Detection of Dirofilaria immitis using microscopic, serological and molecular techniques among dogs in Cabo Frio, RJ, Brazil.

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.

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.

Mosquito abundance in a Dirofilaria immitis hotspot in the eastern state of Rio de Janeiro, Brazil.

Coastal lowlands in the state of Rio de Janeiro, Brazil, support high numbers of heartworm-infected dogs. Microfilariae of heartworm need to be ingested by a potencial mosquito vector in order to develop into infective larvae and infect a new host. Ochlerotatus taeniorhynchus and Ochlerotatus scapularis are the primary vector species in the coastal lowlands of metropolitan Rio de Janeiro; thus, the aim of this study was to investigate whether these two species were abundant enough at the heartworm hotspot in the eastern area of the state to be important to the local parasite's life cycle. The study was conducted at the Massambaba sandbank (22°55'45″S;42°18'51″W), where canine heartworm prevalence was 53.1%. Mosquitoes were captured monthly using two traps with CO2, as well as eventual human landing. A total of 6118 mosquitoes of 16 species were collected. Ochlerotatus taeniorhynchus (37.5%), Culex quinquefasciatus (19.3%), Oc. scapularis (18.2%), and Coquillettidia sp. (10.5%) were the most abundant species. The monthly variation in frequency was marked for Oc. scapularis and Cx. quinquefasciatus and steadier for Oc. taeniorhynchus and Coquillettidia sp. The abundance of the two Ochlerotatus species reaffirms that they are the primary natural vectors for D. immitis in the coastal Rio de Janeiro state lowlands.

Can P-glycoprotein and ß-tubulin polymorphisms be used as genetic markers of resistance in Dirofilaria immitis from Rio de Janeiro, Brazil?

OBJECTIVE: Dirofilaria immitis, the causative agent of canine heartworm infection, is worldwide the most important filarid to affect domestic dogs. Prevention of this infection is done by macrocyclic lactones, but some reports on the lack of efficacy have been published. Although the actual cause of resistance is unknown, single nucleotide polymorphisms (SNPs) on a P-glycoprotein ABC transporter and ß-tubulin genes have been pointed out as candidates for genetic markers of resistance. We conducted a survey to verify the presence of these suggested genetic markers in microfilariae from 30 naturally infected dogs under macrocyclic lactones treatment living in an endemic area in the state of Rio de Janeiro. RESULTS: The analysis of these specific SNPs demonstrated no sign of polymorphism on the P-glycoprotein loci, while 72 and 48% of the samples were polymorphic to the first and second SNPs on ß-tubulin loci, respectively. This work demonstrates that the P-glycoprotein position 11 and 618 were not polymorphic and, therefore, not suitable as a genetic marker of resistance in Rio de Janeiro whereas both ß-tubulin loci were polimorphic. This work points out the difficulty of finding a universal genetic marker for resistance.

Selamectin for the prevention of canine Dirofilaria immitis infection: field efficacy in client-owned dogs in a high risk area.

BACKGROUND: Dog owners and veterinarians in small animal practices began to waive prevention of canine heartworm disease after heartworm infections seemed to have disappeared in Brazil. After 2013, infection rates rebounded, and an evaluation of the efficacy of chemoprophylactic drugs became necessary. Included in this re-evaluation was the efficacy of selamectin in client-owned dogs residing in a high infection-risk area. METHODS: The preventive efficacy of selamectin was evaluated by the topical application of selamectin to 24 client-owned dogs at the recommended rate (minimum of 6 mg/kg) by a veterinarian monthly for 36 months. Blood samples were collected before the first treatment and at the end of the study for testing to detect microfilariae by the modified Knott's test and Dirofilaria immitis antigens using a commercial antigen test. Exposure to risk of heartworm infection was confirmed by the presence of infection in dogs living in low-income communities within a 2 km radius from the homes of dogs in the study. The dogs were managed according to routine practice by the owners within each household throughout the study. RESULTS: All dogs tested negative by both tests after receiving topical treatment with selamectin monthly for 36 months. Testing of 204 dogs from the communities confirmed the presence of heartworm in the area by detection of microfilariae or D. immitis antigen in 44 dogs (21.6 %). CONCLUSIONS: Topical selamectin was 100 % effective for D. immitis prevention in 24 dogs that received monthly treatments by a veterinarian. Detection of heartworm infections in untreated dogs in the area suggests that clients need to be better informed regarding the prevalence of D. immitis and the importance of maintaining regular preventive treatments.

