Dirofilaria immitis and Onchocercidae spp. in wild felids from Brazil.

Among the species described within the Onchocercidae family, Dirofilaria immitis is regarded as the most common worldwide, causing severe and often fatal conditions in dogs, cats, and occasionally humans. Dirofilaria spp. are vectored by mosquitoes, simulids, and culicoids, with their epidemiology dependent on the geographical distribution of competent vectors. Eight species of Dirofilaria have been reported so far in Brazil, of which six parasitize non-human primates, deer, procyonids, and marsupials. Here, we investigated the occurrence of Onchocercidae in wild felids (i.e., Panthera onca, Puma concolor, Herpailurus yagouaroundi, Leopardus geoffroyi, Leopardus guttulus, Leopardus pardalis, Leopardus wiedii, Leopardus munoai) from different locations in Brazil. Overall, 82 samples (n = 63 blood; n = 19 tissues) were molecularly screened for cytochrome c oxidase subunit-1 (cox1) gene. Four (i.e., 4.8%) wild felid samples were positive, and at BLAST analysis, the obtained sequences showed varying percentage of nucleotide identity with the genera Brugia (i.e., 87-88%), Setaria (i.e., 89%), and D. immitis (i.e., 94.4%). Phylogenetic analyses clustered sequences obtained into three distinct clades, one with D. immitis and the remaining two with other Onchocercidae spp. Data herein obtained highlight the need for a more comprehensive understanding of the diversity and biology of Onchocercidae in South America in order to assess the potential impact that these species may have for domestic and wild animals, as well as humans.

Filarial nematode (order: Spirurida) surveillance in urban habitats, in the city of Pécs (Hungary).

As part of the seasonal mosquito control activities in the city of Pécs (Baranya County, Hungary), a total of 1123 adult female mosquitoes belonging to 18 species (including the invasive species Aedes koreicus) were collected from human-inhabited areas, using CO2-baited traps, during two consecutive years. To survey the presence and prevalence of filarial parasites in these mosquitoes, we performed a molecular survey for filarial DNA, attempted by PCR using generic primers (COI), and followed by DNA sequencing. Filaroid nematode DNA was detected in 4% of investigated mosquito pools. Out of 410 pools, 9 pools of mosquitoes were positive for Dirofilaria repens (Aedes vexans, Aedes koreicus, Coquillettidia richiardii), and/or Dirofilaria immitis (Ae. vexans, Cq. richiardii), and further 8 pools were positive for Setaria tundra (Ae. vexans, Cq. richiardii). Our study provides novel insight for prevalence of filaroid nematodes in mosquitoes occurring in close proximity to humans, thereby highlights the possible human and veterinary health importance of these mosquito species, including the recently introduced invasive mosquito Ae. koreicus.

Molecular detection of Dirofilaria immitis in dogs and mosquitoes in Tabasco, Mexico.

BACKGROUND & OBJECTIVES: Dirofilaria immitis is a filarial nematode that causes heartworm disease in domestic as well as wild canines and felines; and cutaneous or pulmonary infections in humans. The purpose of the study was to estimate the prevalence of D. immitis in domestic dogs in Tabasco, Mexico and to assay mosquitoes temporally and spatially associated with dogs for evidence of infection. METHODS: Blood was collected from 1050 dogs in 1039 houses during a random household survey performed in 2016 and 2017. Genomic DNA was extracted and assayed by polymerase chain reaction (PCR) using pan-filarial primers and various species-specific primers. Dog owners were interviewed using a structured questionnaire designed to collect information on factors that may impact the occurrence of filarial infection. The association between canine dirofilariasis prevalence and factors likely to impact infection was determined by univariate logistic regression analysis, followed by multivariate binomial logistic regression analysis. Indoor and outdoor resting mosquitoes were collected from houses by manual aspiration. Mosquitoes were identified according to species, homogenized and tested by PCR for filarial nematodes. RESULTS: A total of 84 (8%) dogs were positive for D. immitis DNA, while 3 (0.3%) dogs contained Acanthocheilonema reconditum DNA. Several factors were significantly associated with D. immitis infection. For example, dogs that lived <100 m from a large source of open standing water were significantly more likely (p = 0.002) to become infected with D. immitis than other dogs. Additionally, dogs with infrequent or no anthelmintic treatment were significantly more likely (p = 0.0) to become infected than dogs that were regularly treated. The entomologic investigation yielded 2618 female mosquitoes from 14 species. Four pools of Culex quinquefasciatus were positive for D. immitis DNA and the minimum infection rate, calculated as the number of positive pools per 1000 mosquitoes tested, was 2.9. INTERPRETATION & CONCLUSION: The study identified several factors positively associated with an increased risk of D. immitis infection in domestic dogs in Tabasco and provides evidence that Cx. quinquefasciatus is potentially an important vector in this region. This information can be used by local veterinarians and dog owners to reduce the burden of D. immitis on canine health.

