EPIDEMIOLOGICAL AND CLINICAL IMPORTANCE OF CANINE DIOCTOPHIMOSIS

Dioctophimosis is an endoparasitism in dogs caused by Dioctophyma renale , a nematode with an indirect life cycle and high zoonotic potential. Infection in dogs occurs through ingestion of transitional/paratenic hosts containing infective larvae. Th e preferred site of parasitism is the right kidney, although the parasite may also be ectopically localized in subcutaneous tissue, internal organs, peritoneum, and mediastinum. Since dogs are usually infected with a small number of parasites, the disease is asymptomatic, making the diagnosis of this disease in clinical veterinary practice rather diffi cult. In addition, as dogs are usually infected with a small number of parasites, the disease is asymptomatic, which makes the diagnosing of this disease in clinical veterinary practice diffi cult. Th e long prepatent period, lasting three to fi ve months, makes it impossible to diagnose the disease in dogs younger than six months. Clinical suspicion of dioctophimosis in dogs is established on the basis of anamnestic/epizootiologic data and the clinical picture, while the fi nal diagnosis is made ante mortem or post mortem on the basis of reliable diagnostic parameters and methods. Th e most practical application in routine diagnosis of canine dioctophimosis is the detection of D


INTRODUCTION
Dioctophyma renale (giant kidney worm) is a widely spread nematode that parasitizes dogs in tropical and subtropical regions and tends to spread to other geographical areas (Taylor et al., 2007;Eiras et al., 2021).
Th e parasite is predominantly localized in the right kidney of dogs but can also parasitize in subcutaneous tissues, mediastinum, peritoneum, and internal organs when it causes an ectopic form of the disease (Taylor et al., 2007;Angelou et al., 2020;Greer et al., 2021). During the indirect developmental cycle and migration of larval stages, D. renale causes nonspecifi c symptoms in the form of lumbar pain, dysuria, hematuria, and general disturbances (Gherman, 2013; Russo et al., 2022). Th e above clinical manifestations are the result of high intensity infections where death due to renal failure is possible (Taylor et al., 2007;Paras et al., 2018).
Larval forms of the parasite cause trauma by migrating through individual tissues and organs -stomach, duodenum, liver, abdominal cavity (Ferreira et al., 2010). Adults infl ict chemical damage to infected tissues and organs and, by mechanical action, cause compression and atrophy of the renal parenchyma (Russo et al., 2022) and obstruction of the ureter with subsequent hydronephrosis (Angelou et al., 2020).
In Serbia, there are conditions for the occurrence, maintenance, and spread of this helminthiasis in carnivores and humans related to the presence of infected intermediate/paratenic hosts (oligochaetes, frogs, and fi sh) and susceptible true hosts (martens, ferrets, foxes, cats, and dogs) in the mentioned epizootic area (Ignjatović, 2022).
Th ere are numerous reports from Serbia related to the problem of intestinal, cardiorespiratory, and ocular parasitic infections in domestic carnivorous animals (Gajić et  Th e aim of this paper is to provide new information on the etiopathogenesis of D. renale and point out the need to investigate the occurrence of this parasite in wild and domesticated carnivores in Serbia. Th e proposed studies are of particular importance because D. renale may pose a threat to human health. Since dioctophimosis was diagnosed in dogs in Greece in 2019, the exacerbation of the eff ects of global warming could be an important predisposing factor for the occurrence of this zoonosis in carnivores in Serbia.

Morphological characteristics of the causative agent
Adult forms of the parasites have a cylindrical bright red body covered with a thin cuticle. Sexual dimorphism is clearly prominent. Females are longer (100-103 cm) and thicker (6-12 mm) compared to males, whose length is about 35 cm, and thickness 3-5 mm. At the anterior end of the body there is a hexagonal shaped mouth, surrounded by 12 papillae arranged concentrically in two rows ( Figure 1A). Th e tale of female parasite is narrow and ends bluntly, while vulva has posterior appearance about the end of oesophagus. Th e tail of the female parasite is narrow and ends bluntly, while the vulva is directed backwards over the end of the oesophagus. At the posterior end of the body of the male worm there is a bell-shaped copulatory bursa with one spicule ( Figure 1B and 2). Th e eggs are oval, yellowish, 74-84 μm in size, unembryonated at the moment of hatching, containing two blastomeres. Th e egg membrane is thick with one mucoid structure at both poles (Taylor et al., 2007;Gherman, 2013).

