Detection of Dirofilaria immitis using microscopic, serological and molecular techniques among dogs in Cabo Frio, RJ, Brazil / Detecção de Dirofilaria immitis utilizando técnicas microscópicas, imunológicas e moleculares em cães de Cabo Frio, RJ

Abstract Heartworm disease is a health problem for dogs and cats, especially in tropical and subtropical coastal regions of the world. Some studies have compared the efficacy of the diagnostic techniques used to detect this parasitosis. Therefore, the aim of this study was to compare parasitological optical microscopy (POM), serological and molecular techniques for diagnosing canine heartworm infection. Samples were collected between July 2015 and April 2016 from 103 dogs in Cabo Frio, RJ, Brazil. The wet fresh blood, thick smears, thin smears and modified Knott's test were used to detect microfilariae. ELISA (Snap™ 4Dx ® IDEXX) was used to detect antigens and the polymerase chain reaction (PCR) was used to detect DNA and enable sequencing for species differentiation and confirmation. 19.4% of samples were positive according to microscopy. Through PCR, 15.5% of the total were positive. Using ELISA, the positivity rate was 29.1%. Occult heartworm infection was detected in 11.6% of the samples. ELISA sensitivity was shown to be higher than PCR or microscopy (P = 0.001). Sequencing of samples confirmed the presence of Dirofilaria immitis and Acanthocheilonema reconditum . ELISA was more effective for serological diagnosis canine heartworm and should be used in clinical and epidemiological studies.

Resumo A dirofilariose é um problema de saúde para cães e gatos, especialmente nas regiões costeiras tropicais e subtropicais do mundo. Alguns estudos compararam a eficácia das técnicas de diagnóstico usadas para detectar esta parasitose. Portanto, o objetivo deste estudo foi comparar a microscopia óptica (OM), técnicas sorológicas e moleculares para o diagnóstico de infecção por Dirofilaria immitis . Foram coletadas, entre julho de 2015 e abril de 2016, amostras de 103 cães em Cabo Frio, RJ, Brasil. O exame direto, distensão espessa, distensão delgada e o teste de Knott modificado foram usados para detectar microfilárias. O ELISA (Snap ™ 4Dx ® IDEXX) foi usado para detectar antígenos e a reação em cadeia da polimerase (PCR) foi usada para detectar DNA e o sequenciamento para diferenciação e confirmação de espécie. Das amostras, 19,4% foram positivas de acordo com a microscopia. Por PCR, 15,5% do total foram positivos. Utilizando o ELISA, a taxa de positividade foi de 29,1%. Dirofilariose oculta foi detectada em 11,6% das amostras. A sensibilidade ao ELISA mostrou-se superior à PCR ou microscopia (P = 0,001). O sequenciamento das amostras confirmou a presença de Dirofilaria immitis e Acanthocheilonema reconditum . O ELISA foi mais eficaz no diagnóstico sorológico de dirofilariose canina e deve ser usado em estudos clínicos e epidemiológicos.

Freqüência de anticorpos anti-Erhlichia canis, Borrelia burgdorferi e antígenos de Dirofilaria immitis em cães na microrregião Ilhéus-Itabuna, Bahia, Brasil / Frequency of antibodies anti-Ehrlichia canis, Borrelia burgdorferi and Dirofilaria immitis antigens in dogs from microrregion Ilhéus-Itabuna, State of Bahia, Brazil

Para avaliação de positividade para antígenos de Dirofilaria immitis, anticorpos anti-Borrelia burgdorferi e anti-Ehrlichia canis foram coletadas 200 amostras de sangue de cães, 100 no município de Ilhéus e 100 no de Itabuna. Foi utilizado o "kit" Snap 3DX (IDEXX Laboratories) para realização das sorologias. Não se observou nenhum animal positivo para antígenos de D. immitis. Apenas dois dos cães estavam positivos para anticorpos anti-B. burgdorferi. Do total de amostras analisadas, 72 (36 por cento) estavam positivas para anticorpos anti-E. canis, sendo 43 em Ilhéus e 29 em Itabuna (p=0,027).

