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Molecular Detection and Therapeutic Management of Malasseziosis in Cattle

P. Ameldev P.V. Tresamol Aarsha Raj Mery Rincy
Vol 8(4), 154-160
DOI- http://dx.doi.org/10.5455/ijlr.20170718043448

Yeast of genus Malassezia inhabit the skin of variety of mammals and birds and are considered as opportunistic pathogens in animals and man. Malassezia dermatitis among three cows from an eco-farm in Thrissur Kerala state, with skin lesions on the dorsal aspect neck and their successful treatment is described. Upon physical examination hyperkeratosis along with circular crusty lesions were noticed. Examination of skin scraping with 10% potassium hydroxide was found to be negative for fungal spores/mites. Hair along with the skin scrapings were cultured in sabouraud’s dextrose agar with chloramphenicol at 37ºC for four weeks and resulted in greyish white, raised convex type colony after 12 hours of incubation. Grams staining of the isolates revealed presence of dark blue coloured budding yeast cells suggestive of Malassezia. No other fungal organisms were isolated during a period of four weeks. DNA was extracted from the isolates by using DNeasy blood and tissue kit (Qiagen) and subjected to a genus specific PCR using primers 5’ AGCGGAGGAAAAGAAACT3’ and 5’GCGCGAAGGTGTCCGAAG. Gel electrophoresis of the PCR products revealed 600bp product suggestive of Malassezia genus. The restriction -endonuclease analysis of PCR products confirmed the species as Malassezia restricta. The case was successfully treated.


Keywords : Cattle Dermatitis Malasseziosis Molecular Diagnosis

Introduction

The yeast of genus Malassezia are commonly isolated from the skin of warm blooded vertebrates. They are normal commensals of cutaneous skin micro-flora but can act as opportunistic pathogen whenever alterations in skin microclimate occur. Genus Malassezia is composed by thirteen species of yeasts M. pachydermatis, M. nana, M. equina and M. caprae could be considered zoophilic (Guého et al., 1996). The lipid dependent species Malassezia globosa, M. sympodialis, M. furfur and M. slooffiae have been associated with bovine parasitic otitis. Malassezia related diseases include pityriasis versicolor, seborrheic dermatitis, folliculitis and systemic infections in humans and otitis externa and seborrheic dermatitis in animals. They are now considered synonymous with those previously named Pityrosporum, which are universally present on human skin and have also been found in numerous other warm blooded animals. The characteristic morphological features of Malassezia yeasts include a thick, multi-layered cell wall and the production of blastoconidia by a process of repetitive monopolar or sympodial budding so leaving a prominent bud scar on the mother cells. They have a distinctive physiological property in that lipids can be utilized as a source of carbon. Malassezial dermatitis is suspected upon clinical evidence and diagnostic methods used to identify overgrowth of Malassezia organisms. The response to treatment with specific antifungal therapy is considered the best tool for a definitive diagnosis. Cytological examination allows to rapidly observe yeasts.

Case History and Observations

Three cows from an eco-farm in Thrissur district of Kerala are reported with recurrent skin lesions and itching since six months. Upon physical examination patchy alopecia with hyperkeratosis and crusty lesions could be noticed in the dorsal aspect of neck and extending towards the thorax (Fig. 1 a, b). Due to severe scratching towards hard objects erythema could be noticed in the periphery of the lesions. Feeding, voiding habits and other vitals are found to be in normal range.

Fig. 1a: Lesion in the lateral neck Fig.1b: Lesion in the dorsal thorax

Materials and Methods

The skin scrapings and impression smears were collected aseptically for the laboratory examinations. Both superficial and deep scrapings were collected with a sterile blade on a glass slide. Hairs along with scabs were also collected in a sterile vial for cultural examination. The skin scrapings were treated with 10% potassium hydroxide (KOH) solution and exam under direct microscopy for detecting the presence of mites or fungus. The skin scrapings along with the scabs were cultured in BHI agar @ 37ºC for 4 weeks. The cultured organism was streaked in urease agar for the detection of the presence of Malassezia species. The culture was inoculated in a urea tube and incubated for 24 hours at 37ºC.

The DNA was extracted from the isolates using commercial DNA extraction kit (Qiagen blood and tissue kit). As per the prescribed protocol, and quality (absorbance ratio 260/280) was measured spectroscopically (Nanodrop, Thermo Scientific, Fisher, USA) for each extracted sample. Extracted DNA samples were eluted in nuclease-free water and stored at−20 ◦C until use. The DNA was subjected to Polymerase chain reaction for the confirmation of the Malassezia organism. The primers (Table 1) and protocol used for PCR are as follows (Guillot et al., 2000).

Table 1: Primers

No. Primers Sequences
1 Mal 1 – Forward 5’AGCGGAAAAGAAACT 3’
2 Mal 2 – Reverse 5’GCGCGAAGGTGTCCGAAG 3’

Primers were selected from the sequences of LSU of rRNA gene of Malassezia species which is highly conserved in all Malassezia yeast and they were considered to be a genus specific PCR. The conventional PCR was performed in a 50µl reaction mix. The protocol for the PCR is described in Table 2.

Table 2: PCR protocol

No. Amplification Process Temperature Time
1. Initial denaturation 94℃ 2 min
2. Denaturation 94ºC 30sec
3. Annealing 55ºC       1 min30 cycles
4. Elongation 72ºC 1 min
5. Final extension 72ºC 10 min

The electrophoresis was carried out in 1.2% agarose gel (Agarose special low EEE, made HIMEDIA®) and documented.

