Case of vector-borne coinfection in a dog

Vector-borne diseases (VBD) in dogs, such as babesiosis, ehrlichiosis, anaplasmosis, and dirofilariasis, pose a significant threat to animal health in many regions worldwide, including Ukraine. These diseases are transmitted through the bites of ectoparasites, such as ticks and mosquitoes. With increasing urbanization, climate change, and population migration, the risk of spreading these diseases is significantly heightened. This article is dedicated to the case analysis of a dog in Ukraine diagnosed with two vector-borne diseases simultaneously, focusing on the clinical manifestations and diagnostic approaches for these conditions. Currently, the prevalence of VBD in dogs in Ukraine is insufficiently studied. There are only sporadic reports of isolated pathogens, with comprehensive information needing to be included. A case of concurrent dirofilariasis and babesiosis in a dog is presented. In the spring of 2024, an 8-year-old intact male German shepherd was brought to a veterinary clinic in Kamianets-Podilskyi due to general weakness. Clinical signs included fever, vomiting, slightly pale mucous membranes, and a mild cough. Following abdominal ultrasound and blood sample collection, anti-inflammatory and antiemetic medications were prescribed. A blood smear revealed Babesia canis and treatment with imidocarb was initiated. However, after a week, the dog's condition did not improve. A repeat blood test detected live microfilariae, and the CaniV-4 test identified antibodies to D. immitis , confirming dirofilariasis. Molecular studies corroborated the diagnosis. Follow-up echocardiography and radiography did not reveal visible adult worms in the heart. The dog was treated with melarsomine and a combination of imidacloprid and moxidectin. Two months later, follow-up blood tests showed no presence of microfilariae, and all blood and biochemical parameters were within normal ranges. The dog was under continuous veterinary supervision for the following months. Overall, there is a noticeable trend in the increase of VBD among dogs in Ukraine, necessitating heightened attention from veterinary services and pet owners. Regular preventive measures, such as ectoparasite control and health monitoring, are crucial for managing the spread of these diseases.


Introduction
Vector-borne diseases (VBD) in dogs, such as babesiosis, ehrlichiosis, anaplasmosis, and dirofilariasis, pose a significant threat to animal health in many regions worldwide, including Ukraine (Baneth, 2014).Given the increasing urbanization, climate change, and population migration, the risk of spreading these diseases has substantially increased.Canine VBD is caused by pathogens transmitted through the bites of ectoparasites, such as ticks and mosquitoes (Gray et al., 2009;Cutler et al., 2021).This article focuses on analyzing a case of a dog in Ukraine simultaneously infected with two VBD, explicitly addressing the clinical manifestations and diagnostic approaches for these conditions.
The prevalence of VBD in dogs in Ukraine is currently underexplored.Reports and studies on the sporadic detection of individual pathogens and disease registration exist; however, comprehensive information still needs to be provided.Dogs infected with various tick-borne pathogens may exhibit diverse clinical signs and pathologies affecting dif-ferent organ systems.However, coinfection with multiple organisms can account for the variation in clinical manifestations observed compared to infections caused by a single pathogen (Kilpatrick & Randolph, 2012;Diuk-Wasser et al., 2016;Sanchez-Vicente & Tokarz, 2023).
Babesiosis is one of the most widespread VBDs in dogs in Ukraine.It is caused by protozoan parasites of the genus Babesia and is transmitted primarily through the bites of Dermacentor reticulatus ticks.The disease is particularly prevalent in Ukraine's forest-steppe and steppe zones, where conditions are favorable for tick reproduction (Onyiche et al., 2021).
Dirofilariasis is caused by nematodes of the genus Dirofilaria, transmitted through mosquito bites.Dirofilariasis is especially dangerous because Dirofilaria immitis can affect the cardiovascular system of dogs.According to the literature, the predominant form of dirofilariasis in Ukraine is the cutaneous type caused by Dirofilaria repens helminths.Cardiopulmonary dirofilariasis, also known as "heartworm disease", is much rarer and is typically observed as isolated autochthonous cases.The prevalence of this disease is in-creasing in the southern and central regions of Ukraine, where climatic conditions are conducive to mosquito breeding.Besides dogs, humans can also contract dirofilariasis, making this disease a significant public health concern.
Overall, there is a trend of increasing cases of VBD among dogs in Ukraine, which requires heightened attention from veterinary services and pet owners (Levytska & Mushinsky, 2019).Regular preventive measures, such as treating animals for ectoparasites and monitoring dog health, are essential for controlling the spread of these diseases.
Considering the limited data available in Ukraine regarding detecting, diagnosing, and treating VBD, this study aimed to examine the clinical manifestations and diagnostic approaches for these diseases, particularly in concurrent infection with multiple pathogens.A case of a dog simultaneously infected with dirofilariasis and babesiosis is presented, along with general approaches to the diagnosis of VBD.

