2.1. Study Area:
The study was conducted in the selected cities of Northern Ethiopia: Mekelle, Kombolcha, Bati, and Kamisse towns where huge numbers of cart-horses are used for the transportation of man and goods. Mekelle is located 783 kms North of Addis Ababa at latitude and longitude of 13o 29N 39o 28E and it is located at 2000-2200 meter above sea level. The weather condition is hot and dry. The mean annual rainfall of the area is 628.8 mm. The annual minimum and maximum temperature is 17°C and 24°C, respectively (1). Respectively Kamisse and Bati are located in North-Eastern and North-central parts of Ethiopia in Amhara National Regional State. Kombolcha, Kamisse, and Bati are located 367, 315 and 406 kms north of Addis Ababa, at latitude and longitude of 11°5′N 39°44′E, 11°11′N 40°1′E, and 10°43′N 39°52′E respectively. The altitudes of Kombolcha, Bati, and Kamisse are 1842 masl, 1502 masl, and 1424 masl, respectively. The topography of the districts generally is marked by the presence of numerous mountains, plateaus, hilly and sloppy area. The average annual rainfall of Kombolcha, Bati, and Kamisse are 1248, 926 and 972.8 mm, respectively. The average annual temperature of Kombolcha, Bati, and Kamisse are 15.9 °C, 20.4 °C and 20.2°C, respectively.
2.2. Study Design
A cross-sectional study was conducted on cart-horses with signs of lymphangitis or lymphadenitis or both (EL typical lesions) and apparently healthy ones sharing a similar husbandry system in the area. Owners were briefed about the purpose and relevance of the study and carthorses from willing owners were recruited in the study. Sampling was purposive to recruit both to include both sick and apparently heathy ones.
2.3 Study Animals and sampling
Working cart-horses in the selected towns with symptoms of EL lesion as described by (14), and apparently healthy cart horses were sampled. EL horse cases were defined as a naturally infected horse with symptoms of lymphangitis or lymphadenitis or both upon clinical examination. Apparently healthy horse cases, in this study, were horses that did not demonstrate any sign of EL lesions upon clinical examination. From each study animal, body condition, age, and clinical condition were recorded. Dentation and owner history were used to classify the age as young (< six years old) and adult (> six years old).
2.4. Sample Collection and Transport
From each study animal, blood samples from jugular vein were collected in 10 ml Vacutainer tubes containing EDTA anticoagulant. The blood was centrifuged at 3000 rpm for 5 minutes thereafter buffy coat was extracted for later use in molecular tests. From horses with un-ruptured nodules or lesion of EL, pus samples were aseptically collected with sterile syringe and needle and striped into two universal bottle containing Sabarouds Dextrose Agar (SDA) enriched with horse serum (each for isolation of yeast and mycelial forms of HCF at varying temperatures). Part of the pus was smeared on glass slide for gram staining. Specimens were transported at +4 ℃ cool transport box to the College of Veterinary Sciences, Mekelle University. Buffy coat samples were kept in -20 ℃ until use. The SDA culture media inoculated with pus was incubated at room temperature and 37 ℃ and 5% Co2, respectively to isolate mycelial and yeast form. A total of 191 cart-horses were sampled. Out of the 70 samples taken from cart-horses with visible EL lesion, only 32 pus samples of closed lesions were collected for gram stain and culture (Table 1).
2.5. Smear Preparation and Gram Staining of Pus Sample Collected from Closed Lesion of EL
Pus smears were prepared in field from 32 cart-horses. The smears were fixed with methanol for 3 minute, and stained with Gram’s stain for the identification of the yeast form of HCF. Examination was made using oil immersion at 1000x magnification (100 objective*10 ocular). Search was conducted for typical yeast form of the organism, which should appear as Gram-positive, pleomorphic, ovoid to globose structures, approximately 2–5 µm in diameter. They may occur singly or in groups, and either extracellularly or within macrophages. A halo around the organisms (unstained capsule) is frequently observed (5).
2.6. Fungal Culture and Isolation
Collected pus samples were inoculated aseptically onto slants of SDA containing chloramphenicol (0.5g/ liter) and enriched with 2.5% glycerol. For the isolation of the mycelia form, incubation was made at 26 °C while isolation of the yeast form was made at 37 °C with 5% CO2 tension and high humidity (14;15). The culture was checked periodically and Gram-stained preparations were made from suspicious growth (2).