In order to detect the positivity to antigens of Dirofilaria immitis, antibodies anti-Borrelia burgdorferi and anti-Ehrlichia canis, 200 canine blood samples were collected as followed: 100 from the municipality of Ilhéus and 100 from Itabuna, State of Bahia. The kit Snap 3DX (IDEXX Laboratories) was used to performe serology. None of the tested animals were positive for antigens of D. immitis. Only two dogs of them were positive for antibodies anti-B. burgdorferi. From all the samples analyzed, 72 (36 percent) were positive for antibodies anti-E. canis, 43 from Ilhéus and 29 from Itabuna (p=0,027).

Focos de dirofilariose canina na Ilha do Marajó: um fator de risco para a saúde humana / Focus of canine heartworm disease in Marajó Island, North of Brazil: a risk factor for human health

A ocorrência de dirofilariose pulmonar humana relaciona-se com a prevalência de infecção por Dirofilaria immitis na população canina. Várias espécies de mosquitos são vetores desse nematóide. Analisaram-se amostras de sangue canino coletados nas vilas Pingo d'Agua e União, município de Salvaterra (Ilha do Marajó, PA), em junho, 2004 (n=34) e abril, 2005 (N=90). Os diagnósticos parasitológico e imunológico (ELISA - kit SNAP® 3DX™, Biobrasil) foram comparados (chi2, alfa=0,05) no exame de 34 amostras. A prevalência na população (N=90) foi avaliada pelo ELISA. O ELISA revelou mais positivos (25/34; 73,5 por cento) que a gota espessa (23/34, 67,6 por cento) e o Knott (21/34, 61,8 por cento), mas a diferença não foi significativa (p>0,05). A freqüência de infecção por D. immitis na faixa de 0 a 2 anos foi 58 por cento, enquanto em cães mais velhos foi 100 por cento. A prevalência da dirofilariose canina em Pingo d'Agua e Vila União foi alta (53,5 por cento), indicando risco de transmissão do parasito às pessoas nessa área.

The occurrence of human pulmonary dirofilariasis maintains a relation with the prevalence of Dirofilaria immitis infection in the canine population. Several mosquito species are vectors of this nematode. Canine blood samples collected in Pingo d'Agua and União villages, Salvaterra municipality (Marajó Island, Pará), in June, 2004 (n=34) and April, 2005 (N=90) were analyzed. Parasitological and immunological (ELISA - kit SNAP® 3DX™, Biobrasil) diagnoses were compared following the examination of 34 samples. The prevalence in the population (N=90) was evaluated by means of ELISA. ELISA revealed more positive samples (25/34; 73.5 percent) than thick smears (23/34, 67.6 percent) or Knott (21/34, 61.8 percent), but the differences were not significant (p>0.05). The frequency of D. immitis infection was 58 percent in dogs ranging from 0-2 years old, whereas in older dogs it was 100 percent. The prevalence of canine dirofilariasis was high in Pingo d'Agua and Vila União (53.5 percent), indicating the risk of parasite transmission to the people in this area.

Detection of IgG antibodies of Brugian filariasis with crude male and female antigens of Dirofilaria immitis.