Life cycle
Adult forms of D. renale are mainly localized in the renal pelvis of the right kidney, while secondary forms can be found in the renal parenchyma, subcutaneous tissue, and abdominal cavity (Gherman, 2013). Th e prepatent period lasts for approximately 135-155 days, and the complete life cycle is completed in 2 years (Ferreira et al., 2010). Adult female worms hatch eggs, which are ejected-by the urine of infected animals. At an optimal temperature of 25-300C, fi rst stage larvae (L1) are developed in intermediate hosts for about one month. Low temperatures signifi cantly extend this period (Freitas, 1980).
Th is parasite has a complex life cycle that includes the presence of intermediate and paratenous hosts. Th e fi rst intermediate hosts are aquatic oligochaetes (Lumbricus variegatus), in which the larvae of the fi rst and second stages are formed (L2). Th ere are three diff erent ways of continuing the transmission. In the fi rst case, infective third (L3) and fourth stage (L4) larvae are formed in the same host. In second, paratenous hosts (fi sh or frogs) per os ingest oligochaetes containing fi rst larval stage forms, which develop in infectious larvae in tissues. A third way of transmission occurs when fi sh or frogs eat crayfi sh species from the Cambarus genus where infected oligochaetes parasites are present (Angelou et al., 2020). Dogs become infected by ingesting some intermediate hosts (oligochaetes, fi sh, or frogs), which contain infective larvae. In the pathogenesis of dioctophimosis, the most important event is the migration of larval forms of parasites, which penetrate the intestinal wall and then reach the abdominal cavity, from where they migrate to the kidneys. Th e primary place of localization and maturation of the parasite into its adult form is the right renal pelvis, due to its anatomical connection with the duodenum (Ferreira et al., 2010). Th e adult forms of D. renale live for 3 years in a defi nitive host, while eggs in optimal environmental conditions can survive for 5 years (Angelou et al., 2020).
During the migration through various organs of the infected dog, D. renale causes mechanical damage, especially in the right kidney by parenchymal compression. Apart from the mechanical eff ects, parasites have esophageal glands, whose secretion of lipolytic and proteolytic enzymes causes chemical damage to infected tissues and organs (Russo et al., 2022).

GEOGRAPHICAL DISTRIBUTION AND EPIZOOTIOLOGICAL CHARACTERISTICS OF D. RENALE
Dioctophyma renale is very widespread in tropical and subtropical regions with a tendency to spread to other geographical areas. Th is phenomenon mainly occurs due to transport of dogs from endemic areas where they get infected by eating meat from intermediate hosts containing infective larvae (Measures, 2001;Russo et al., 2022). It is most commonly diagnosed in North and South America, Asia, and sporadically in Europe (Taylor et al., 2007). According to the literature, D. renale has not been diagnosed in Africa and Oceania yet (Acha and Szyfres 1986;Measures, 2001).
Th e highest prevalence of dioctophimosis is found in mammals from South America (37-42.1%), where the disease has been reported in thirtythree countries with most clinical cases in Argentina (1,414). Due to tropical ecosystems, which are suitable for the coexistence of intermediate and defi nitive hosts, there is an increasing likelihood of the occurrence, maintenance, and spread of the infection, while humidity and suitable temperature favourably aff ect the development and vitality of eggs (Russo et al., 2022).
Although the disease in dogs is sporadically present in Europe, the reported prevalence in Poland is 10.5%, indicating a risk of spreading to surrounding countries (Eiras et al., 2021). Dioctophimosis has not yet been diagnosed in dogs from Serbia. Sporadic cases in some countries in Europe, whose number is continually rising, sudden climate changes, increased commercial and uncommercial dog transport worldwide are the factors which contribute to preconditions for occurrence of this parasitosis in Serbia as well (Ignjatović, 2022). Th e intensity of infection is quite variable and depends on host species and localization of helminths in their organism. High intensity infection considered to be the one with at least twenty adult forms of parasite per one individual (Eiras et al., 2021).
Despite the fact that high temperatures are appropriate for egg development, dioctophimosis is also present in areas with colder weather, which is suitable for intermediate hosts. Th e factors like ways of keeping and feeding animals, weather disasters (fl oods) can signifi cantly increase the risk for occurrence of the infection. Th at is especially true for dogs who live in yards and have close contact with watercourses and are fed raw fi sh or frogs. Since immunosuppression occurs due to frequentative pregnancies (especially in stray bitches), females are more commonly aff ected than males. However, males are more territorial with regard to females, and infected prey is more accessible to them. Despite the stated assumptions, gender and age predisposition for dioctophimosis isn't defi ned in dogs (Pedrassani et al., 2017).
Lack of reports on the prevalence of D. renale infections in animals from diff erent regions is a result of insuffi cient epidemiological studies or the low prevalence and complex life cycle of the parasite (Eiras et al., 2021).

