Collection of Hair, Crusts, and Skin in the horses

In: A Manual of Equine Diagnostic Procedures, Schumacher J. and Moll H.D. (Eds.). Publisher:
Teton NewMedia, Jackson, WY, USA (www.tetonnm.com/). Internet Publisher: International
Veterinary Information Service, Ithaca NY (www.ivis.org), Last updated: 16-Jul-2010; A5406.0710

Collection of Hair, Crusts, and Skin
J. Schumacher1 and H.D. Moll2
1College of Veterinary Medicine Auburn University Auburn, AL, USA and 2Center for Veterinary Health Sciences,
Oklahoma State University, Stillwater, OK, USA.

Skin Scrapings
Skin scrapings in the horse are primarily of value for the diagnosis of microscopic ectoparasitism. Surface and burrowing
mites, and some nematode larva can be identified during examination of skin scrapings. Because ectoparasitism in the
horse is uncommon, and often identified by alternative methods, skin scrapings are often omitted from the routine
dermatological examination.
Indications
For investigation of pruritic skin disease typical of a parasitic infestation. Pruritic skin disease of the limb should be
investigated with skin scrapings, because chorioptic mange is occasionally a cause of skin disease involving the distal
portion of the limbs of horses.
•
Many equine dermatologists co • nsider skin scrapings to be part of a routine dermatological examination.
Materials
• Hair clippers (optional)
#10 or 22 scalpel blade to decrease the chance of lacerating the skin, the blade should be dulled before use.
Alternatively, a bone curette can be used for scraping (Fig. 4.1).
•
• Glass microscope slides, coverslips and a container for transport of the slides
Mineral oil, to immobilize mange mites making them easier to identify, an insecticide can be added to the mineral oil
to further immobilize mites.
•
Figure 4.1. A bone curette can be used to collect a skin scraping. - To view this image in full size go to
the IVIS website at www.ivis.org . -
Procedure
• A site at the periphery of an unexcoriated lesion is selected.
• Hair that may interfere with the scraping can be removed with a clipper.
• Mineral oil is applied to the area of skin selected for scraping.
The scalpel blade is held perpendicular to the surface of the skin and a large area is superficially scraped. Then a
smaller area of skin within the scraped area is scraped until capillary bleeding is observed. Squeezing the skin prior
to, and during deep scraping may improve collection of parasites.
•
The tissue collected is transferred to a glass slide and a cover slip is applied, or alternatively, transferred to a container
for transport.
•
Samples should be examined as soon as possible to avoid distortion or escape of collected parasites. At least five
samples should be examined before a negative diagnosis can be made with any confidence.
•
To increase diagnostic effectiveness, material from a scraping can be added to a saturated salt solution for flotation or
centrifugation. Mites and ova, which rise to the top of the solution, can be found by microscopic examination of
several drops of the surface solution.
•
Collection of Hair and Crusts for Dermatophyte Culture
Diagnosis of dermatophytosis is best confirmed by fungal culture of hair, scales or crusts. Direct microscopic examination
of hair, scales or crusts from affected and adjacent skin may reveal microorganisms. Because dematophytes that commonly
affect horses do not fluoresce, a Wood’s lamp examination is not a reliable method of diagnosis.
Indications
Any focal or generalized expanding area of alopecia should be examined for dermatophytes especially if the lesions involve
the head, neck, saddle or girth areas, or limbs of horses, especially young horses. Dermatophyte lesions often contain scales
or crusts and may or may not be pruritic.
Materials
• Clean hemostats
• An envelope for collection of hair and crusts
A clearing agent such as 10 to 20% solution of potassium hydroxide (KOH) (Remel, Inc. 12076 Sante Fe Drive,
Lenexa, KS 66215). Clearing agents digest hair and debris so that fungal elements can be seen more clearly. For
identifying equine dermatophytes, using a clearing agent is not essential.
•
• A light microscope and microscope slides and coverslips
• 70% Isopropyl alcohol or a non-antiseptic soap
Dermatophyte testing medium (DTM) (Sab-Duets plates, Bacti-Labs, Mountain View, CA; Derma Tube, Remel, Inc.
