ACCIDENTAL FINDING OF DIROFILARIA REPENS IN DOG DURING THE QUALITY CONTROL OF SEMEN– CASE REPORT

A nine-year-old male dog of Doberman breed was taken to the laboratory for animal reproduction of the Scientifi c Veterinary Institute „Novi Sad“ for semen quality control. Th e sample of the ejaculate was taken without diffi culty; however, the sample contained a signifi cant amount of fresh blood (total ejaculate volume was 5 ml). Th e assessment of semen quality from such sample was not possible because blood components mask the spermatozoa. Nevertheless, the sample of ejaculate drop was placed for microscopic observation and analysis. In the semen, a lot of blood cells and only a few spermatozoids (mostly not moving) were found; however, something else was observed the presence of a live motile organism, longshaped, looking very much like a larval stage of a parasite Dirofi laria sp. Next day, the blood sample was taken for the analysis for dirofi lariosis. Th e result of the ELISA test for Dirofi laria immitis antigen was negative indicating the absence of adult worms of Dirofi laria immitis. Th e result of modifi ed Knott test showed the presence of larval stages of Dirofi laria repens.


INTRODUCTION
Today, there is a growing demand for dogs of diff erent breeds with superior genetic traits meaning the good exterior traits (constitution, color, temper, etc.) and health status in order to obtain a better off spring. Th is especially means the exclusion of hereditary diseases in these animals, such as dysplasia, blood coagulation disorders, diseases of the eye and the heart and other health problems that can cause a lot of pain to dogs and their owners / breeders, a lot of worry and economic costs.
Quality control of the semen in dogs has been showing increasing tendency among the dog keepers and breeders. More oft en, dog owners or breeders decide to test the semen of males before natural mating or artifi cial insemination of bitches. Over the time, dog breeding has become a lucrative business. Th erefore, before the purchase of the male dog, some customers require from the breeder/owner to provide the semen analysis results obtained in accredited laboratories. Since 2016, Scientifi c Veterinary Institute "Novi Sad" (NIV-NS), laboratory for domestic animal reproduction, is accredited for the assessment of animal semen quality, using CASA -Computer Assisted Sperm Analysis. On this occasion, the owner brought a dog to determine the quality of the semen and received a result on dirofi lariosis.
Dirofi lariosis is a vector borne disease, transmitted by a mosquito bite. Disease reservoirs are dogs and wild canids but the disease can also be transmitted to humans. Climatic, ecological, and many other factors have an infl uence on Dirofi laria sp. transmission. Dogs and wild canids can be defi nitive hosts for both types of Dirofi laria sp. On the other side, less well adapted or "aberrant" hosts are cats, wild felids and other mammalians (Potkonjak et al., 2020). Th ere are two forms of dirofi lariosis. One is caused by Dirofi laria immitis (Heartworm disease) where the adult worms migrate into the heart of a dog and cause serious problems, very oft en leading to the death of a dog. Th e other form caused by Dirofi laria repens is much milder form of the disease yet much more diffi cult to identify (Capelli et al., 2018). Th e disease caused by D. repens has almost no symptoms in dogs. Adult worms are imbedded under the skin and can persist there for a long time producing new larvae. Dogs can express nervousness or itchy feeling, if anything at all. Rarely, some cutaneous manifestations such as pruritus, dermal swelling and subcutaneous nodules can occur, or ocular conjunctivitis can be observed depending on the site where adult Dirofi laria are imbedded. It is important to emphasize that D. repens infection can be transmitted to people causing a zoonotic form of dirofi lariosis (Genchi and Kramer, 2017).
Both forms of dirofi lariosis have been identifi ed and reported by many authors in Serbia