Chemoprophylaxis of Dirofilaria immitis (Leidy 1856) infection at a high challenge environment.

BACKGROUND: The frequency of canine heartworm infection in the state of Rio de Janeiro, Brazil was high before chemoprophylactic treatment was available, with one of the highest rates of infection (52.5 %) found among dogs living on the eastern shore of the state. Following the launch of a chemoprophylactic product, the rate of infection gradually decreased, and new infections were rarely reported. After 2005, outbreaks reported at the eastern shore as well as for new infections in other areas of high infection frequency were considered to possibly be related to reduced efficacy of macrocyclic lactones. Therefore, this study aimed to evaluate the efficacy of topical heartworm preventatives from different drug families at the high challenge area of the state of Rio de Janeiro. METHODS: A total of 46 dogs, including animals negative for Dirofilaria immitis microfilariae and antigen (Snap 4 Dx, IDEXX Laboratories, USA) at the initial screening were randomly allocated to two monthly treatment groups. Dogs in one group received topical moxidectin + imidacloprid and dogs in the other group received topical selamectin for eight consecutive months. Blood samples were obtained for microfilariae and antigen detection until the eleventh month after the first treatment. Dogs becoming microfilaremic or antigenemic on or before day 180 were considered to be infected prior to the first dose and were excluded from the study. RESULTS: A total of 29 dogs completed the study, including 14 treated with moxidectin + imidacloprid and 15 treated with selamectin. No dogs treated with moxidectin + imidacloprid (0/14) became infected during the treatment period, whereas four dogs of the selamectin group (4/15) became infected. CONCLUSION: Topical moxidectin + imidacloprid is 100 % effective in preventing D. immitis infections in dogs living in a high challenge natural environment.

Toward the formation of a Companion Animal Parasite Council for the Tropics (CAPCT).

This letter advises the imminent formation of the Companion Animal Parasites Council for the Tropics (CAPCT). The CAPCT consists of region-specific (e.g., Asia-Pacific, Latin America and Caribbean, Africa) experts comprising academics, veterinarians, parasitologists, physicians and allied industry partners that will work together to inform, guide and develop best-practice recommendations for the optimal diagnosis, treatment and control of companion animal parasites in the tropics, with the aim of protecting the health of pets and that of the public.

Updated canine infection rates for Dirofilaria immitis in areas of Brazil previously identified as having a high incidence of heartworm-infected dogs.

BACKGROUND: Canine heartworm infections were frequently diagnosed in Brazil before the new millennium. After the year 2000, the frequency of diagnosis showed a sharp decline; however, a few years later, new evidence indicated that the parasite was still present and that canine infection rates seemed to be increasing. Therefore, an updated survey of canine heartworm prevalence was conducted in several locations in south, southeast, and northeast Brazil. METHODS: Dogs from 15 locations having previously reported a high prevalence of heartworm infection were included in the survey according to defined criteria, including the absence of treatment with a macrocyclic lactone for at least 1 year. Blood samples from 1531 dogs were evaluated by an in-clinic immunochromatography test kit (Witness® Heartworm, Zoetis, USA) for detection of Dirofilaria immitis antigen. At each location, epidemiologic data, including physical characteristics and clinical signs reported by owners or observed by veterinarians, were recorded on prepared forms for tabulation of results by location, clinical signs, and physical characteristics. RESULTS: The overall prevalence of canine heartworm infection was 23.1%, with evidence of heartworm-infected dogs detected in all 15 locations studied. There was a tendency for higher prevalence rates in environmentally protected areas, despite some locations having less-than-ideal environmental temperatures for survival of vector mosquitoes. Among physical characteristics, it was noted that dogs with predominantly white hair coats and residing in areas with a high (≥20%) prevalence of heartworm were less likely to have heartworm infection detected by a commercial heartworm antigen test kit than were dogs with other coat colors. In general, dogs older than 2 years were more frequently positive for D. immitis antigen than were younger dogs. Clinical signs of heartworm infections were rare or owners were unable to detect them, and could not be used for reliable prediction of the presence of heartworm. CONCLUSIONS: These results indicate that the prevalence of D. immitis has increased in these areas of Brazil over the past few years. Small animal practitioners in these areas should include routine screening tests for heartworm infections in every dog's annual evaluation protocol and make sure to have uninfected dogs on prevention.

[Dirofilaria immitis (Leidy, 1856) in the neighborhood of a feline case: a study about the transmission]. / Dirofilaria immitis (Leidy, 1856) no entorno de um caso felino: um estudo sobre sua transmissão.

