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.

Molecular Characterization of a Dirofilaria immitis Cysteine Protease Inhibitor (Cystatin) and Its Possible Role in Filarial Immune Evasion.

Infection with canine heartworm (Dirofilaria immitis), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di-CPI from D. immitis. Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di-CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-rDi-CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di-CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di-CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di-CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di-CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.

Feline heartworm (Dirofilaria immitis) infection: first case report of serological diagnosis in Brazil, confirmed by molecular assay.

The clinical importance of heartworm infection in cats has indeed increased in recent years. Dirofilaria immitis infection has been reported worldwide in cats and continues to be regularly diagnosed in endemic areas. The diagnosis can be overlooked easily, especially in Brazil, where there is not a specific feline immunodiagnostic test, forcing the veterinarians to use a test made for the canine host. In 2015, a 10-year-old female neutered cat was diagnosed with D. immitis using an antigen serological test, based on imunocromatography and designed for dogs. The modified Knott test was negative. As the disease progressed, the cat showed clinical signals of respiratory distress, such as dyspnoea and polypnea in addition to prostration and emaciation, and died a few weeks after the diagnosis. During necropsy, one adult nematode was found in the pulmonary artery. D. immitis infection was confirmed by molecular amplification, performed in the worm fragment. This is the first report of serological diagnosis of feline dirofilariasis in Brazil. A chemoprophylaxis routine in cats should be done, as is done in dogs from endemic areas.

Genetic profiles of ten Dirofilaria immitis isolates susceptible or resistant to macrocyclic lactone heartworm preventives.

BACKGROUND: For dogs and cats, chemoprophylaxis with macrocyclic lactone (ML) preventives for heartworm disease is widely used in the United States and other countries. Since 2005, cases of loss of efficacy (LOE) of heartworm preventives have been reported in the U.S. More recently, ML-resistant D. immitis isolates were confirmed. Previous work identified 42 genetic markers that could predict ML response in individual samples. For field surveillance, it would be more appropriate to work on microfilarial pools from individual dogs with a smaller subset of genetic markers. METHODS: MiSeq technology was used to identify allele frequencies with the 42 genetic markers previously reported. Microfilaria from ten well-characterized new isolates called ZoeKY, ZoeMI, ZoeGCFL, ZoeAL, ZoeMP3, ZoeMO, ZoeAMAL, ZoeLA, ZoeJYD-34, and Metairie were extracted from fresh blood from dogs. DNA were extracted and sequenced with MiSeq technology. Allele frequencies were calculated and compared with the previously reported susceptible, LOE, and resistant D. immitis populations. RESULTS: The allele frequencies identified in the current resistant and susceptible isolates were in accordance with the allele frequencies previously reported in related phenotypes. The ZoeMO population, a subset of the ZoeJYD-34 population, showed a genetic profile that was consistent with some reversion towards susceptibility compared with the parental ZoeJYD-34 population. The Random Forest algorithm was used to create a predictive model using different SNPs. The model with a combination of three SNPs (NODE_42411_RC, NODE_21554_RC, and NODE_45689) appears to be suitable for future monitoring. CONCLUSIONS: MiSeq technology provided a suitable methodology to work with the microfilarial samples. The list of SNPs that showed good predictability for ML resistance was narrowed. Additional phenotypically well characterized D. immitis isolates are required to finalize the best set of SNPs to be used for large scale ML resistance screening.

Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of D. immitis and D. repens in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species. METHODS: Overall, four dogs harbouring natural co-infection with D. immitis and D. repens were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott's test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted. RESULTS: The dynamics of microfilaremia showed similar patterns for both Dirofilaria species. In all four dogs, D. immitis was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for D. repens, one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples, while duplex PCR failed to amplify D. repens DNA in many cases. CONCLUSIONS: Both Dirofilaria immitis and D. repens microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with D. repens in co-infected dogs when the ratio of microfilaremia is in favour of D. immitis.

Clinical and pathological effects of Dirofilaria repens and Dirofilaria immitis in a dog with a natural co-infection.

Canine dirofilarioses are mosquito-borne zoonotic diseases with a continuous expansion of their geographical distribution, as a consequence of different climatic and ecological factors. Dirofilaria immitis, the aetiological agent of heartworm disease, has gained a major veterinary interest, mainly due to its severe clinical implication. In the last decades, D. repens, despite of being regarded as a less pathogenic species, regained attention due to its recognized zoonotic potential. Romania has been traditionally regarded as a non-endemic country, but recent epidemiological surveys are highlighting the presence of both D. repens and D. immitis. The present case report describes the clinical and pathological features of a natural co-infection with D. repens and D. immitis in 5year old male Boxer from north-western Romania. Based on clinical and laboratory investigations, a diagnosis of kidney failure and chronical cystitis was established. The parasites were identified by morphology and confirmed by PCR and sequencing. To the best of our knowledge this is the first worldwide report of the ectopic presence of D. repens adults in the pelvic cavity and mesentery by natural infection and first report of the ocular localization of this parasite for Romania. The clinical and paraclinical findings suggest the implication of D. repens microfilariae in the aetiology of histopathological lesions. Further investigations are needed in order to establish the actual pathogenic potential of D. repens, a frequently neglected parasite.

