Frequency of Dirofilaria immitis infection in blood donor dogs of the Rio de Janeiro state

Abstract Dirofilariasis, a parasitic disease caused by the nematode Dirofilaria immitis, commonly known as heartworm, primarily inhabits the pulmonary artery and right heart of dogs and other animals. The disease is transmitted through diptera, predominantly from the Culex, Aedes, and Anopheles genera. Dirofilariasis is cosmopolitan in nature, endemic in coastal regions and tropical climates. Factors such as temperature, humidity, vector density, and the presence of definitive hosts significantly contribute to the spread of this parasitic disease. In the state of Rio de Janeiro, a prevalence of 58.6% of D. immitis infected animals has been recorded in municipalities like Niterói. Given that blood transfusions are routine clinical procedures and blood bags are not always accurately evaluated, an investigation into D. immitis infection in blood donor dogs from the metropolitan region of Rio de Janeiro was conducted. A total of 1044 blood donor dog files from a blood bank in Niterói, RJ, collected from January 2019 to December 2022, were analyzed. These samples, originating from kennels in various municipalities in the Metropolitan Region, were tested for the presence of microfilariae through direct examination using tubes and microhematocrit evaluated in optical microscopy. Additionally, the search for antigens was conducted using the enzyme-linked immunosorbent assay technique. Out of the 1044 records evaluated, 17.8% (186/1044) were positive for heartworm infection, with 2% (21/1044) samples positive for microfilariae and 14.8% (154/1044) positive for D. immitis antigens. The high prevalence rate indicates that canine D. immitis infection remains prevalent in the state of Rio de Janeiro, necessitating effective guidelines for prescribing preventive medications by veterinarians and an increase in epidemiological surveillance in the region.


Introduction
Canine heartworm, a global affliction caused by the parasite Dirofilaria immitis, is typically found in regions with hot climates and environments conducive to the growth of culicid vector species (Dantas-Torres & Otranto, 2013).Coastal regions, which possess these characteristics, often report the highest incidence of this parasitic disease (Genchi et al., 2011).Dogs with microfilaremia are the primary source of parasitosis dispersion, as they often travel with their owners and can infect local vectors susceptible to the disease (Guerrero et al., 1992).A study by Labarthe et al. (2014) revealed a national prevalence of 23.1% in the Brazilian states examined.In the municipality of Niterói, this rate escalated to 58.6%.According to Silva et al. (2019), the prevalence of this parasitosis in the state of Rio de Janeiro was 24.1%.
In dogs, the impact of infection by this nematode is contingent upon several factors: the parasitic load, duration of infection, host immune response, and level of physical activity.Most patients remain asymptomatic (Ames & Atkins, 2020).However, when symptoms do manifest, the most common clinical signs include exercise intolerance (observed in approximately 46% of infected dogs), unproductive cough (42%), and loss of appetite (25%) (Polizopoulou et al., 2000).In extreme cases, the infection can lead to right congestive heart failure, syncope, vena cava syndrome, and even death (Atwell et al., 1988).
Infection of canines by D. immitis can be detected through the identification of microfilariae or antigens of adult parasites in blood samples (Batista et al., 2008).The investigation and identification of microfilariae are typically conducted using the Knott (1939) technique, as modified by Newton and Wright (1956), a method that is globally recognized (Rawlings, 1986;Soulsby, 1968).Antigen detection is accomplished through enzyme-linked immunosorbent assay (ELISA) or immunochromatographic methods, utilizing various commercial tests.These tests have demonstrated high specificity, with sensitivity contingent on the number of female parasites the host carries.Some tests are capable of detecting a single female D. immitis (Henry et al., 2018;Nelson et al., 2018).
Given the high incidence of dirofilariasis in Rio de Janeiro, this study aimed to document the infection rate of D. immitis among blood donor dogs.This was achieved by analyzing records from a blood bank situated in the Niterói/RJ municipality.

Characterization of the study area
The state of Rio de Janeiro, along with three other states, constitutes the Southeast region of Brazil.Characterized by a tropical climate, high temperatures, Atlantic Forest formations, and mangroves, it also boasts an extensive coastline along the Atlantic Ocean.

Analysis of the files
We reviewed the records of canine blood donors, all over one year of age, whose samples were collected between January 2019 and December 2022.These samples were obtained from a private blood bank situated in Niterói, RJ.

Processing of blood samples
Prior to blood donation, all dogs were assessed for D. immitis infection via microfilariae and D. immitis antigen investigations.