CLINICAL DIAGNOSTIC PARAMETERS OF DIOCTOPHIMOSIS IN CARNIVORES
Long prepatent period (3-5 months) complicates diagnosis of the disease in dogs under six months of age. Clinical suspicion of dioctophimosis in dogs can be established on the basis of anamnestic or epizootiological data and a clinical picture.

Anamnestic and epizootiological data
During the collection of anamnestic data, it is signifi cant to ask dog owners about feeding habits, contact with water surfaces or aquatic animals (such as frogs or fi sh), and the possibility of roaming around without owner's supervision (Russo et al., 2022).
Th e data on lifestyle and quality of life of carnivores collected from owners and available data on climate parameters, transport of dogs and cats in endemic areas of dioctophimosis, or buying pets from risky epizootiological localities, could be very signifi cant for raising suspicion about this nematode (Ignjatović, 2022).

Clinical picture
Nonspecifi c clinical symptoms of dioctophimosis are not suffi cient to make a f diagnosis, but they may serve to raise a reasonable suspicion of this disease.
In the clinical picture of dioctophimosis, signs of renal insuffi ciency are dominant (Gherman, 2013). Symptoms depend on the intensity of the infection and the localization of the causative agent. In low-grade infections, the disease is asymptomatic because one kidney is aff ected while the other compensates for its impaired function. Th en non-specifi c clinical symptoms like fever, convulsions, abdominal distension, inappetence, and consequently loss of body mass are dominant. In high-grade infections, animals experience haematuria at the end of urination, and pyuria, while the abdomen is painful on palpation in the renal region (Russo et al., 2022).
In the case of an ectopic form of disease, oedema can occur in the inguinal region (it looks like a stab wound with a fi stula containing serohemorrhagic exudate), proliferation of surrounding tissue (due to chronic peritonitis), and urethral obstruction (the consequences of which are anuria, uremic coma, and death) ( Haematological analysis in the aff ected dog shows anaemia, eosinophilia, and lymphopenia, while microscopic examination of the blood smear reveals toxic granulation of neutrophils. Th ese fi ndings suggest the presence of acute infl ammation. Blood biochemistry results indicate increased blood urea nitrogen and creatinine values. On physicochemical examination, the urine sample is blurred, red, with an alkaline pH value of 8 and contains proteins. Aft er the centrifugation, there is a large amount of urinary sediment (Gherman, 2013;Russo et al., 2022).
A valid ante mortem diagnosis is established by parasitological examination of urine sediment, ultrasonography, contrast radiography, computerized tomography, and serological methods of diagnostics (indirect ELISA).

Parasitological diagnostics from urine sediment
Eggs detection in the urine of an aff ected dog represents the gold standard in diagnosing this nematode. Th e urine sample obtained by cystocentesis or catheterisation is centrifuged on 400 spins in 5 minutes with the aim of getting the sediment. Aft er that, the sediment is examined by optical microscope using 40x magnifi cation, and based on morphological characteristics of eggs, D. renale can be diagnosed (Figure 1). An advantage of this method is low price, high specifi city and sensitivity. However, eggs absence cannot eliminate suspicion of this disease with certainty, especially in cases of ectopic forms, the absence of females, or presence of male adults (Pedrassani et al., 2017). Eiras et al. (2021) report about the rare but possible fi nding of eggs in animal faeces, which seems to be a result of urine contamination.

Ultrasonography
Ultrasonography is a fast, eff ective, and non-invasive technique for diagnosing multifocal circulatory structures that represent longitudinal and transversal sections of a parasite located in the right kidney. Within the ultrasonographic diagnostics it is useful to apply doppler ultrasonography technique that can detect reduction or complete absence of its signal, indicating

Native radiography
Native radiography does not provide valid results, and therefore it is applied in combination with contrast urography, which detects deviations in kid-ney size, shape, and topography. Based on the elimination of contrast medium, the preservation of kidney function is estimated, which is a signifi cant parameter for the selection of an adequate therapy procedure (Eiras et al., 2021;Russo et al., 2022).

Computerised tomography
CT scanner is a valid method of diagnostics. Due to its cost, it is primarily used in human medicine, and rarely in aff ected dogs. Circulatory structures with contrast medium deposits in peripheral parts of tissue are considered positive fi ndings (Eiras et al., 2021).