2.7. PCR Based Detection of HCF
2.7.1. DNA Extraction from Buffy Coat and Isolated Yeast Form
DNA extraction from buffy coat samples and isolated yeast form were made with little modifications in the recommended procedures indicated in Qiagen DNeasy blood and Tissue Kit (Lot 157043215, QIAGEN, Hilden, Germany). Buffy coat sample (100 µl) or pieces of cultured cells were transferred into a 1.5 ml micro centrifuge tube. Because we noted, compact nature of the stored buffy coat sample or solid nature of yeast cell culture, tissue lysis buffer (ATL, 100 µl) was applied and incubated at 56oC for 2 hours by frequent vortexing every 15 minutes. Thereafter, 20 µl proteinase K and 200 µl buffer AL (lysis buffer) were added, mixed thoroughly by vortexing, incubated at 56oC for 10 minutes. A 200 µl of 99% ethanol was added and mixed thoroughly by vortexing. To avoid occlusion of columns, the mix was centrifuged at 1000 rpm for 5 minutes. The supernatant was transferred into a DNeasy Mini spin column placed in 2 ml collection tube and centrifuged at 10000 rpm for 5 minute. After discarding the flow through and collection tube, the spin column was placed in a new 2 ml collection tube; 500 µl buffer AW1 (wash buffer) was added and centrifuged at 10000 rpm for 3 minute. The flow through and collection tube were discarded and the spin column was placed in a new 2 ml collection tube then 500 µl buffer AW2 (wash buffer) was added and centrifuged at 13000 rpm for 4 minute. The flow through and collection tube was discarded while the spin column was transferred into a new 1.5 ml micro centrifuge tube and to elute the DNA, 100 µl buffer AE was added into the spin column and incubate for 2 minute at room temperature and centrifuged 10000 rpm for 2 minute. DNA yield was assessed using agarose gel electrophoresis method. 10µl sample of the isolated DNA and 2µl loading dye was loaded into a well of the 2% agarose gel pre-stained in Ethidium bromide, electrophoresed for 45 minutes with voltage of 92 and visualized in UV light. Extracted DNA was stored in -20oC until use.
2.7.2. PCR Protocol Please reduce the size of this section by citing references
Except little modification in the cycling conditions, primers and PCR protocols were adopted from (13). Primers were sequenced in genetic facility of Iowa State University, Ames, Iowa, USA. PCR amplifications were carried out in 200 μL thin wall PCR tubes (KASVI RCR tube) in a thermocycler (Tianlong PCR thermal cycler, China). The first-round PCR was performed using P3/2R8 primers (primer P3 5’-3’, CGGAAGGATCATTACCACGCCG and primer 2R8 5’-3’, CAGCGGGTATCCCTACCTGATC) and cycling conditions of 95°C for 10 min (activation) and then 39 cycles of 94°C for 1 min (denaturation), 49°C for 1 min (annealing), and 72°C for 1 min (elongation) and followed by a final extension cycle of 72°C for 10 min. The product from the first round was expected to be 587 bp. A 1-in-10 (vol/vol) dilution of the product from this first reaction was added to fresh master mix including primers of F5 and 2R5 (primer F5 5’-3’, CTACCCGGCCACCCTTGTCTAC and primer 2R5 5’-3’, CCTACCTGATCCAGTCAACC). The thermocycler program for the second round was the same as that for the first round, except that the annealing temperature was raised to 55°C for 1 min. The ladder used in this protocol was 100 base-pair DNA Ladder (HIMEDIA™, MBTO049). The expected product was 514 bp and was visualized via electrophoresis at 92 V for 45 min on a 2% (wt/vol) agarose gel stained with Ethidium bromide (13).
2.8. Data Processing and Analysis
After data collection, the corresponding code number was written carefully at each margin. The data generated was entered in to the Microsoft excel. The data was imported to and analyzed using Statistical Package for Social Sciences (SPSS) software version 21.0. Descriptive statistics was computed and categorical result was presented using Tables. The association between exposure and outcome variables was assessed using binary logistic regression analysis. Binomial logistic regression through crude odds ratio (COR) was used to asses strength of association of EL with body condition score, age and study area. For those variables that showed significant difference through COR, to avoid confounding factors, multiple logistic regression through adjusted odds ratio (AOR) was computed. Test agreement between two diagnostic tests was computed by Kappa test statistics. The level of agreement between the diagnostic tests was determined using Cohen’s kappa coefficient (16;17). Probability (p) values < 0.05 were considered as significant.