Dirofilaria immitis in a child from the Russian Federation.

An immature female worm, Dirofilaria immitis, was isolated from the scrotum of a 14-month-old child. This is the first identification of human dirofilariosis caused by D. immitis in a relatively Northern region (Moscow) of the Russian Federation. The parasite was diagnosed by means of morphological examination of the worm, confirmed by PCR assay. This case raises questions about the range of the D. immitis distribution among humans in Russia. To better understand the geographical distribution of dirofilarioses, detailed clinical and epidemiological information should be collected from human cases with appropriate laboratory confirmation.

Rapid Differentiation of Filariae in Unstained and Stained Paraffin-Embedded Sections by a High-Resolution Melting Analysis PCR Assay.

BACKGROUND: Apart from infection with human filariae, zoonotic filariasis also occurs worldwide, and the numbers of cases have been increasing steadily. Diagnosis of intact filariae in tissues or organs depends on histological identification. The morphology of parasites in tissue-embedded sections is poor and shows high levels of homoplasy. Thus, the use of morphological characteristics in taxonomic studies is difficult and may not allow a specific diagnosis. METHODS: Here we report the use of real-time PCR with high-resolution melting analysis (HRM) to detect and identify Brugia malayi, Brugia pahangi, Wuchereria bancrofti, and Dirofilaria immitis in paraffin-embedded sections. Assay specificity was determined using other tissue-dwelling parasites, Angiostrongylus cantonensis, Gnathostoma spinigerum, and Cysticercus cellulosae. We also developed a quick paraffin removal protocol. RESULTS: Both human and animal filariae in formalin-fixed paraffin-embedded sections (FFPES) were diagnosed and identified rapidly, whereas other parasites were negative. There was no difference in the melting temperature of products amplified from filarial DNA obtained from unstained FFPES and Hematoxylin & Eosin-stained sections. Therefore, the DNA extraction protocols developed in this study could be used for real-time PCR with HRM. CONCLUSIONS: We report the successful application of a HRM-PCR assay to differentiate four filarial parasites in FFPES, thus providing the pathologist with an effective alternative diagnostic procedure. Furthermore, the quick paraffin removal protocol developed could shorten the duration and number of steps required for paraffin removal using a standard protocol.

Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance.

Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom(®) SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms.

[Human dirofilariasis: clinical and diagnostic signs and diagnostic methods].

The clinical and diagnostic signs caused by the tissue location and migration of adult Dirofilaria in the human body determine the use of different methods for the diagnosis of dirofilariasis. During their investigations, the authors modified polymerase chain reaction (PCR): they chose and synthesized primers and selected amplification regimens for them and obtained agarose gel bands that corresponded to PCR fragment length nucleotide sequences that were equal to 245 bp for D. (N.) repens and 656 bp for D. immitis. There was 100% agreement in the results of PCR and microscopic examination of sera from 32 dogs and 1 female patient with low parasitemia and in the blood nucleotide sequence characteristic of D. repens.

Expression of translationally controlled tumor protein (TCTP) gene of Dirofilaria immitis guided by transcriptomic screening.

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.

Canine heartworm disease in Porto Velho: first record, distribution map and occurrence of positive mosquitoes / Dirofilariose canina em Porto Velho: primeiro registro, mapa de distribuicao e ocorrencia de mosquitos positivos

The aim of this study was to make the first report on canine heartworm disease in the state of Rondônia and confirm its transmission in this state. Blood samples were randomly collected from 727 dogs in the city of Porto Velho. The samples were analyzed to search for microfilariae and circulating antigens, using three different techniques: optical microscopy on thick blood smears stained with Giemsa; immunochromatography; and PCR. Mosquitoes were collected inside and outside the homes of all the cases of positive dogs and were tested using PCR to search for DNA of Dirofilaria immitis. Ninety-three blood samples out of 727 (12.8%) were positive according to the immunoassay technique and none according to the thick smear method. Among the 93 positive dogs, 89 (95.7%) were born in Porto Velho. No difference in the frequency of infection was observed between dogs raised indoors and in the yard. PCR on the mosquitoes resulted in only one positive pool. This result shows that the transmission of canine heartworm disease is occurring in the city of Porto Velho and that there is moderate prevalence among the dogs. The techniques of immunochromatography and PCR were more effective for detecting canine heartworm than thick blood smears. The confirmation of canine heartworm disease transmission in Porto Velho places this disease in the ranking for differential diagnosis of pulmonary nodules in humans in Rondônia.