Serological methods
In cases of ectopic forms of disease, absence of D. renale eggs in urine, or presence of immature females or only males, alternative diagnostic procedures for detecting dioctophimosis include indirect ELISA as well. Th is test has high specifi city of 93 %, it consists of soluble antigens that are obtained by extraction from oesophagus of parasite and it is based on specifi c antibodies (IgG) detection in blood serum (Pedrassani et al., 2017).
Post mortem diagnosis is established on the basis of parasitological sections and histopathology examinations (Ferreira et al., 2010).

Parasitological section
Since the infections by D. renale nematode in cats and dogs are usually low intensity, pathomophological changes could be less prominent or are completely absent.
In high intensity infections, necropsy has the following fi ndings: thicker kidney capsule, connective tissue infi ltration with signs of cystic degeneration. As a result of compressive atrophy on transversal sections, thinned regions of cortex and medulla are common. As a result of urethral obstruction by adult parasite forms, the renal pelvis is highly dilated due to hydronephrosis and fi lled with haemorrhagic exudate. Due to compensatory hypertrophy and glomerulonephritis, left kidney is bigger compared to the right (Hallberg, 1953;Angelou et al., 2020).
Ectopic migration oft en occurs when larvae or adult helminth forms are found in the abdominal cavity (Shahbazi et al., 2017), causing peritonitis and perihepatitis (Taylor et al., 2007), or rarely in the thoracic cavity, like perforation of the mediastinal part of the pleura (Russo et al., 2022). Interference of parasites around hepatic lobes is possibly resulting in capsule erosions, bleeding and rupture (Angelou et al., 2020). In cases of extrarenal localization, larval forms can be encysted in subcutaneous tissue, the spleen, the uterus, the mammary gland, the ovaries, the scrotum, and the testes (Greer et al., 2021), when necropsy is the most appropriate method (Ferreira et al., 2010;Eiras et al., 2021).
In some cases of dioctophimosis, subcutaneous changes are reported. Bitencourt Vidal et al. (2021) report on the accidental fi nding of nodular structures in the inguinal region of cats, during routine spaying. Parasitological examination of nodular content established the presence of D. renale, while histopathological fi ndings confi rmed granulomatous infl ammation.
Due to clinical signifi cance of this disease and increasing prevalence during last decade in dogs of surrounding countries, the list of diff erential diagnosis of dioctophymiasis needs to include the following: nematode Capillaria plica (Ilić et al., 2021), chronic nephritis, renal fi brosis and functional kidney disorders of diff erent etiology (Gherman, 2013), as well as polycystic kidney disease of cats and canine leptospirosis (Mesquita et al., 2014).

THERAPEUTIC AND PROPHYLACTIC APPROACH
Th e most common approach in therapy of dioctophimosis is surgical removal of pathologically changed kidney. In cases of extrarenal localization, laparoscopy is used for larval removal and adult nematode forms. In cases of rare fi nding of D. renale in both kidneys, the only therapeutic method is helminths' extraction from renal pelvises, with uncertain prognosis and survival time from six months to three years (Greer et al., 2021;Eiras et al., 2021).
Medication therapy is extremely limited and uncertain, but avermectine derivatives that can be used are moxidectine and doramectine (Russo et al., 2022). One of eff ective therapeutic protocols implies intramuscular application of 5 % enrofl oxacine (1 ml/10 kg) for fi ve days, then doramectine (1 ml/20 kg) for three days. Seven days later, tablets of enrofl oxacine are applicated for three days per os and doramectine injection once a week (Eiras et al., 2021). Greer et al. (2021) designed a protocol that, beside diagnostic methods, also gives information about treatment options for aff ected dogs according to the results of specialized clinical examination methods (Figure 3). If urinalysis detects D. renale eggs, a patient is a candidate for surgical removal of the aff ected kidney. Before this invasive intervention, it is necessary to examine blood biochemistry parameters that indicate the degree of preservation of kidney function (blood urea nitrogen, creatinine, and electrolytes). If they are within reference values, the patient undergoes surgical intervention and aft er the surgery is treated with moxidectine (2,5 mg/kg monthly). If kidney function isn't preserved, euthanasia is justifi ed. When there is absence of eggs in urine sediment, ultrasonography is performed. If adult parasite forms are found in the renal pelvis, the previously mentioned procedure is implemented. Figure 3. Protocol for diagnosing and treating dogs older than six months infected by D. renale (Greer et al., 2021) Experimental investigations indicate that fungal proteases and chitinases extracted from Pochonia chlamydisporia have a harmful eff ect on the causative agent's eggs. Eiras et al. (2021) suggests the potential possibility of biological control of dioctophimosis and the need for additional investigations.
Control of dioctophimosis is accompanied by the use of general prophylactic measures that are educational for owners because specifi c measures like vaccination aren't available. Due to the high zoonotic potential of D. renale nematode, prevention of infection is especially important for stray dogs because of their uncontrolled movement and contact with ecosystems that represent the source of this parasitosis (Gherman, 2013;Russo et al., 2022). It is necessary to limit the movement of pet dogs or keep them monitored and under supervision in order to avoid contact with water courses, which are habitats for intermediate and paratenic hosts for D. renale. It is required to limit the feeding of dogs with raw fi sh, amphibians, or other intermediate hosts (Gherman, 2013;Russo et al., 2022).
Th e therapeutic prophylactic approach is very limited by the progressive development of parasitic resistance to some anthelminthic chemical groups.