Results

Direct microscopic examinations of skin scrapings were found to be negative for the fungal elements and mites. A greyish white raised convex colony could be detected after 24hours incubation at 37ºC (Fig. 3). Grams stained culture smear revealed the presence of Gram positive budding yeast cells which is suggestive of Malassezia spp. (Fig. 4). Pink colour was developed in all three samples after 24hour incubation, thus revealed the positive urease test (Fig. 4). In Polymerase chain reaction all three test samples yielded amplicons of 600bp and no product were amplified in negative control (Fig. 5).

 

 

 

 

 

 

 

 

 

 

Fig. 2: Culture growth

 

 

 

 

 

 

 

Fig. 3: Stained yeast cells

 

 

 

 

 

 

 

Fig.4: Urease test

 

 

 

 

 

 

 

 

 

 

 

 

1.      100bp ladder

2.      Sample I

3.      Sample II

4.      Sample III

5.      Negative control

6.      Nil

7.      Nil

8.      Nil

 

600bp

 

 

 

 

 

 

 

 

 

 

Fig. 5: PCR amplified product 600bp

Therapeutic Management

The animals were treated with external application of 2% miconozole (Micogel) for a period of 30 days. The animal was supported with herbal immunity boosters and multivitamin supplements. Clinical recovery was noticed after 30 days of antifungal therapy (Fig. 6a and 6b).

 

 

 

 

 

 

 

Fig. 6a: After treatment                               Fig. 6b: After treatment

Discussion

Malassezia dermatitis is an opportunistic yeast infection affecting the skin of all warm blooded animals. The genus malassezia was revised on the basis of morphology, physiology and rRNA gene sequencing studies and has classified as lipid depended and lipid non-depended. Malassezia sympodialis, Malassezia fufur, and Malassezia globosa are the species which are capable of causing infection in external ear of cattle with otitis (Duarte et al., 2001).  Very few cases has been reported regarding malassezia dermatitis in cattle. The present study reports the molecular confirmation of malassezia dermatitis in cattle and its therapeutic management. Machado et al. (2010) reported that primary lesion in Malassezia was commonly associated with pruritus and secondary changes were erythema, alopecia, excoriation and hyperpigmentation, similar symptoms were noticed in all three affected animals in the present study. In this study, the skin scrapings were cultured in saboraud dextrose – chloramphenicol agar, olive oil were added for the better growth of lipophilic Malassezia. The creamy white raised colony were microscopically visible after 24 hour incubation and dark blue coloured footprint shaped organisms was seen on microscopic examination, this was in accordance with the finding of  David et al. (2003). All Malassezia species uses urea for their metabolism and it breaks down urea to ammonia and Carbon dioxide.  Positive urease test obtained in the present study is an indication of presence of Malassezia. Further we have confirmed the diagnosis using polymerase chain reaction. Successful amplification of 600bp products confirmed the presence of Malassezia genus as reported by Guillot et al. (2010). Miconozole and ketoconazole is the drug of choice for the treatment of Malassezia dermatitis (Rosenkrantz, 2006 and Scott et al., 2011). In the present study animals were treated with external application of miconazole in gel base for better penetration.

Conclusion

Very few studies reported incidence of Malassezia related otitis in cow and nowhere is it reported that it can be act as primary pathogen for dermatitis in cow. The present study investigates the role of Malassezia spp. causing dermatitis in cow. The Microscopical, molecular and biochemical confirmation of Malassezia spp causing dermatitis in cattle give lights to veterinarians to include Malasseziosis in the differential diagnosis of Dermatitis in large animals.

References

  1. David M, Gabriel M, and Kopecka, M, 2003. Unusual ultrastructural characteristics of the yeast Malassezia pachydermatis.Scripta Medica (BRNO)76: 173-176.
  2. Duarte ER, Batista RD, Hahn RC and Hamdan JS, 2003. Factors associated with the prevalence of Malassezia species in the external ears of cattle from the state of Minas Gerais, Brazil.Medical mycology, 41(2): 137-142.
  3. Gueho E, Midgley, G, and Guillot J, 1996. The genus Malassezia with description of four new species. Antonie Leeuwenhoek, 69: 337-355.
  4. Guillot J, Deville M, Berthelemy M, Provost F and Guého E 2000. A single PCR‐restriction endonuclease analysis for rapid identification of Malassezia species.Letters in applied microbiology, 31(5).
  5. Machado ML, Ferreiro L, Ferreira, RR, Corbellini LG., Deville M, Berthelemy, M. and Guillot, J. 2011. Malassezia dermatitis in dogs in Brazil: diagnosis, evaluation of clinical signs and molecular identification.Veterinary dermatology, 22:.46-52.
  6. Midreuil, F, Guillot, J, Guého E, Renaud F, Mallié M. and Bastide JM, 1999. Genetic diversity in the yeast species Malassezia pachydermatis analysed by multilocus enzyme electrophoresis.International Journal of Systematic and Evolutionary Microbiology, 49(3): 1287-1294.
  7. Rosenkrantz W, 2006. Practical applications of topical therapy for allergic, infectious, and seborrheic disorders.Clinical techniques in small animal practice, 21: 106-116.
  8. Srivastava AK, 2008. Modern concepts of Malasseziosis in dogs.Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases, 29:1-8.
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