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Frequency of Dirofilaria immitis infection in blood donor dogs of the Rio de Janeiro state Microfilariae were identified through direct examination, utilizing a microhematocrit capillary tube and an optical microscope with a 100× magnification (De Carli, 2001).Antigen detection was performed using the ELISA technique with commercial kits (Snap™ 4Dx® -IDEXX), which identify the presence of antigens in female D. immitis older than 8 months.
The animal count and the percentage of positive cases were analyzed for each mesoregion of the State.These regions included North Fluminense, Northwest Fluminense, Baixada Fluminense, Metropolitan Region, Center Fluminense, and South Fluminense.

Statistical analyses
The breed, age, sex, and region of residence for each parasite, along with the number of positive or negative samples, were recorded in a Microsoft Excel spreadsheet.
The analysis of the records revealed that 17.71% (185/1044) of the samples tested positive for D. immitis.Out of the 185 diagnosed dogs, 111 were female (60%) and 74 were male (40%).In the antigen-only test, a total of 154 samples (154/185 -83.24%) were positive.However, microfilariae were only detected in 21 samples (21/185-11.35%)upon direct examination.When considering both the presence of microfilariae and antigen detection, only 10 samples (10/185 -5.41%) tested positive.The results obtained from the mesoregions are presented in Table 1.

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Frequency of Dirofilaria immitis infection in blood donor dogs of the Rio de Janeiro state The animal count and the proportion of positive cases from each mesoregion of the State were analyzed.These regions included North Fluminense, Northwest Fluminense, Baixadas, Metropolitan Region, Center Fluminense, and South Fluminense (Figure 1).*Presence of one or more samples testing positive for both examinations.N: number of samples, Ag +: number of samples with antigen for D. immitis, Mf +: number of samples with microfilariae, Total +: number of samples that contain both microfilariae and the antigen for D. immitis.

Discussion
The obtained results, indicating 17.71% of animals infected, align with the prevalence data reported by Silva et al. in 2019 (24.1%) for the state of Rio de Janeiro.This likely stems from the climatic and environmental conditions within the state, which are conducive to the vector's development.The high canine population, both domiciled and otherwise, also contributes to this prevalence (Klinge et al., 2006).

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Frequency of Dirofilaria immitis infection in blood donor dogs of the Rio de Janeiro state The higher prevalence of purebred animals may be attributed to the fact that the blood bag donors were dogs from commercial breeding kennels.This could also account for the high incidence in specific regions, contingent on the kennel's location.
The higher percentage of animals diagnosed through antigen research (83.24%) as opposed to microfilariae research (11.35%) can be attributed to the superior sensitivity of the ELISA technique (Das et al., 2023;Rawlings et al., 1982).
In the current study, approximately 88% of the animals were amicrofilaremic, a significant deviation from the literature, which typically reports amicrofilaremic infections in 30% of infected animals.This discrepancy may be attributed to the fact that the search for microfilariae in the microhematocrit capillary is not the gold standard for detecting microfilariae.Instead, concentration techniques, such as the Knott technique, should be employed (Das et al., 2023;Trancoso et al., 2020).
Nonetheless, 11.35% of the animals tested positive solely in the microfilariae test, indicating that the antigen test's sensitivity is not absolute and could be influenced by the formation of immune complexes and the unique characteristics of the infection in each patient (Ferrer Montaño et al., 2002;Genchi et al., 2011;Vieira et al., 2014).Velasquez et al. (2014) highlighted the significance of preheating serum samples to diminish immune complexes for a more accurate diagnosis.
The presence of both circulating antigens and microfilariae in only 5.41% of the samples (10/185) underscores the necessity of conducting both tests, rather than relying on a single one, for accurate infection diagnosis (Nelson et al., 2018).

Conclusions
The current study suggests that heartworm remains a significant disease in the state of Rio de Janeiro.Veterinarians in the region must maintain vigilance in accurately diagnosing the disease, employing at least two tests to confirm infection and intensifying preventive measures.

Figure 1 .
Figure 1.Rio de Janeiro State map: mesoregions and their canine D. immitis infection positive sample percentages.Note.In the Rio de Janeiro region, positive animal cases were as follows: 20% (131/647) in the North, 15.2% (48/316) in the Metropolitan Region, 11.4% (5/44) in the Center, and 5.4% (2/37) in the South.Samples from the Northwest Fluminense and Baixada samples were not analyzed.

Table 1 .
Canine Dirofilaria immitis infection diagnosis in blood donor dogs across Rio de Janeiro's mesoregions: antigens and microfilariae investigation.
*Presence of one or more samples testing positive for both examinations.N: number of samples, Ag +: number of samples with antigen for D. immitis, Mf +: number of samples with microfilariae, Total +: number of samples that contain both microfilariae and the antigen for D. immitis.