Introduction

Filarioid nematodes are worldwide parasites transmitted by ' 'invertebrate's host causing filariasis in numerous vertebrate hosts except fish (1 , 2) . Filarioid worms fall under the class of Nematoda, order Spirurida, above the family Filarioidae, which includes the families Filariidae and Onchocercidae, and within these two families are many genera that infect humans and animals (3) . Generally, Parafilaria and Stephanofilaria are small to medium-sized parasites, and the genus Onchocerca are medium-sized fibroids (4) . The larvae of these worms are called microfilariae(mf), which are define as serpentine organisms covered by sheath and contain many cell nuclei their length varies from 231-300 μm and the width ranges from 5-7 μm (5) . They circulate in the peripheral blood and infect tissues and body cavities of vertebrate hosts (6) and larvae could be found in various body parts, including the peritoneal cavity, arteries, and subcutaneous tissue; they can cause skin filariasis, which is characterized by lesions that resemble scabies; they can also spread to the nervous system through blood circulation, resulting in neurological disorders; and when they get to the eye, they can cause blindness (7) . Although adult nematodes are thought to be non-pathogenic in their natural hosts, hematophagous vectors can spread infectious stages to non-permissive hosts like sheep, goats, horses, cattle, and humans, leading to severe and frequently fatal neuropathological disorders known as cerebrospinal filariasis (8) . (9) diagnosed for the first report of lumber paralysis due to infection of sheep with the larval stage of Setaria spp, in Iran. (10) showed in the brain cross-section of sheep the presence of nematode larvae with encephalomyelitis, lymphocytic, eosinophilic, and moderate leptomeningitis with many hemorrhagic tracts, degeneration, and necrosis brought on by larval migraines. The adult worm causes a syndrome called "Kumri", meaning weak back characterized by In coordination and a swinging back gait caused by paralysis of the lumbar region and one or both fore-hind limbs (11) . This study aimed to use conventional methods to explore microfilariasis in sheep.

Materials and methods

Ethical approve: The institutional animal care approved the study approved the study and use committee of Veterinary Medicine College, University of Mosul (UM.VET.2024.034 decision number in 1/9/2024)

Three hundred sheep samples, aged five months to seven years, were randomly selected from various Mosul city regions for blood collection. The samples were examined in the college of Veterinary Medicine University of Mosul during the period from August 2024 to October 2024 for the detection of microfilariae infection after taking a case history from the owners' animals that included information about (age, sex, treated or not, location of the animal, type of nutrition and rearing) took 3ml of blood samples from sheep appeared many clinical symptoms, including pneumonia, evidence of locomotor dysfunction, weakness, depression, and fluctuating appetite) Blood samples were collected from the jugular veins in EDTA tubes. We used the Modified Knott technique, M G G quick stain, and Acridine orange stain to detect microfilaria. The morphometric study-based diagnosis of microfilariae species was determined using (12,13).

1-Modified Knott's technique: After placing one milliliter of the sample blood and nine milliliters of 2% formalin in a tube and centrifuging the tube for five minutes at 1500 rpm, a drop of sediment was placed on a glass slide and a drop of 1% con. Methylene blue dye was added to the sediment drop using a pipette. The coverslip was then placed on the slide and examined with an ocular micrometer and an X 10 magnification power in 22 x 22 mm fields to determine the quantity and measurement of microfilariae (13).

2-May Grunwald Giemsa stain (M G G quick stain): After placing a single drop of blood on a glass slide, a light blood smear was created, allowed to dry for three minutes, fixed with methyl alcohol for five minutes, and then stained with M G G quick stain for two minutes. It was then rinsed under tap water, allowed to dry, and examined using an ocular micrometer and an (X40) or (X100) magnification power under fields to determine the quantity and measurement of microfilariae

3-Acridine orange stain: After thoroughly dried in the staining solution, the blood smears were fixed with 100% methyl alcohol and left in the Acridine orange solution (0.01%) for two minutes. After carefully washing the dyed smears with regular water and letting them air dry completely, they were inspected using a fluorescence microscope with several filters (B BF G). The BF filter was used for the examination. (15)

Statistical analysis: The results were analyzed using the IBM SPSS version 22 statistical program, the two-sided Chi-square test, and the Fischer test to determine the significant differences between the factors and the infection rate. The Kappa value was also used to establish the agreement between the different tests used in this study, namely, microscopic examination and modified nucleotide technology (16) .

Results

The present study's findings demonstrated that, throughout the inquiry using the modified Knott approach, 168 sheep samples tested positive for microfilariae under a microscope, yielding a total percentage of 56%. in table 1.