First molecular identification of Dirofilaria spp. (Onchocercidae) in mosquitoes from Serbia.

Dirofilariosis is a common and widespread veterinary health issue in several European countries with notable zoonotic potential. The causative agents are Dirofilaria immitis and Dirofilaria repens nematoda species which are transmitted by different mosquito vectors. Similar to other mosquito-borne infections, the knowledge about mosquito species involved in disease transmission is crucial for the complex understanding of local transmission cycles. Since there is no available data on mosquito species, potentially involved in disease transmission from Serbia, 6369 female mosquito individuals were retrospectively tested for Dirofilaria nematodes, collected from 13 localities in Vojvodina province, Serbia, in 2013. Altogether, 8.33 % of tested pools showed positivity, composed of five mosquito species, mainly, Culex pipiens and Aedes vexans. D. immitis and D. repens were both detected from multiple localities, during the whole period of mosquito breeding season, which provides the first data on local transmission characteristics regarding mosquitoes from the Balkans.

Indigenous Dirofilaria immitis in Bangladesh.

Dirofilaria immitis is a parasite of domestic and wild canids and felids in tropical, subtropical and temperate regions throughout the world. The canine heartworm (D. immitis) is the causative agent of canine and feline cardiopulmonary dirofilariasis. This parasite is known to cause a zoonotic disease, namely human pulmonary dirofilariasis. D. immitis is known to be endemic in several South and Southeast Asian countries (e.g. India and Malaysia), but there has previously been no information about the presence of this pathogen in Bangladesh. We present a case of canine dirofilariasis caused by D. immitis in rural southeastern Bangladesh. A male filaroid nematode (95 mm in length and 1.94 mm in width) was identified in the heart of a dog. Species classification was performed by microscopy and molecular tools. Sequence analysis revealed a 100 % identity within the mitochondrial cytochrome c oxidase I (CO1) gene to two Chinese and one Australian D. immitis samples. Usually, dogs stay outside overnight with a high risk to get infected with D. immitis via nocturnal mosquito vectors, which may lead to high prevalences of this pathogen in the canine population and thus increase the risk of human infections with this neglected parasitic disease.

Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

[Helminth fauna of the wolf (Canis lupus Linnaeus, 1758) in Belorussian Polesie].

The results of our own long-term helminthological investigations (1980-2010) of wolves in Belorussian Polesie are presented. 87.2% of wolves were infected by helminths according to helminthological autopsy, and 98.5% by coproscopical data. 26 species of helminths were found: 5 trematode species, 9 cestode species, 11 nematode species and 1 acanthocephalan species. The wolf is a new host for nematode Dirofilaria immitis (Leidy, 1856) in Belarus.

Detection of heartworm antigen without cross-reactivity to helminths and protozoa following heat treatment of canine serum.