O objetivo deste estudo foi de registrar pela primeira vez a dirofilariose canina no estado de Rondônia e confirmar sua transmissão neste estado. Amostras de sangue de 727 cães foram colhidas aleatoriamente na cidade de Porto Velho. As amostras foram analisadas em busca de microfilárias e antígenos circulantes usando três técnicas diferentes: microscopia ótica de gota espessa corada com Giemsa e imunocromatografia de fluxo lateral e PCR. Mosquitos foram colhidos no domicilio e peridomicílio de todos os casos de cães positivos, estes mosquitos foram testados pela PCR na detecção de DNA de Dirofilaria immitis. Noventa e três das 727 amostras de sangue foram positivas na técnica de imunoensaio (12,8%). Nenhuma amostra foi positiva na gota espessa. Entre os 93 cães positivos, 89 (95,7%) foram nascidos em Porto Velho. Nenhuma diferença na frequencia de infecção foi observada entre cães criados dentro da casa ou no quintal. O PCR dos mosquitos resultou em apenas um pool positivo. Este resultado mostra que a transmissão de dirofilariose canina está ocorrendo na cidade de Porto Velho e a frequência que ocorre nos cães é considerada moderada. As técnicas de imunocromatografia e PCR são mais eficazes na detecção de dirofilariose comparadas a gota espessa. A confirmação de transmissão de dirofilariose canina em Porto Velho, coloca esta doença no ranking de diagnóstico diferencial de nódulos pulmonares em seres humanos em Rondônia.

Dirofilariosis in the Americas: a more virulent Dirofilaria immitis?

Dirofilarioses are widespread diseases caused by filarioid nematodes (superfamily Filarioidea) of the genus Dirofilaria, which are transmitted by a plethora of mosquito species. The principal agent of canine dirofilariosis in the Americas is Dirofilaria immitis, which may also occasionally infest humans, resulting in pulmonary nodules that may be confounded with malignant lung tumours. Because human cases of dirofilariosis by D. immitis are relatively frequent in the Americas and rare in Europe and other eastern countries, where Dirofilaria repens is the main causative agent, the existence of a more virulent strain of D. immitis in the Americas has been speculated. Recently, a case of human ocular infestation by Dirofilaria sp. was diagnosed in Pará State, northern Brazil, where canine heartworm dirofilariosis is endemic. The nematode was shown to be morphologically and phylogenetically related to D. immitis but it was genetically distinct from reference sequences, including those of D. immitis infesting dogs in the same geographical area. This finding raised questions regarding the aetiology of human dirofilariosis in the Americas, since information on the genetic makeup of filarioids infesting dogs and humans is meagre. Further studies would be needed to better characterize filarioids infesting dogs, wild animals, and humans in the Americas and to assess the existence of a more virulent D. immitis strain in this continent. Finally, the competence of different culicid species/strains from Europe and the Americas as vectors of Dirofilaria species should be investigated. Such studies would help us to understand possible variations in transmission patterns and even to predict possible scenarios that may emerge in the future, with the introduction of non-endemic Dirofilaria species/strains in free areas through importation of infested animals, vectors, or both.

First report of Dirofilaria immitis in the Republic of Cape Verde.

In Maio Island, Republic of Cape Verde, a seven-year old mongrel female dog exhibiting severe generalized adenomegaly and a poor body condition was examined during an animal welfare campaign. A blood smear was drawn from peripheral blood collection and several organisms consistent with Dirofilaria immitis microfilariae were identified. Both the antigen test conducted from plasma and the RT-PCR test performed from the blood smear sample were positive for D. immitis. This is, to the best of our knowledge, the first report of D. immitis in Cape Verde. The fact that the dog was autochthonous and had never left the island strongly suggests there might be other animals infected with the parasite. Our finding confirms the existence of the parasite in the canine population and necessarily implies the presence of a competent vector. As a serious cardiopulmonary disease and with the risk of the pathogen spreading rapidly, broader epidemiological studies need to be conducted to determine D. immitis prevalence in the canine population of Maio Island.

Canine Dirofilaria infections in two uninvestigated areas of Serbia: epidemiological and genetic aspects.