12076 Sante Fe Drive, Lenexa, KS 66215; or Dermatophyte Test Medium, Blue Ridge Biologicals, Inc. Box 634
Hickory, NC 28603) (Fig. 4.2)
•
Figure 4.2. Dermatophyte testing medium. - To view this image in full size go to the IVIS website at
www.ivis.org . -
Examination for Dermatophytes
Procedure
The lesion can be wiped with alcohol or washed with a non-antiseptic soap to decrease the growth of contaminants
before hairs are collected for culture. The lesion should be dry when the specimen is collected (to decrease growth of
contaminants).
•
Using a clean hemostat, broken hairs from the periphery of the lesion are plucked in the direction of growth. Several
lesions should be sampled to increase the likelihood of isolating a dermatophyte.
•
For direct microscopic examination, hair and crusts are suspended in a clearing solution. If a clearing solution is
unavailable, samples can be viewed in mineral oil or water. Hair and crusts are examined for hyphae and
arthroconidia, using the 10 and 40x objective of the microscope. Clinicians inexperienced at direct examination of
hair for identification of dermatophytes should submit specimens to a laboratory with experienced personnel.
•
For inoculation of DTM, clumps of hair should be teased apart and a few affected hairs are gently pressed onto the
medium. Avoid penetrating the surface of the medium with the samples. Inoculation of the dermatophyte-testing
medium with an excessive amount of hair may result in over-growth of contaminants.
•
When using DTM in a vial, the cap of the vial should not be tightened because fungi require oxygen for growth.
Innoculated samples should be incubated at room temperature (for growth of Trichophyton equinum, Microsporum
canis, or Microsporum gypseum) and at 37°C (for growth of Trichophyton verrucosum).
•
Identification of dermatophyte colonies is aided by microscopic examination (10X). A slide can be prepared by
touching the colony with transparent cellophane tape and then applying the tape to a microscope slide to which a drop
of water or lactophenol cotton blue has been added.
•
Interpretation
Even experienced technicians often fail to identify dermatophytes during direct examination of infected hair. Hairs
infected with dermatophytes are often fragmented, pale or swollen. Fungal spores are very small, round to oval, and
often grouped as chains along the hair shaft (Fig. 4.3).
•
Dermatophyte colonies grow in approximately three to 14 days. Prior treatment of the horse with antifungal agents
may delay growth of dermatophytes. Cultures from treated horses should be incubated for 21 days.
•
Dermatophytes grow as white-, beige-, or cream-colored colonies. Contaminant colonies are often black, green, gray
or brown.
•
Growth of dermatophytes causes a rise in pH, which causes the medium to change from yellow to red around the area
of colony growth. The color changes simultaneously with colony growth (Fig. 4.4). Contaminants may also cause a
•
red-color change in the medium, but the change is usually delayed and does not occur simultaneously with colony
growth (Fig. 4.5). To correctly interpret test results, the samples must be examined daily.
Identification of dermatophytes requires microscopic examination of the colonies grown on DTM. Most Microsporum
species can be identified by recognizing characteristic macroconidia spores. Speciation of Trychophyton species
requires additional testing that involves determining specific growth requirements. Most microbiology texts include
information concerning dermatophyte identification.
•
Figure 4.3. Dermatophytes are rarely identified, even by experienced technicians during direct
examination of infected hair. - To view this image in full size go to the IVIS website at
www.ivis.org . -
Figure 4.4. Dermatophytes cause the medium to change from yellow to red around the area of colony
growth. The color change is simultaneous with colony growth. - To view this image in full size go to
the IVIS website at www.ivis.org . -
Figure 4.5. Contaminants may also cause a red-color change in the medium, but the change is usually
delayed and not simultaneous with colony growth. - To view this image in full size go to the IVIS
website at www.ivis.org . -
Skin Biopsy
Skin biopsies are most helpful in the diagnosis of nodular skin disease. Some diffuse skin diseases cannot be diagnosed
without histological examination of affected skin. A skin biopsy taken early from an untraumatized site is much more
valuable than a biopsy from a chronic lesion. Multiple skin biopsies increase the chance for an accurate diagnosis.
Indications
• Suspected neoplastic lesions
• Non-healing ulcers
• Any dermatosis not responding as expected to treatment
• Any dermatosis with an unusual appearance
• Definitive diagnosis of skin disease for which treatment will be expensive, dangerous, or time consuming
Contraindications
None, although the location of the lesion may influence the type of biopsy taken. For example, an excisional or
incisional biopsy (see Fig. 1.1) of a lesion on the back of a riding horse may be contraindicated, unless results of a
less invasive biopsy technique (i.e., a needle or aspiration biopsy) indicate that an incisional biopsy is necessary for
diagnosis or that excision is necessary for resolution of the lesion.
