) Laser vaporization: A 256-281. novel treatment of botryomycosis. J. Dermatol. Surg. Oncol. 15, Bader, H. and Busch, W. (2006) Erkrankungen der Milchdrüse. In: 703-705. Handbuch Pferdepraxis, 3rd edn., Eds: O. Dietz and B. Huskamp, Lous, F., Mariotti, F., Magi, G.E., Spaziante, D., Fugazzola, M.C. and Tesei, Enke Verlag, Stuttgart. pp 618-619. B. (2009) Mammary carcinoma in a mare: Clinical, histopathological Pferdeheilkunde 25 Biemans, A.A.M., Waalboer-Spruij, R., Koljenovic, S. and Thio, H.B. (2010) and steroid hormone receptor status. , 18-21. Botryomycosis, responding well to antibiotics. Nederl. Tijdschr. Machado, C.R., Schubach, A.O., Conceiçào-Silva, F., Quintella, L.P., Dermatologie En Venereologie 20, 581-583. Lourenço, M.C.S., Carregal, E. and Francesconi do Valle, A.C. (2005) Botryomycosis. Dermatology 211, 303-304. Böhm, K.H., Klug, E. and Jacobs, B.J. (2009) Mastitis in the mare – A long term study on the incidence, clinical symptoms, diagnostics, Madlon-Kay, D.J. (1986) ‘Witch’s Milk’. Galactorrhea in the newborn. microbiology, therapy and economic importance, as well as Am. J. Dis. Child. 140, 252-253. recommendations for veterinary practice. Prakt. Tierarzt. 90, Magrou, J. (1919) Les formes actinomycotiques du staphylococque. 842-849. Ann. Inst Pasteur Lille 33, 344-374. Bollinger, O. (1870) Mycosis der Lunge beim Pferde. Virchows Arch. 49, Makama, J.Z., Khan, N., Makhanya, N.Z. and Motswaledi, H. (2006) 583-586. Botryomycosis. S. Afr. J. Radiol. 10, 14-15. Bonifaz, A. and Carrasco, E. (1996) Botryomycosis. Int. J. Dermatol. 35, McCue, P.M. and Wilson, W.D. (1989) Equine mastitis–areview of 28 381-388. cases. Equine Vet. J. 21, 351-353. Bostedt, H., Lehmann, B. and Peip, D. (1988) The problems of mastitis in McGladdery, A.J. (1998) Differential diagnosis and treatment of mares. Tierarztl. Prax. 16, 367-371. diseases of the equine mammary gland. Equine Vet. Educ. 10, Bostrom, R.E., Huckins, J.G., Kroe, D.J., Lawson, N.S., Martin, J.E., Ferrell, 266-268. J.F. and Whitney, R.A., Jr. (1969) Atypical fatal pulmonary Mehregan, D.A., Daniel Su, W. and Anhalt, J.P. (1991) Cutaneous botryomycosis in two guinea pigs due to Pseudomonas aeruginosa. botryomycosis. J. Am. Acad. Dermatol. 24, 393-396. J. Am. Vet. Med. Ass. 155, 1195-1199. Mendes, L., de Araujo, M., Bovino, F., Rozza, D., Machado, G., Cadioli, F., Brendemuehl, J.P. (2008) Mammary gland enlargement in the mare. Feitosa, F. and Peiró, J. (2011) Clinical, histological and Equine Vet. Educ. 20, 8-9. immunophenotypic findings in a mare with a mammary lymphoma Brito, M.F., Seppa, G.S., Teixeira, L.G., Rocha, T.G., França, T.N., Hess, T.M. associated with anaemia and pruritus. Equine Vet. Educ. 23, and Peixoto, P.V. (2008) Mammary adenocarcinoma in a mare. 177-183. Ciência Rural. 38, 556-560. Miller, M.A., Fales, W.H., Tyler, J.W. and Suedmeyer, W.K. (2001) Brunken, R.C., Lichon-Chao, N. and van den Broek, H. (1983) Pulmonary botryomycosis in a Scottish highland steer. J. Vet. Diagn. Immunologic abnormalities in botryomycosis: A case report with Invest. 13, 74-76. review of the literature. J. Am. Acad. Dermatol. 9, 428-434. Munson, L. (1987) Carcinoma of the mammary gland in a mare. J. Am. Casamián-Sorrosal, D., Fournier, D., Shippam, J., Woodward, B. and Vet. Med. Ass. 191, 71-72. Tennant, K. (2008) Septic pericardial effusion associated with Prendergast, M., Bassett, H. and Larkin, H. (1999) Mammary carcinoma pulmonary and pericardial botryomycosis in a dog. J. Small Anim. in three mares. Vet. Rec. 144, 731-732. Pract. 