Crude antigens from male and female Dirofilaria immitis were used to detect antibody to Brugian filariasis in humans by indirect ELISA. Both antigens were tested with 42 cases of Brugian filariasis, 131 cases of 20 heterologous infections and 35 healthy controls. The results--using male and female antigens--showed sensitivity of 88.1% and 88.1%, and specificities of 64.1% and 51.8%, respectively. Cross-reaction from other helminthic infections using crude male antigen gave false-positives with 48 sera from 13 heterologous diseases at the threshold value of 0.180, while the female antigen gave 63 sera from 15 diseases, at 0.309. Serum antibodies from patients with other helminthic infections--gnathostomiasis, strongyloidiasis, hookworm infections, trichinellosis, capillariasis, angiostrongyliasis, ascariasis, trichuriasis, toxocariasis, neurocysticercosis, cystic echinococcosis, taeniasis and opisthorchiasis--resulted in false-positives with both male and female antigens. One each of sparganosis and paragonimiasis heterotremus sera cross-reacted with only crude female antigen and their OD values were close to the threshold value. Although crude male antigen showed better specificity than crude female antigen, both female and male worms are sources of antigens needed for further purification. This study provides baseline data for further serodiagnosis of Brugian filariasis using dirofilaria antigen.

Evaluation of crude antigen of Dirofilaria immitis third-stage larva for detection of antibody against Wuchereria bancrofti infection by indirect ELISA.

Dirofilaria immitis is an important heart worm in dogs. An immunodiagnostic test is frequently applied to use an alternative antigen from other parasites. A crude antigen from infective third stage larva (L3) of D. immitis was employed in detecting the antibody to Bancroftian filariasis in humans by indirect ELISA. It was shown that 25 cases of Bancroftian filariasis (76%) at a cut-off value of 0.230, were positive. Cross-reactivity was tested using available sera of other helminthic infections. These sera were 47% (23/49) positive. They comprised a major intestinal helminthic infection, 7 from 15 (46%) strongyloidiasis sera, none from 5 (0%) hookworm infection sera, 6 from 10 (60%) trichinosis sera, 2 from 10 (20%) cysticercosis sera and 8 from 9 (88%) gnathostomiasis sera. The mean OD of sera from Bancroftian filariasis patients was not significantly different from that of the other helminthic infections (p>0.05). In this study, crude antigen may be valuable for the serodiagnosis of Wuchereria bancrofti when subjects do not have tissue helminth infections. However, the crude antigen should be purified to obtain a better sensitivity and specificity of the test.

Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis

This study was performed to investigate whethere there is cross-reactivity between Dirofilaria immitis and three intestinal nematodes of dogs. In ELISA, D. immitis-infected dog sera obtained at the 4th molting stage (9-11 weeks) and microfilaremic stage (25-30 weeks) were shown to be highly reactive with crude extract of T. canis. In immunoblotting, some antigenic fractions, 44, 57, 88, 100 kDa of crude extract of T. canis, were found to be positive reaction with sera of dogs infected with D. immitis. However, little or no cross-reaction were observed between sera of D. immitis-infected dogs and crude extract antigen of T. vulpis or A. caninum. These result suggest that there are partial cross reaction between sera of D. immitis-infected dogs and the antigen of T. canis.

IgG- and IgG4-detected antigens of Dirofilaria immitis adult worms for bancroftian filariasis by enzyme-linked immunoelectrotransfer blot.

In Thailand, Wuchereria bancrofti filariasis has persisted along the border between Thailand and Myanmar, its dynamic distribution caused by the infected transmigrants between neighboring countries, and the availability of susceptible mosquito vectors. Dirofilaria immitis adult worm was used as a source of antigens, excretory-secretory (ES) and partial surface extracts, to detect human filariasis. ES products showed several stained bands with Coomassie brilliant blue ranging from 14.5-93 kDa and mostly being glycoproteins as shown by concurrent reaction with Concanavalin A, except those at 18, 16 and 14.5 kDa which stained only with Coomassie brilliant blue. Surface proteins of 33.5-91.5 kDa were stained with Coomassie brilliant blue and showed smear bands with Concanavalin A. By enzyme-linked immunoelectrotransfer blot, Bancroftian filariasis sera gave specific reactions with glycoprotein ES antigens at MW 20.5 kDa against anti-human IgG. A prominent band of 18 kDa appeared consistently with the IgG4-ES antigen system. Surface extracts reacting with IgG and IgG4 were considered to be unsuitable as antibodies from all cases of filariasis could not detect any bands.