CASE PRESENTATION
Male dog of Doberman breed, 9 years old, was brought in for semen collection and quality control. By observing the general condition of the dog, the weakness of posterior legs was noticed. Th e dog had a strong libido and the process of taking ejaculate passed without diffi culty. However, the ejaculate was full of substantial amount of fresh blood (total ejaculate volume was 5 ml). Th e assumption was that a small blood vessel was hurt during the process of semen intake. It was explained to the dog owner that it is not possible to assess the semen quality from that sample because the blood cells mask the spermatozoa and white blood cells weaken the sperm. Th ey release substances, which destroy microorganisms that cause infection but also aff ect the sperm by destroying the sperm membrane, impair sperm movement, and damage sperm DNA. Th e result obtained from such kind of sample would not be valid. Nevertheless, the sample of ejaculate drop was placed under the microscope for observation and analysis (USB 200i light microscope at 100× magnifi cation -Proiser, Paterna, Spain).
On the next day, blood sample was taken from the dog for analysis to dirofi lariosis using modifi ed Knott test and ELISA antigen test for Dirofi laria immitis. Modifi ed Knott test was used for the detection of Dirofi laria sp. circulating microfi lariae (Bazzocchi et al., 2008). Morphological characteristics of microfi lariae (cephalic and caudal ends) were used in order to diff erentiate D.immitis and D.repens microfi lariae of the two Dirofi laria species (Genchi et al., 2007). ELISA test was used for the detection of Dirofi laria immitis female adults' antigen.
Th e semen contained a lot of blood cells and just a few spermatozoids (mostly not moving). Th e whole semen sample was of dark red color resulting from excess blood in the sample. In the same semen sample, something else was seen -a live motile organism, long-shaped, looking very much like a larval stage of a parasite Dirofi laria sp. (Figure 1). Th e owner was immediately notifi ed that his dog is suspected for dirofi lariosis and that it would be recommendable to bring the animal for additional testing. Th e very next day, a blood sample was taken and analysed in the laboratory of Scientifi c Veterinary Institute "Novi Sad". Th e result of the analysis performed using ELISA test for   immitis and Dipetalonema reconditum in dogs in the same region. Th ere is no doubt that the region from which the dog from our case study originates is endemic for dirofi lariosis. Dirofi laria repens in dogs is pretty oft en neglected. A lot of the owners have only heard about Dirofi laria immitis as the causative agent of a heartworm disease because of its "deadly nature" for dogs. As compared with the disease caused by Dirofi laria immitis, dirofi lariosis caused by Dirofi laria repens is most frequently asymptomatic and remains undiagnosed because the dog-owners do not recognize that there is something wrong. Another problem is the lack of commercial test for testing the dogs for the presence of Dirofi laria repens. Th ere are no ELISA or fast/snap tests that can be used for Dirofi laria repens. Most commonly, the diagnosis is established unintentionally and the only option is to perform modifi ed Knott test on blood sample for the identifi cation or (when possible) a PCR for defi nite confi rmation of the pathogen.
Th e reports of human ocular and subcutaneous dirofi lariosis in Serbia published so far (Džamić et al., 2004) have confi rmed the importance of dirofi lariosis caused by Dirofi laria repens in human medicine in our country. Some of the authors identifi ed adult D. repens worms in peritoneal location, that is, in the scrotum of dogs. During castration procedure, they were found protruding out from incised tunica vaginalis. Th e worm infected dogs had veins congestions, widening of the cavernous spaces of testes and epididymis, as well as thickened and enlarged epididymis along with interductal fi brosis. No signifi cant eff ects on the process of spermatogenesis were observed (Ravindran et al., 2016). In a dog from our case study, there were no clinical symptoms of the same kind. However, the general condition of the dog was changed; the dog was nervous, uncomfortable, with week posterior legs. Th e semen sample showed very few spermatozoids with a very low, almost none activity. At this point, it cannot be stated whether the clinical status of the dog is a consequence of Dirofi laria repens infection. Th e dog will be subjected to therapy and observed during next several months in order to confi rm the clinical status.

AKNOWLEDGEMENT
Th is work was supported by the Ministry of Science and Technological Development of the Republic of Serbia, grants TR 31084.