49, 655-659. Reesink, H., Parente, E., Sertich, P. and Engiles, J. (2009) Malignant Cheng, A.G., Kim, H.K., Burts, M.L., Krausz, T., Schneewind, O. and fibrous histiocytoma of the mammary gland in a mare. Equine Vet. Missiakas, D.M. (2009) Genetic requirements for Staphylococcus Educ. 21, 467-472. aureus abscess formation and persistence in host tissues. FASEB J. 23, Reppas, G., McClintock, S., Canfield, P. and Watson, G. (1996) Papillary 3393-3404. ductal adenocarcinoma in the mammary glands of two horses. Vet. Coulibaly, B., Géraldine, H., Le Hémon, A., Gouriet, F. and Marie-Anne, Rec. 138, 518-519. C. (2008) Staphylococcus aureus isolated from a case of lymph Saadat, P., Ram, R., Sohrabian, S. and Vadmal, M. (2007) Botryomycosis node botryomycosis. Arch. Pediatr. 15, 1440-1442. caused by Staphylococcus aureus and Pneumocystis carinii in a Ellenberger, C., Schoon, D. and Schoon, H.A. (2006) Exceptional patient with acquired immunodeficiency disease. Clin. Exp. diagnostic findings in uterine biopsies of the mare. Pferdeheilkunde Dermatol. 33, 266-269. 22, 171-176. Schiemann, V. and Bartmann, C.P. (2004) Mastectomia in the horse - Eshar, D., Mayer, J. and Keating, J.H. (2010) Dermatitis in a Siberian indication and surgical procedure. Pferdeheilkunde 20, 506-510. hamster (Phodopus sungorus). Lab. Anim. 39, 71. Schlossberg, D., Pandey, M. and Reddy, R. (1998) The Findlay, G. and Vismer, H. (1990) Botryomycosis. Int. J. Dermatol. 29, Splendore-Hoeppli phenomenon in hepatic botryomycosis. J. Clin. 340-344. Pathol. 51, 399-400. Foreman, J.H., Weidner, J.P., Parry, B.W. and Hargis, A. (1990) Pleural Scott, D.W. (2007) Cutaneous bacterial pseudomycetoma effusion secondary to thoracic metastatic mammary (botryomycosis) in dogs: Two new case reports and a review of the adenocarcinoma in a mare. J. Am. Vet. Med. Ass. 197, 1193-1195. literature. Jpn. J. Vet. Dermatol. 13, 135-140. Hirayama, K., Honda, Y., Sako, T., Okamoto, M., Tsunoda, N., Tagami, M. Scott, D.W. and Miller, W.H. (2003) Bacterial pseudomycetoma. In: and Taniyama, H. (2003) Invasive ductal carcinoma of the Equine Dermatology, Eds: D.W. Scott and W.H. Miller, Saunders mammary gland in a mare. Vet. Pathol. 40, 86-91. Elsevier Science, St. Louis. pp 245-249. Hussein, M.R. (2008) Mucocutaneous Splendore-Hoeppli phenomenon. Shank, A. (2009) Mare mammary neoplasia: Difficulties in diagnosis and J. Cutan. Pathol. 35, 979-988. treatment. Equine Vet. Educ. 21, 475-477. Kimmelstiel, P. and Easley, C. Jr (1940) Experimental botryomycosis. Am. Sharma, S.P. and Iyer, P.K.R. (1974) Pathology of chronic lesions in J. Pathol. 16, 95-101. mammary glands of goats (Capra hircus): Nocardiosis and Knottenbelt, D.C. (2003) The mammary gland. In: Equine Stud Farm botryomycosis. Indian J. Anim. Sci. 44, 41-45. Medicine and Surgery, Eds: D.C. Knottenbelt, M. Le Blanc, C. Lopate Sheikh-Omar, A.R. and Abdullah, A.S. (1985) Perforated gastric ulcer and R.R. Pascoe, Saunders Elsevier, Edinburgh. pp 343-352. associated with disseminated staphylococcal granuloma Knottenbelt, D.C. (2009) Bacterial granuloma (botryomycosis / (botryomycosis) in a cat. Vet. Rec. 117, 131. staphylococcal pseudomycetoma / deep pyoderma). In: Pascoe’s Sleeman, J., Clyde, V., Finnegan, M., Ramsay, E. and Shires, M. (2003) Principles and Practice of Equine Dermatology, 2nd edn., Ed: D.C. Mammary botryomycosis and mastectomy in an African elephant Knottenbelt, Saunders Elsevier, Edinburgh. pp 148-150. (Loxodonta africana). Vet. Rec. 152, 54-55. Continued on page 376 © 2012 EVJ Ltd