BACKGROUND: Detection of Dirofilaria immitis, or heartworm, through antigen in sera is the primary means of diagnosing infections in dogs. In recent years, the practice of heat-treating serum prior to antigen testing has demonstrated improved detection of heartworm infection. While the practice of heat-treating serum has resulted in earlier detection and improved sensitivity for heartworm infections, it has been suggested that heat treatment may cause cross reactivity with A. reconditum and intestinal helminth infections of dogs. No studies have assessed the potential cross-reactivity of these parasites with heartworm tests before and after heat treatment using blood products and an appropriate gold standard reference. METHODS: Canine sera (n=163) was used to evaluate a heartworm antigen-ELISA (DiroCHEK®) and potential cross-reactivity with common parasitic infections. The heartworm status and additional parasite infections were confirmed by necropsy and adult helminth species verified morphologically or by PCR, and feces evaluated by centrifugal fecal flotation. RESULTS: Intestinal parasites were confirmed in 140 of the dogs by necropsy, and 130 by fecal flotation. Acanthocheilonema reconditum microfilariae were confirmed in 22 dogs. Prevalence of heartworm infection confirmed by necropsy was 35.6% (58/163). In the 105 dogs without heartworms, specificity remained unchanged at 100% both before and after heat treatment despite confirmed infections with A. reconditum, Ancylostoma caninum, Ancylostoma brasiliense, Trichuris vulpis, Toxocara canis, Dipylidium caninum, Spirometra mansonoides, Macracanthorynchus ingens, Cystoisospora sp., Giardia sp., and Sarcocystis sp. CONCLUSIONS: These findings suggest that the use of heat treatment improves sensitivity of heartworm tests and is unlikely to cause false positive antigen results due to Acanthocheilonema reconditum, intestinal helminths, and protozoal parasites in dogs.

Ocular Dirofilariasis Case in Thailand Confirmed by Molecular Analysis to Be Caused by Dirofilaria immitis.

A rare ocular dirofilariasis case along with the clinical characteristics, treatment, and outcome is reported. A whitish roundworm (10.6 cm long and 0.5 mm width) emerged from the pterygium, a triangular tissue growth on the cornea of the eye, of a male patient. The worm had a rounded anterior part, mouth without lips, smooth cuticular surface, and short rounded posterior tail with spicules: these features suggested that it was a male Dirofilaria sp. Molecular identification confirmed that the worm belonged to Dirofilaria immitis. This is the first molecular confirmation that D. immitis is a causative agent of ocular dirofilariasis in Thailand: dirofilariasis is a newly emerging zoonotic disease. Physicians should be alert to zoonotic filarial worms and knowledgeable about treatment of this disease.

Molecular survey of Dirofilaria species in stray dogs, red foxes and golden jackals from Vojvodina, Serbia.

Cardiopulmonary dirofilariosis in dogs and other carnivores is caused by Dirofilaria immitis, while Dirofilaria repens usually causes a subcutaneous infection. The importance of red foxes and golden jackals in the epidemiology of dirofilariosis remains unknown. Thus, the aim of this study was to conduct a cross-sectional molecular survey of Dirofilaria species in stray dogs, red foxes and golden jackals from the endemic region of Vojvodina, Serbia, in order to determine and update data on their prevalence and provide insight into the epidemiological importance of wild canids. A total of 59 blood samples from stray dogs, 94 from red foxes and 32 from golden jackals were collected and screened by real-time PCR targeting a 115-bp fragment of the mitochondrial 12S gene of filarioids and by conventional PCR assay targeting a 484-524-bp fragment of 5.8S-ITS2-28S locus of filarioids. The cross-sectional molecular survey detected the filarioid mitochondrial 12S gene fragment in stray dogs (27.1 %), red foxes (8.5 %) and golden jackals (6.3 %) in the same endemic region of Vojvodina, Serbia. Only D. immitis was detected in stray dogs, while both D. immitis and D. repens were detected in populations of red foxes and golden jackals. These results outline a possible interaction of D. immitis infection between the dog population and the wild canid populations, while D. repens was found to circulate mostly in golden jackals and red foxes populations.