SIGNIFICANCE OF DIOCTOPHYMA RENALE NEMATODE FOR PUBLIC HEALTH
Humans can become infected with the D. renale nematode aft er eating raw fi sh or frogs containing infective larvae or by ingesting aquatic oligochaetes of the Annelida genus (Chauhan et al., 2016). Due to its nonspecifi c clinical presentation and rarely diagnosed cases of the disease, this parasite is oft en neglected in human medicine (Yang et al., 2019). Th e disease is common in people from Asia (China, Indonesia, Iran, Th ailand, and Japan), Europe (Greece and Serbia), North America, and Australia. Most cases are diagnosed in China, which is attributed to their traditional diet and consumption of raw meat (Yang et al., 2019).
Previous studies have found a slightly higher prevalence in males compared to females (59.5% and 40.5%) (Perera et al., 2021). Nematodes can survive in the kidneys for up to fi ve years and cause obstruction, hydronephrosis, and destruction of the renal parenchyma (Chauhan et al., 2016). Aff ected patients usually have nonspecifi c symptoms suggestive of nephritis: Haematuria and pain in the lumbar region, while fever, abdominal pain, anaemia, and loss of body mass are less commonly reported. More severe cases are confi rmed to be fatal (Perera et al., 2021).
Th e diagnosis of dioctophimosis in humans is made on the basis of the detection of eggs and adult forms of D. renale in a urine, parasitological dissection, and histopathological examination of infected tissue from the right kidney. Th is fi nding is explained by the fact that the right kidney is closer to the stomach. According to Yang et al (2019), the parasite presence in the renal pelvis destroys the integrity of the renal tissue and leads to metaplastic changes. Th is is the reason why the parasite fi nding is related to the occurrence of renal tumours. Th e occurrence of ectopic parasitism should not be neglected when D. renale larvae are localized in the subcutaneous tissue and retroperitoneal cavity and adult forms are localized in the thoracic cavity, causing infl ammatory nodules and skin papules characterized by marked pruritus (Yang et al., 2019). Th e fi rst diagnosed case of subcutaneous dioctophimosis in a Chinese man who has lived in Japan for 15 years was confi rmed by molecular identification of D. renale larvae (Tokiwa et al., 2014).
Th ere is still no adequate treatment protocol for human patients, and nephrectomy is considered too invasive. Although there are confi rmed successful treatments with ivermectin and albendazole, the use of anthelminthics is still not accepted as a safe therapeutic option. Th erefore, in human clinical practice, it is desirable to include this parasitosis in the list of diff erential diagnoses for patients with nonspecifi c symptoms such as haematuria and lumbar pain (Yang et al., 2019). Th e most eff ective way of prevention is to avoid eating raw fi sh or frog meat (Chauhan et al., 2016).
Th us far, one case of dioctophimosis has been reported in Serbia in a person who consumed minced fi sh meat during a trip. Upon his return to Serbia, the patient exhibited the following symptoms: fever, haematuria, dysuria and pain in the lumbar region. Th e diagnosis was confi rmed aft er surgical intervention and pathohistological examination of the extirpated material (Nikolić Svetozarević et al., 2001).

CONCLUSION
Fro m a veterinary point of view, dioctopimosis has a high value in clinical practice because dogs are usually aff ected by only a few parasites. Th e fact that the disease is oft en asymptomatic complicates its diagnosis. For this reason, only a reasonable suspicion can be made during the clinical examination of dogs, while it is essential to include the disease in the list of diff erential diagnoses of urinary tract diseases in carnivores. Because of the high zoonotic potential of the D. renale nematode, eff ective control and management of this helminthosis is crucial. It is also necessary that all fi elds of human and veterinary medicine work closely together within the concept of "one health".

ACKNOWLEDGMENTS
Th e study was supported by the Minister of Science, Technological Development and Innovation of the Republic of Serbia (Contract number 451-03-47/2023-01/200143).