Materials and methods
The study was conducted in 2024 at the branch of the Department of Infectious and Invasive Diseases of the PDU Higher Education Institution, Fauna-Service Veterinary Medicine Clinic Kamianets-Podilskyi. Diagnosis was established comprehensively based on epizootiological, clinical, and laboratory methods.A microscopic examination of peripheral blood smears was performed to diagnose babesiosis, with samples collected from the tip of the ear (Konus 5605 Biorex-3).Smears were stained using the Leukodyph-200 kit.To assess hematological parameters in dogs, blood samples were collected in sterile EDTA tubes containing 2 ml of anticoagulant.Examinations were conducted according to standard methodologies.Hematological analyses were performed using semi-automatic analyzers Micro CC-20 Plus (HTI, USA) and BioChem SA (HTI, USA).
The presence of VBD pathogens was confirmed by laboratory tests, which included hematological parameters (morphological and biochemical), CaniV-4 rapid tests (Vet Expert, Poland), and PCR testing.Additionally, Knott's method was employed to establish the diagnosis of dirofilariasis.One milliliter of blood was mixed with 9 ml of 4 % formalin and centrifuged for 10 minutes at 500 g.The supernatant was removed, and the sediment was stained with 1% methylene blue.A drop was placed on a microscope slide and examined under a microscope at magnifications of ×10 and ×40.