•
• Incisional biopsy of a sarcoid may initiate aggressive growth of the tumor.
• Sites over vessels, joints, and bony prominences are best avoided.
Materials
• Local anesthetic solution, syringe, and a 20- to 25-ga (0.9- to 0.5-mm) needle
• A 4- to 10-mm skin biopsy punch (i.e., AcuPunch Biopsy Punch, Acuderm Inc., Ft. Lauderdale, FL 33309). Or
• A scalpel blade, thumb forceps, needle holders, suture material (or staples), and a tongue depressor or cardboard
• Fixative (usually 10% formalin)
Procedure
Skin should be minimally prepared so that superficial lesions are not removed during preparation. Hair can be
clipped, and the site rinsed with alcohol, but skin should not be shaved or scrubbed. Use of iodine prep may interfere
with staining.
•
Regional anesthesia with a local anesthetic agent is ideal, when possible (e.g., limbs or the perineal region), or local
anesthetic agent can be injected subcutaneously directly under the site of biopsy or injected around the lesion as a ring
block.
•
A circular piece of skin and subcutaneous tissue can be removed by continuous circular motion (in one direction only)
of a skin biopsy punch (Fig. 4.6). Most pathologists prefer samples >6mm. For deep lesions, the biopsy punch can be
reinserted into a site to remove more tissue. Or
•
Using a scalpel blade, an elliptical piece of skin and subcutaneous tissue that contains both grossly normal and
abnormal skin can be removed. Biopsies of ulcerative lesions should always include an edge of normal epithelium.
Vesicular and bullous lesions should be biopsied in their entirety.
•
After the skin is cut with a punch or scalpel blade, the subcutaneous tissue and the skin is grasped and lifted delicately
with a forceps (alternatively, the biopsy specimen is held and lifted with a small gauge needle pushed through an edge
of the specimen) and cut free with a scalpel blade. Some pathologists emphasize that, to avoid artifactual
histopathogical changes, the biopsy specimen not be freed with a scissors. Excess blood should be removed from the
sample by blotting with a gauze sponge prior to fixing.
•
Touch preps for cytological evaluation can be made at this time by gently rolling and pressing the sample against a
microscope slide.
•
Elliptical biopsies should be mounted so they don’t curl during fixation. The subcutaneous side of the specimen is
placed on a tongue depressor or piece of cardboard and gently pressed so that it adheres to the surface.
•
10% formalin is the usual fixative for skin. Bouin’s solution is also suitable, but only for small or thin biopsies
because it poorly penetrates tissue. Skin should not be stored in Bouin’s solution for over 24 hours. Michel’s fixative
is sometimes used to fix skin for immunoflorescence studies when immune-mediated skin disease is suspected, but
formalin-fixed skin may also be suitable for diagnosis of immune-mediated skin disease. When in doubt concerning
fixation of tissue, the pathologist should be consulted before biopsies are taken.
•
Elliptical biopsy sites are closed with sutures or staples. Punch biopsy sites are often closed with a single suture or
staple or are left to heal as an open wound.
•
Figure 4.6. A circular piece of diseased skin surrounding normal skin and subcutaneous tissue can be
removed by continuous circular motion of an AcuPunch skin biopsy punch. - To view this image in
full size go to the IVIS website at www.ivis.org . -
Interpretation
A pathologist with knowledge of equine skin disease should interpret samples submitted for histological examination. To
aid in interpretation, a history, description of physical findings, and suspected diagnosis should always accompany the
specimen.
Suggested Reading
Evans AG, Stannard AA. Diagnostic approach to equine skin disease. Compendium on Continuing Education for the
Practicing Veterinarian. 8:652-660, 1986.
Pascoe RRR, Knottenbelt DC. Manual of Equine Dermatology. New York: WB Saunders Co; 1999:21-34.
Kowalski JJ. Bacterial and mycotic infections. In: Reed SM and Bayly WM, eds. Equine Internal Medicine. Philadelphia:
WB Saunders Co; 1998:61-93.
Scott DW, Miller WH. Jr. Equine Dermatology. St. Louis: Elsevier Science; 2003:59-162.
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