Dirofilaria immitis preferably infects canids and can, in lower frequency, be found in cats (Felis catus Linnaeus, 1758). The parasite may be transmitted by various Culicidae species. Motivated by an autochtone feline heartworm case diagnosed in the region of Engenho Novo - RJ (S 22 degrees 90' e WO 43 degrees 27'), the following issues were surveyed: 1) the local Culicidae fauna and; 2) the ocurrence of microfilaremic dogs. The mosquito study was conducted from April 2003 through May 2004, by peri-domiciliary and intra-domiciliary captures. A total of 672 mosquitoes (female) of the following species were caught: Aedes aegypti (Linnaeus) (561/672-83.5%); Culex quinquefasciatus Say (96/672-14.3%); Aedes (Oc.) scapularis (Rondani) (12/672-1.8%) and Aedes albopictus (Skuse) (3/672-0.4%). Only A. aegypti and C. quinquefasciatus specimens were captured year-round. A total of 235 canine blood samples were collected during the Campanha de Vacinação "Rio Sem Raiva" in October 2003. No microfilaria could be found in any of the examined samples (Knott s and NAN techniques), even though only 3.4% of the dogs received chemoprophylaxis and the majority of them frequently traveled to endemic areas. The presence of vectors associated to a possible dog mobility, may have provided the right scenario for the feline D. immitis infection to happen in the studied area.

Dirofilaria immitis (Leidy, 1856) no entorno de um caso felino: um estudo sobre sua transmissão / Dirofilaria immitis (Leidy, 1856) in the neighborhood of a feline case: a study about the transmission

A espécie Dirofilaria immitis parasita principalmente canídeos, podendo infectar também gatos embora raramente (Felis catus Linnaeus, 1758). Seus hospedeiros intermediários podem ser diferentes espécies de culicídeos. Motivados por um caso autóctone de dirofilariose felina, encontrado na região do Engenho Novo - RJ (S 22º 90' e WO 43º 27'), estudou-se: 1) a fauna culicídica local e; 2) a ocorrência de cães portadores de microfilárias. Os estudos sobre a fauna culicídica foram conduzidos, de abril de 2003 a maio de 2004, realizando-se capturas peridomiciliares e intradomiciliares. Foram capturados 672 indivíduos (fêmeas) das espécies Aedes aegypti (Linnaeus) (561/672 - 83,5%), Culex quinquefasciatus Say (96/672 - 14,3%), Aedes (Oc.) scapularis (Rondani) (12/672 - 1,8%) e Aedes albopictus (Skuse) (3/672 - 0,4%). Apenas as espécies A. aegypti e C. quinquefasciatus foram capturadas em todos os meses do ano. Foram coletadas 235 amostras de sangue canino, durante a Campanha de Vacinação "Rio Sem Raiva", em outubro de 2003. Nenhuma amostra examinada continha microfilárias (técnicas de Knott e NAN), apesar de apenas 3,4% dos cães receberem quimioprofilaxia. A presença de culicídeos vetores, associada à possibilidade de mobilidade dos cães, pode ter favorecido o aparecimento da infecção felina na região.

Dirofilaria immitis preferably infects canids and can, in lower frequency, be found in cats (Felis catus Linnaeus, 1758). The parasite may be transmitted by various Culicidae species. Motivated by an autochtone feline heartworm case diagnosed in the region of Engenho Novo - RJ (S 22º 90' e WO 43º 27'), the following issues were surveyed: 1) the local Culicidae fauna and; 2) the ocurrence of microfilaremic dogs. The mosquito study was conducted from April 2003 through May 2004, by peri-domiciliary and intra-domiciliary captures. A total of 672 mosquitoes (female) of the following species were caught: Aedes aegypti (Linnaeus) (561/672 - 83.5%); Culex quinquefasciatus Say (96/672 - 14.3%); Aedes (Oc.) scapularis (Rondani) (12/672 - 1.8%) and Aedes albopictus (Skuse) (3/672 - 0.4%). Only A. aegypti and C. quinquefasciatus specimens were captured year-round. A total of 235 canine blood samples were collected during the Campanha de Vacinação "Rio Sem Raiva" in October 2003. No microfilaria could be found in any of the examined samples (Knott´s and NAN techniques), even though only 3.4% of the dogs received chemoprophylaxis and the majority of them frequently traveled to endemic areas. The presence of vectors associated to a possible dog mobility, may have provided the right scenario for the feline D. immitis infection to happen in the studied area.

