Identification of Fungi from Police Dogs in Borri, Khartoum, Sudan

  

    
Under year (puppies)
    
2-5 years
    
5-10 years
    
Above 10 years
  
  

    
11 
    
  49 
    
  18 
    
  5 
  

Table 2:  Shows age groups.

Collection of samples

The dogs with lesions were sampled by plucking hairs with sterile forceps and by scraping epidermal scales with a sterile surgical blade from the affected areas. The samples from each dog were placed in separate sterile Petri dishes. Each animal (with or without lesions) was sampled by brushing its fur with a sterilized nailbrush using the following standard protocol. The animals were brushed from the head down to the tail and down the flanks and the legs. After specimen collection, the nailbrushes were placed in sterile Petri dishes for transport to the laboratory. Samples collected by brushing were inoculated on plates of Sabouraud dextrose agar (Difco) with chloramphenicol (400 ppm) (sca) supplemented with 0.5% cycloheximide.

Plucked hairs and scraped scales, originating from dogs with lesions, were examined for fungal elements by direct microscopy in 20% potassium hydroxide, covered with cover-slip and heated mildly above the flame (3min), after 10min they were diagnosed microscopically by potassium hydroxide mounts and by culture on the above mentioned media. The plates were incubated at 28°C and regularly examined for a month. Taxonomic identification of all mycelial colonies considered different was based on thorough macroscopic and microscopic studies. Shape dimensions, arrangement of macroconidia and other parts of isolated cultures. Suspected dermatophytes were identified to species level, and most of the remaining fungi were identified to genus level.

Potassium hydroxide mounts

A drop of 20% KOH was added on a clean glass slide, the sample (hair and skin) was placed in KOH drop and slide passed through a burner flame to hasten keratolysis. When keratolysis softened the sample, a clean glass cover slip was slowly placed at inclined angle on the sample and slightly pressed, preventing the formation of the air bubbles. The sample was kept in 20% KOH for a variable duration ranging from 5 minutes to 20 minutes, depending upon the thickness of the scales and examined every 5 minutes. Each slide was thoroughly examined for the presence of filamentous, septate, branched hyphae with or without arthrospores crossing the margins of the squamous epithelial cells of the skin, in case of hair, type and arrangement of the spore was noticed.

Processing of samples

The skin scraping and the hair were collected in the sterile plastic Petri-dishes to prevent contamination in transit to the laboratory.

The samples were kept at temperature in the laboratory room. The agar plates were arranged around the flame, and then with help of inoculation straight wire all the agar plates were inoculated.

Agar plates were then inoculated upside down at 28°C degrees centigrade for 4 weeks before each was discarded.

Agar plates were frequently removed from incubator and observed (checked) for fungal (i.e. dermatophytes) growth.

Fungal (dermatophyte) growth was checked for colonial characteristics and pigmentation.

Results were recorded straight on the data sheet.

Inoculation of culture media

prior to inoculation , the media were dried in oven for about 15 minutes, after which the plates were arranged around the flame and flamed straight wire was used to inoculate all the Petri dishes aseptically in order to prevent contamination of cultures, specimens and safety of personal as well as the environment. All sterile technique measures were strictly observed.

The temperature of the incubator was adjusted at 28°C (degrees centigrade) where all the cultures were maintained for one month before they are discarded or subcultured.

Microscopic examination procedure

During microscopic procedure two different techniques were used for the determination of fungal morphology, spores, shapes, size, chlamydospores and irregular hyphae.

Needle mount technique

Clean sterile glass slide and cover slip lactophenol cotton blue (LPCB) stain.

Positive pure agar plates were arranged around the flame on the work bench.

Needle wire was sterilized by heating until it becomes hot red.

Few drops of lactophenol cotton blue (LPCB) stain was added onto glass slide.

Needle wire was used to pick few colonies from the culture plates then placed onto glass slide with lactophenole cotton blue (LPCB) stain.

The cover slip was gently placed at inclined angle on the glass slide, in order to prevent the formation of air bubbles.

Microscopic observation was carried out using l ower power of 10^x and 40^x objectives to determine the colony morphology, size, shape, chlamydospores and irregular hyphae.

Result was inserted straight onto data sheet.

Slide culture technique

About 6-8 mm square block of Sabouraud dextrose agar (SDA) medium was on to a sterile slide and sub-inoculated at four sides with the pure culture, the inoculated block is then covered with sterile cover glass and placed in a petri dish supported on glass rod. Small amount of sterile water was added to a filter paper at the bottom of dish to that prevented drying of the agar media. Then the preparation was for a week at 28°C after which the cover slip was removed and mounted in the drop of lactophenol cotton blue (LPCB).

The block was then discarded into disinfectant. Few drop of lactophenol cotton blue (LPCB) was added on to the growth on the slide and it was covered with a cover glass. Where both preparations were then examined microscopically using the 10x and 40x objectives with the condenser iris diaphragm adjusted to give maximum contrast.

Results

The study was carried out on 83 dogs in the police dogs Department in Borri-Khartoum-Sudan. Dogs were examined to detect dogs with suspected Dermatophytosis. Only a few dogs (n=2) presented skin lesions with focal or diffuse hair loss and/or scaling. A representative number of dogs without visible skin lesions (n=81), were used to carry out the Dermatophyte of their fur. The approximate age was between (1-12) years, breed was German-Shepherd and genders of the dogs were male (70) and female (13).

Out of (83) samples (76) of hair samples (91.5%) yielded positive growth for fungal colony, while (5) samples (6%) - 3 hair samples and 2 skin scrapings were negative for fungal growth in (SDA) with Cycloheximide. The species isolated were from hair samples, and were able to grow in (SDA) supplemented with (0.05%) Chloramphenicol and (0.5% Cycloheximide) at 28°C. The isolated species were in two hair samples Microsporum-canis was in (one samples), and M.gypseum (one sample).

Microscopic diagnostic results were positive for 2 samples (2.40%). and negative result were 76 samples (91.5%) from the number of the colonies tested, while 5 samples (6%) didn’t show any fungal growth.

The most common fungi isolated from skin of dogs with susceptible Mycoses were saprobe, especially Aspergillus spp (72%), Penicillium spp (12%), and Altenaria spp (7.2%). Cultural Characteristic of Isolated Species is arranged in Table 3.

  

    
Species 
    
Growth rate 
    
Colonial appearance 
    
Microscopic appearance 
  
  

    
Microsporum canis
    
4-7 days
    
white and fluffy center;    border  closely spaced radial grooves
    
knob end and spiny with a    rough, thick wall  6-15  cells
  
  

    
Microsporum gypseum
    
4-9 days
    
buff (yellowish-brown) with    white border  rapidly spreading    mycelium
    
many, spiny thin wall with 6 to    15 cells, rounded ends
  

Table 3:  Cultural Characteristic of Isolated Species.