The intensity and prevalence of microfilariae infection in sheep was found to be mild, moderate, and severe, with percentage rates of 85.1%, 10.7%, and 4.1%, respectively. This indicates a significant difference in infection severity below the probability level (P < 0.05) table (2). The effect of risk factors on the intensity of the infection, like age, revealed significant differences. We noticed a high percentage of infection in animals over three years old (93.1%) while low rats in sheep below one year old (27.1%) table 3.

The percentage of infection in sheep males (54%) and females (58%) did not significantly differ from one another table 4 . The relationship between animal husbandry and infection rates indicated an increase level of infection in door systems (61.6%) than in indoor systems (45.7%) in table 5.

The present study used different stains for staining microfilariae, like Methylene blue stain, M G G quick stain, and Acridine orange stain. The uses of these stains revealed the presence of sheathed and non-sheathed microfilaria figures (A,B,C).

Fig (A).Blood smear stained with M.G.Quick stain (Giemsa Stain) showing microfilaria SH: Sheathing, NR: Nerve ring, EP: Excretory pore, BN: Body nuclei, AP: Anal pore, under power magnification (X100), by using a digital camera.

Fig (B).Microfilaria (Setaria spp.) stained with methylene blue stain under the power of magnification (X10) by using a digital camera.

Fig (C).Blood smear stained with Acridine orange fluorochrome showing microfilaria under magnification (X4 ) power by using digital camera.

Discussion

Sheep in Mosul city had a 56% overall infection rate with microfilaria, which indicated an increase in the disease's incidence. This high infection rate may be attributed to the availability of intermediate hosts, such as mosquitoes and flies, which are responsible for transmitting microfilaria (18) . Our results were in agreement with (11) and the study of (18), who recorded a total infection rate of microfilariae in sheep (60% ,65%), respectively. Also, (19) , (20) , (21) recorded high percentage rates of microfilaria in farm animals (54.28% ,55%, 70%), respectively. Numerous researches abort incidences of microfilaria in Mosul city have documented variation in infection rates in farm animals ranging from 0% to 100% (23 - 31) . The parasitism in various parts of the world could be explained by variations in the number of samples analyzed, the physiological state of the animal, and the time of sampling, as these larvae are characterized by a cyclic nocturnal-diurnal tendency (13) . Studying the severity of the infection with microfilariae revealed three forms of infection, mild, moderate, and severe infection with microfilariae in sheep with percentages rates (85.1%,10.7%, and 4.1%) respectively by using Modified Knott Technique, these differences in the results may be attributed to the difference in immunological states of the animal. The study showed that people of all ages are susceptible to infection, with a significant difference in infection rates. We noticed a low rat in sheep below one year old (27.1%) while a high percentage rate of infection in animals over three years old (93.1%). The reason may also be attributed to the long life span of these parasites inside their final host, like a period of larval development until they reach adult stages and shed microfilaria inside the host's body, consuming several months (13) . This is consistent with what was mentioned by (24) , (25) , (31) , (32) . who indicated that the infection rate with microfilaria increases with the animal's age. As for the gender factor, no significant difference was observed in the infection rate between males (54%) and females (58%). This result agreed with (33,34), who stated that the infection has occurred in both sexes equally, while our study was in agreement with (24) , (25) , (19) , (35) , (36) . There were significant differences between animal husbandry and infection rates, a higher rate in outdoor systems (61.6%) than indoor systems (45.7%). Also, there were significant differences between the breed of the animals and the infection rates. The reason may be attributed to the inhibition of animal immune responses and increase their sensitivity to the microfilariae infection, as well as the effects of environmental conditions, breeding conditions, herd infection, field cleanliness, and the number of sucking insects present in the field. This study used Acridine orange and M G G quick stain to identify microfilaria in sheep blood. Both stains were characterized by high sensitivity for diagnosing this parasite at different microscopic magnifications of 4X, 10X, and 40X. also, it was easy and speed to identify infection with the microfilaria because of very short time during staining and examination not more than 5 minutes. (18 , 37 , 44) . This suggests that M.G G quick stain and Acridine orange are reliable in detecting microfilariae infection in sheep.

Conclusion

Modified Knott's technique is a suitable procedure for the detection of microfilariae, but it is time-consuming and requires a centrifuge during the test. M.G quick stain (Giemsa) is a good standard quick technique for the detection of microfilariae and discrimination its morphological characteristics. This stain is fast and consumes less time, and it is beneficial in epidemiological studies in which the number of samples is large. The Acridine orange stain has high sensitivity in identifying microfilariae at microscopic magnifications of 4X and 10X.

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical Clearance

This work is approved by The Research Ethical Committee.

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