Results and discussion
In the spring of 2024, an 8-year-old intact male German Shepherd was brought to a veterinary clinic in Kamianets-Podilskyi, Khmelnytskyi region, for evaluation due to general weakness.The clinical signs included fever (39.5 ℃), vomiting, mildly pale mucous membranes, and a mild cough.Peripheral lymphadenopathy was not observed, and an abdominal ultrasound revealed no abnormalities.A blood sample was collected for morphological and biochemical analysis, following which anti-inflammatory and antiemetic medications were prescribed.The results are presented in Table 1.Blood smear analysis initially identified protozoa consistent with Babesia canis, and treatment with imidocarb was administered twice according to the manufacturer's guidelines.Despite this intervention, no significant improvement was noted in the dog's condition after one week.A follow-up blood sample was collected for additional morphological analysis and blood smear examination.The dog's extensive outdoor activity during peak ixodid tick season warranted further testing for vector-borne diseases (VBD).The CaniV-4 test (Vetexpert) was positive for antibodies against D. immitis.While the follow-up smear did not reveal Babesia parasites, live microfilariae were observed in the fresh blood sample.The Knott's test identified nematodes with characteristic features of Dirofilaria spp., and molecular assays confirmed the presence of D. immitis microfilariae.Follow-up echocardiography did not reveal any adult helminths in the heart, and thoracic radiographs in both dorsoventral and lateral views showed no abnormalities.The dog's owner reported that the animal had never traveled abroad.
Treatment with immiticide was administered twice with a 24-hour interval, and the dog was also treated with a combination of imidacloprid and moxidectin in topical drops (Advocate, Bayer) according to the following regimen: four doses were administered at four-week intervals.Two months later, a follow-up blood test showed no presence of microfilariae, and all hematological and biochemical parameters were within normal ranges.The dog remained under continuous veterinary supervision in the following months.
Coinfection with multiple pathogens of vector-borne diseases is not uncommon, and similar cases have been documented in Europe, Asia, and the United States (Lindgren et al., 2000;Steiner et al., 2008;Chen et al., 2014;Lu et al., 2016;Moutailler et al., 2016;Razanske et al., 2019).In recent years, numerous reports have emerged in the literature describing clinical cases of mixed infections in dogs, including Hepatozoon canis and Ehrlichia canis, Babesia spp, as well as D. immitis, E. canis, and Borrelia burgdorferi (Krause et al., 1996;Mitchell et al., 1996;Niwetpathomwat et al., 2006).
An unprecedented level of simultaneous infection with multiple tick-borne pathogens has been documented using serological and molecular methods in the United States.Approximately 40 % of the 27 dogs studied showed serological evidence of infection by organisms from four genera, and an additional 52 % had serological evidence of infection with three co-members (Ehrlichia sp., Babesia canis, Bartonella vinsonii, Rickettsia rickettsii).According to PCR data, nearly half of the dogs had evidence of infection with four pathogens (Kordick et al., 1999).Moreover, in a U.S. study, coinfections with Borrelia burgdorferi, Babesia spp., and Ehrlichia spp.have also been serologically confirmed in humans (Magnarelli et al., 1995).
In another study conducted in Germany and Latvia, 39 % of humans were co-infected with tick-borne pathogens, and 81 % of ticks were co-infected with B. burgdorferi and Babesia (Süss et al., 2002).Studies conducted in Ukraine in recent years have also revealed multiple pathogens, including Rickettsia spp., Neoehrlichia mikurensis, Anaplasma phagocytophilum, and Bartonella spp., in 22.7 % of I. ricinus ticks and 18.1 % of D. reticulatus ticks (Levytska, 2021).
Additionally, coinfections with two species of Dirofilaria have been reported in Romania, accounting for 23.9 % of all positive samples (Ionica et al., 2015).Dirofilaria species are widely distributed across Europe, though their prevalence varies by region.Infections with D. repens are widespread throughout Europe and endemic in many Southern and Central European countries.Moreover, the risk of the emergence of new endemic regions is increasing in Northern Europe and the Baltic countries, where cases have become more frequent in recent years (Alsarraf et al., 2021;Jensen et al., 2023).Animal infections are predominantly observed in Southern Europe, particularly in the hyperendemic Mediterranean region.Infections are only occasionally reported in the rest of Europe, especially in the central part.However, according to Morchón et al., the epidemiological situation continuously evolves.Over the past 10 years, the prevalence of the disease has not only increased in previously endemic areas but has also spread to new non-endemic regions.Isolated cases, or the appearance of infected mosquitoes, have even been reported in northern European countries such as Norway and Denmark (Vatne, 2015;Fuehrer et al., 2021).
Climate change contributes to the emergence of vectors capable of transmitting infections and extends the period of possible transmission, allowing more generations of Dirofilaria to develop in a season.The development of mosquito larvae, which depends on temperature, progresses most rapidly at 28-30 °C, taking 8-9 days for D. immitis, with a threshold of 14 °C below which Dirofilaria does not develop (Sevimli et al., 2007).This information led to creating a seasonal transmission model for "heartworm" disease, allowing for the prediction of cases.The necessary conditions have recently been observed in northern European countries such as Sweden, Norway, Finland, and Denmark (Fuehrer et al., 2021).Human activities also play a crucial role in transmitting the disease to non-endemic regions.Travel with inadequately protected and improperly screened pets contributes to the emergence of new outbreaks, which, within a short period, can lead to the establishment of endemic areas in new regions.
The absence of adult helminths during clinical examination may indicate a low intensity of infestation (ESDA, 2017;AHS, 2018).Echocardiography is crucial for assessing the severity of the disease; however, it can be misleading, particularly in cases of mild infestation in dogs where the helminths may be out of view.Sporadically, D. immitis nematodes have also been detected in atypical locations.As reported, patients did not exhibit any specific symptoms of cardiac involvement, with only occasional coughing and general weakness observed (Penezić et al., 2022).
Despite the increased awareness of dirofilariasis in recent years and the growing body of epidemiological data, several questions still need to be answered.One of these is molecular xenomonitoring, which has recently been improved in many European countries, and specialized studies have been developed (Pękacz et al., 2024).In Ukraine, only one study has been conducted in this area.Mosquitoes of the genera Aedes spp.and Culex spp.were found to be infected with Dirofilaria spp. in 8 % of cases, consisting of D. repens DNA, with 5.3 % also testing positive for D. immitis (Poljuhovych, 2024).These studies are complex and costly, but they provide valuable information about the risk of infection and the actual epizootiological situation.
Although Ukraine is not considered an endemic country, dirofilariasis may be underestimated, leading to potentially undetected or misdiagnosed cases.Ukraine borders at least two endemic countries (Slovakia, Poland).Therefore, it can be anticipated that more reports of the disease will emerge (Demiaszkiewicz et al., 2014).A recent study in Poland demonstrated that the genetic diversity among dog populations infected with D. repens and D. immitis reflects the geographical origin and spread of the disease from neighboring countries (Alsarraf et al., 2023).Further development in this research area could significantly contribute to understanding the disease's spread and monitoring potential migration between populations.
In conclusion, adhering to the OneHealth approach, it is important to monitor the epizootiological situation in dogs and humans closely.

Conclusions
Vector-borne diseases in dogs, such as babesiosis, ehrlichiosis, anaplasmosis, and dirofilariasis, pose a significant threat to animal health in Ukraine and other regions of the world due to increasing urbanization, climate change, and population migration.The described case of simultaneous infection of a dog with dirofilariasis and babesiosis highlights the complexity of diagnosing and treating these diseases.In this case, the dog exhibited general weakness, fever, and vomiting symptoms.Laboratory tests revealed the presence of D. immitis microfilariae and B. canis parasites.Treatment, which included immiticide and microfilaricidal drugs, yielded positive results, and follow-up tests showed no pathology.
Prospects for further research: Future studies should focus on systematically monitoring the spread of vector-borne diseases in dogs in Ukraine, improving diagnostic and therapeutic methods, and examining the impact of climate change on the distribution of ectoparasites and disease spread.Additionally, raising awareness among pet owners about the risks of vector-borne diseases and preventive measures to prevent their spread is crucial.

Table 1
Clinical blood analysis of a sick dog