Técnica de centrífugo-flutuação com sulfato de zinco no diagnóstico de helmintos gastrintestinais de gatos domésticos / Zinc sulfate centrifugal flotation technique on the diagnosis of gastrointestinal helminthes of domestic cats

O diagnóstico coproparasitológico é o recurso laboratorial mais utilizado para detecção de infecções parasitárias gastrintestinais. A técnica de centrífugo-flutuação com sulfato de zinco (FAUST et al., 1938) é técnica de eleição no diagnóstico de estruturas parasitárias leves, podendo também ser usada para detecção de estruturas pesadas. Para avaliar o desempenho dessa técnica no diagnóstico das helmintoses gastrintestinais de gatos domésticos, foram realizados exames coproparasitológicos de 13 gatos domésticos 15 dias antes de suas mortes. A necropsia, os helmintos adultos encontrados no tubo digestivo e fígado foram contados, fixados e identificados. A técnica utilizada mostrou-se capaz de recuperar ovos de todos os nematóides e trematódeos encontrados, mas não foi adequada para a recuperação de cestóides.

Fecal examinations are the most commonly used laboratory tool for diagnosis of gastrointestinal nematode parasitic infections. Zinc centrifugal floatation technique (FAUST et al., 1938) is not only considered to be efficient in detecting light parasitic structures, but it is also used for recovery of heavy structures. In order to evaluate the performance of that technique in the diagnosis of domestic cats' gastrointestinal helminthiasis, fecal examinations of 13 animals were carried out 15 days prior to the animals' deaths. At necropsy, adult gastrointestinal parasites found were collected, counted, fixed and identified. The studied technique was able to recover eggs from all detected nematodes and trematodes, but appeared inappropriate for recovery of cestode eggs.

Infecçäo pelo Parvovírus Canino no Rio de Janeiro: um estudo de cinco anos / Canine parvovirus infection in Rio de Janeiro: a five-year study

Amostras fecais de cäes com até seis meses de idade, com gastrenterite, foram testadas para a presença do parvovírus canino (CPV) pelo teste de hemaglutinaçäo (HA) e confirmadas como positivas pelo teste de inibiçäo da hemaglutinaçäo. Noventa e duas das 208 amostras recebidas no período de abril de 1995 a novembro de 2000 foram positivas. Aproximadamente, 76 por cento das amostras foram obtidas de cäes entre dois e quatro meses de idade. Entre os 92 animais positivos para CPV-2,28 tinham sido vacinados, e para 11 destes o resultado positivo do HA poderia ser devido a detecçäo do vírus vacinal. Através da reaçäo em cadeia pela polimerase, pode-se confirmar a infecçäo pelo vírus selvagem em nove dos 11 animais vacinados. Neste estudo näo foi possível observar que fatores como sexo ou raça possam ser importantes no desenvolvimento da doença. No período estudado, o parvovírus canino pode ser detectado, em todos os meses do ano, näo apresentando sazonalidade definida.

Lesöes histopatológicas renais causadas por Dirofilaria immitis (Leidy, 1856) em cäes infectados experimentalmente / Renal histopathology lesion by Dirofilaria immitis (Leidy, 1856) in dogs experimentally infected

Foram estudadas lesöes renais de 17 cäes da raça Beaglo, infectados experimentalmente com o parasita Dirofilaria immitis através de transplante de parasita adulto ou por injeçäo subcutânea de larva infectante, com o período de infecçäo variando de 111 a 923 dias. Os animais foram separados em seis grupos de acordo com o tempo de duraçäo e tipo de infecçäo e um grupo controle composto por 11 cäes da mesma raça. As amostras renais foram obtidas por necropsia e examinadas através da microscopia óptica pelas coloraçöes de hematoxilina-eosina, ácido periódico de Schiff, prata-metanamina e tricrômico de Masson. Nos grupos de cäes infectados a alteraçäo glomerular mais freqüente foi o aumento da celularidade mesangial, associado ou näo à presença de microfilárias intracapilares ou intersticiais. Nos grupos com tempo de infecçäo acima de 365 dias foram observadas alteraçöes glomerulares como espessamento de membrana basal glomerular e nefrite intersticial. Esses achados preliminares confirmam relatos anteriores sugerindo que a glomerulonefrite e a nefrite intersticial säo as principais lesöes renais associadas a cäes infectados por D. immitis.