A comparison between different laboratory methods and stains for detection microfilaremic dogs

Abstract


Introduction
Dirofilaria immitis infection or canine cardiac dirofilariasis (heartworm disease) is a zoonotic nematode that infects dogs' hearts, pulmonary arteries, and other canids (1). It causes respiratory (pulmonary hypertension, embolization, allergic pneumonitis, eosinophilic granulomas), cardiac (Myocardial hypertrophy, in some animals, congestive heart failure due to high right ventricle afterload), and immune complex glomerulonephropathy (2,3). D. immitis is transmitted by various mosquitoes of the genera Culex, Aedes, Anopheles and Ochlerotatus (4)(5)(6). The laboratory diagnosis of canine cardiac dirofilariasisin live animals is made through the finding of microfilariae (Direct and concentration methods) or direct ELISA or heartworm antigen test kits (7)(8)(9) and eventually confirmed by molecular analyses (10,11). The modified Knott's technique is based on the concentration method, and it is a test of choice for the differentiation of species (12). Modified Knott's technique is performed by diluting 1 ml of blood in 9ml of 2% formalin, which breaks down erythrocytes for better readability and maintains microfilariae for morphometric methods (13). The microfilariae of D. Units are currently differentiated by their size in Knott's preparations (7). Previous studies suggest that 2% acetic acid can be used in place of the 2% formalin fixative for a more straightforward disposal of liquid waste and lower potential health risks (14,15). Acetic acid-like formalin has hemolytic properties; it is used as a hemolytic reagent in Türk's solution for the total white blood cell count (16). Acridine orange is an inexpensive fluorescence dye used in laboratory investigation and research applications (17)(18)(19)(20). Due to the high sensitivity of the acridine orange stain, it has been widely used for rapid screening of biological samples having external contamination with microorganisms such as bacterial cells in cerebrospinal fluid (21), and malaria in the blood smear (22). The present study aimed to compare modified Knott's technique, serum concentration, and Türk's solution concentration method for detection of microfilaria of D. immitis in dogs and to investigate the suitability of Giemsa and acridine orange stains as a substitute to methylene blue in the modified Knott's technique for discovery of microfilaremic animals.

Ethical approve
The study was approved by the institutional animal care and use committee of Veterinary Medicine College, University of Mosul (UM.VET.2021.17 decision number in 6/9/2021).

Sample collection
During the period from October 2021 to January 2022, a five ml of blood was sampled via the cephalic vein from a ten naturally infected local stray dogs (positive with the canine heartworm Ag ELISA kit, DRG International Inc., USA, according to manufacturer's instructions) with an age range of 1 to 7 years. Three milliliters of blood were transferred to a tube containing ethylenediaminetetraacetic acid. The remaining blood was emptied into a non-EDTA (plain tube) and centrifuged at 1500 rpm for5 minutes to obtain serum.

Modified Knott's technique
Sirois et al. (23) described the following modifications, briefly, two milliliters of blood were mixed with 18 milliliters of 2% formalin, then shaken thoroughly to hemolyze the blood and centrifuged at 3000 × g for 5 minutes. The supernatant was discarded, and 3 thin smears were prepared from sediment. The smears were air-dried and fixed in anhydrous (absolute) methanol for 2 minutes, and the first and second smears were stained with methylene blue as mentioned in Coles (13), and Giemsa stains 10% as mentioned in Newton and Wright (24), respectively. The third smear was stained with acridine orange for 2 minutes, as mentioned in Gray et al. (25). The first and second smears were examined under a light microscope at 100× and 400× magnifications, but regarding smear was examined under a fluorescent microscope at 100× and 400×magnifications.

Türk'ssolution concentration test
One milliliter of blood was added to 9 milliliters of Türk's solution (0.2 milliliters of gentian violet 1% + 0.3 milliliters of acetic acid 100 % to 0.950 milliliters of distilled water) (13), mixed by inversion and centrifuged at 3000 × g for 5 minutes. The supernatant was discarded. The sediment was observed under a light microscope at 100× and 400× magnifications.

Serum concentration test
A thin smear was prepared, air-dried, fixed with absolute methanol for 2 minutes, then stained with Giemsa stain 10 % for 10 minutes, and then examined under a light microscope at 100× and 400× magnifications Hadi and Al-Zahawi (26). Body length (from the front end to the tail) and diameter with an ocular micrometer of 10 microfilariae in each smear were determined Magnis et al. (27), Heidari et al. (28).

Statistical analysis
It was performed by two-way analysis of variance to detect the changes in the length and width of microfilariae in the different laboratory tests, and the difference in the percentages between the various stains was assessed by using the Chi-square test using SPSS version 11.5 software for windows. A statistically significant association between variables exists when P<0.05 and at the 95 % confidence level.

Results
All concentration tests applied in the current study were positive for 10 samples. Slides prepared using the Türk's solution concentration test were clearer and freer of cellular debris than slides prepared by the modified Knott's technique (Figures 1 and 2). Slides prepared from serum appeared to be easy to examine (without erythrocytes) (Figure 3).   There were no significant changes in the length and width of the microfilariae detected by the three methods ( Table 1). Examination of stained smears prepared with modified Knott's technique sediment revealed that 100% of the microfilariae were stained with acridine orange (Figure 4), while 70 and 40% of the microfilariae were stained with Giemsa and methylene blue stains, respectively.

Discussion
Accurate diagnosis of D.immitis infestation is essential for disease management and control transmission to other animals and humans. The current guidelines from the American Heartworm Society (7) recommend using the heartworm antigen test and concentration test to detect microfilariae to confirm canine cardiac dirofilariasis.
In the current study, smears prepared by Türk's solution concentration test were clearer and freer of cellular debris than slides prepared using the modified Knott's technique, while the slides prepared from concentrated serum appeared to be easier to examine. In contrast, based on previous studies, the modified Knott's technique remains more valuable than the buffy coat method, wet mount, and thick and thin blood smears in diagnosing canine cardiac dirofilariasis (29,30). In the modified Knott's technique, Ohishiet al. (31) examined acetic acid, acetone, saponin, formalin, benzalkonium hydrochloride, and distilled water for their hemolytic action and established that acetone was most excellent. Recently, acetic acid (15) and glacial acetic acid (32) have become safe and suitable substitutes for formalin.
Türk's solution consists of acetic acid, which is responsible for the hemolysis, and crystal violet to stain the remaining leukocytes, making them easier to see and count (16,33,34). Therefore, Türk's solution concentration test appeared to be an appropriate substitute to modify Knott's technique. Hemolysis is an advantage of the modified Knott's technique due to the hypotonicity of 2% formalin and 2% acetic acid. This improves slide readability by removing most erythrocytes from the sample (13,35). Therefore, the slides prepared from concentrated serum appeared easy to examine (No erythrocytes).
In the modified Knott's technique, acridine orange staining appears to be a favorable alternative to methylene blue. Hansen et al. (36) described acridine orange staining as a fluorochrome stain for the detection of deoxyribonucleic acid (DNA) in bovine babesiosis (37,38). Ravindran et al. (39) described the acridine orange staining technique as an accurate, simple, and rapid screening of many blood samples. It was also reported that time for identification of first parasite was only 1/3 by acridine orange staining as compared to giemsa-stained smears (40,41). This suggests that acridine orange staining is a reliable way to detect cardiac microfilaremia in dogs.

Conclusions
The current study provides uncomplicated, economical, and quick methods (Türk's solution concentration test and the serum concentration test) to detect the microfilaremic dogs, and acridine orange staining is a suitable substitute for methylene blue and Giemsa stains in the modified Knott's technique.