In 2009 canine filarial infections were investigated in two northern areas of Serbia (Pancevo and Veliko Gradiste), applying morphometry, biochemical staining, and immunological kit to detect Dirofilaria immitis antigens, and two home-made ELISAs to detect antibodies to D. repens and D. immitis somatic/metabolic polyproteins. Moreover, molecular tools were applied to analyze the phylogenetic relationships of the isolates. The microfilariae detected in 21/122 dogs (17.2%) were identified as D. repens (n=21) and D. immitis (n=2). D. immitis antigens were found in another 13 animals with occult infection. All of the 15 heartworm-positive dogs also had antibodies to this parasite, which were detected in another 13 subjects, indicating an overall D. immitis seroprevalence rate of 22.9%. Serology for D. repens revealed evidence of antibodies in 42.6% of the dogs, but was negative for 4 microfilaremic dogs. As for the two different areas, the prevalence of microfilariae and/or D. immitis antigens, mainly due to D. repens microfilaremic animals, was not significantly higher in Veliko Gradiste (33.3%) than in Pancevo (22%). However, serology showed a different epidemiological picture. Heartworm infection occurred more often in both areas, and antibodies to dirofilarial nematodes were detected in 72.9% of dogs living in Pancevo, a rate higher than in those living in Veliko Gradiste (57.1%). No risk factors for infection were found, confirming the uselessness of prophylactic drugs against D. repens, and suggesting the presence in these areas of sunrise- or sunset-biting mosquitoes as important vectors. The results indicate the need for both appropriate entomological studies and further research on the intra-species variability shown by D. repens.

Dirofilaria immitis: identification of a novel ligand-gated ion channel-related polypeptide.

We have isolated a cDNA from Dirofilaria immitis that encodes a predicted ion channel subunit, Di-LGR-1. Secondary structure predictions and database searches reveal that Di-LGR-1 is distantly related to ligand-gated anion channels, such as the GABA(A) receptors, though there are marked differences in the sequences of the putative channel forming regions. Di-LGR-1 has 52% sequence identity to the Caenorhabditis elegans predicted polypeptide, T27A1.4: neighbour-joining trees show that these two polypeptides are the most divergent members of the nematode ligand-gated anion channel family. No close homologues are present in vertebrates, suggesting that their function may be specific to nematodes. RNAi experiments using a fragment of T27A1.4 with C. elegans failed to reveal any obvious phenotype, so the function of these channels remains unknown.

Dirofilaria immitis infection of a snow leopard (Uncia uncia) in a Japanese zoo with mitochondrial DNA analysis.

Three dog heartworms (Dirofilaria immitis) were detected in the lumen of the right cardiac ventriculus and of the pulmonary artery of a captive female snow leopard (Uncia uncia) that died of pancreatic carcinoma at a zoo in Japan. Neither clinical respiratory nor circulatory symptoms caused by the heartworm infection were observed. The filarial worms were identified as D. immitis from the morphologic characteristics of the esophagus, the presence of faint longitudinal ridges on the cuticular surface, the situation of vulva posterior to the esophagus, and the measurements of the body. The heartworms from the snow leopard were identical to that of D. immitis from dogs in the sequence of the cytochrome oxidase I region in the mitochondrial DNA. This host record is the first of D. immitis in U. uncia.

Case report: Unusual location of Dirofilaria immitis in a 28-year-old man necessitates orchiectomy.

The fourth case of zoonotic Dirofilaria immitis infection in a California resident is reported. This case was unique in that it involved the spermatic cord, produced significant symptoms, and upon physical examination was suspected of being an incarcerated inguinal hernia. At surgery frozen sections were inconclusive regarding neoplasia and cord involvement precluded removal of the mass without orchiectomy, which was done. Histopathology revealed a well-preserved immature male D. immitis, the canine heartworm, in a branch of the spermatic artery. The morphological identification of the worm was confirmed by polymerase chain reaction (PCR) studies. This case illustrates that D. immitis infections can complicate differential diagnosis of extrapulmonary lesions and suggests that the public health significance of this vector-borne filarial worm needs to be evaluated through appropriate epidemiological studies in enzootic areas.

Dirofilaria immitis: molecular cloning and expression of a cDNA encoding a selenium-independent secreted glutathione peroxidase.

A cDNA clone, Di29, encoding a homolog of glutathione peroxidase, was isolated from a Dirofilaria immitis adult female cDNA expression library by a combination of polymerase chain reaction amplification with primers designed from the Brugia pahangi glutathione peroxidase gene sequence and hybridization screening of D. immitis cDNA libraries. The Di29 nucleotide and deduced amino acid sequences were very similar to those described for lymphatic filariae and predicted a secreted form of glutathione peroxidase with a cysteine residue substituted for selenocysteine in the active site. The cDNA clone was expressed in Escherichia coli and Spodoptera frugiperda Sf9 insect cells, and the resulting recombinant proteins were purified for antibody production and assessment of enzymatic properties, respectively. An antiserum generated against the E. coli-expressed protein detected a protein of 29 kDa in D. immitis via immunoblotting. This protein is expressed in adult worms (both sexes) and fourth stage larvae generated via 6 days of in vitro culture, but was undetectable in microfilariae, and third stage larvae obtained either directly from mosquitoes or following 2 days of culture. The Di29-encoded recombinant protein was secreted from Sf9 insect cells and displayed low-level glutathione peroxidase activity against a range of hydroperoxide substrates, including hydrogen peroxide.

Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis.

A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.