The application of proteomic methods (MALDI-toff MS) for studying protein profiles of some nematodes (dirofilaria and ascaris) for differentiating species.

INTRODUCTION: Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-toff MS) is a reliable method for diagnosing a number of bacterial and fungal infections. It is also effective as a method of rapid diagnosis of several parasitic agents. We used MALDI-toff MS to study the protein profiles of four nematodes: Dirofilaria repens, Dirofilaria. immitis, Ascaris suum and Ascaris lumbricoides. METHODS: We studied the protein profiles of dirofilaria (five of each species: D. repens and D. immitis) and ascaris (five of each species: A. suum and A. lumbricoides), using a proteomic analysis based on MALDI-toff MS. RESULTS: Analysis of protein extracts of dirofilaria and ascaris showed spectra with high-intensity peaks in the range of 2-20 kDa. The quality of the spectra (clear graphical reflection of mass/charge to luminous intensity, consistent in repeated analyzes) and the intensity of the spectral peaks were consistent in all samples of the same species. The spectra profiles of D. repens and D. immitis differed in eight major peaks which makes it possible to differentiate species according to the protein profile. The spectra profiles obtained from A. suum and A. lumbricoides proteins differed slightly in 3 major peaks in both species and were discovered in m/z 13000; 13400 and 14400. The protein peaks in diapason 3000 kD-7300 kD specific for all genus ascaris are constant. CONCLUSIONS: MALDI-toff MS-based proteomic analysis can serve as an effective taxonomic tool for parasitological studies.

PREVALENCE AND MOLECULAR CHARACTERIZATION OF DIROFILARIA IMMITIS (FILARIOIDEA: ONCHOCERCIDAE) IN DOGS FROM ENDEMIC AREAS OF RIO DE JANEIRO STATE, BRAZIL.

Canine dirofilariasis is common in Brazil, but molecular diagnosis is rare even though molecular studies increase our knowledge about molecular epidemiology and circulating genotypes from helminths worldwide. This study aims to estimate the prevalence of infection with a modified Knott's test and to perform molecular characterization of Dirofilaria immitis (Leidy, 1856) Railliet and Henry, 1911, in dogs from endemic areas of Maricá and Niterói municipalities, Rio de Janeiro State, Brazil. Molecular characterization was performed in 33 blood samples from dogs positive for microfilariae and 4 adult worms obtained from 2 other dogs. DNA extraction followed by PCR for mitochondrial target 12S rDNA and cytochrome oxidase subunit 1 (COI) of D. immitis were performed, and the amplified products were sequenced. All sequences were identical for both gene targets and showed 100% identity with D. immitis sequences from different animal species from various countries. The study concluded that this genotype of D. immitis might be dispersed worldwide.

Dirofilaria immitis JYD-34 isolate: whole genome analysis.

BACKGROUND: Macrocyclic lactone (ML) anthelmintics are used for chemoprophylaxis for heartworm infection in dogs and cats. Cases of dogs becoming infected with heartworms, despite apparent compliance to recommended chemoprophylaxis with approved preventives, has led to such cases being considered as suspected lack of efficacy (LOE). Recently, microfilariae collected from a small number of LOE isolates were used as a source of infection of new host dogs and confirmed to have reduced susceptibility to ML in controlled efficacy studies using L3 challenge in dogs. A specific Dirofilaria immitis laboratory isolate named JYD-34 has also been confirmed to have less than 100% susceptibility to ML-based preventives. For preventive claims against heartworm disease, evidence of 100% efficacy is required by FDA-CVM. It was therefore of interest to determine whether JYD-34 has a genetic profile similar to other documented LOE and confirmed reduced susceptibility isolates or has a genetic profile similar to known ML-susceptible isolates. METHODS: In this study, the 90Mbp whole genome of the JYD-34 strain was sequenced. This genome was compared using bioinformatics tools to pooled whole genomes of four well-characterized susceptible D. immitis populations, one susceptible Missouri laboratory isolate, as well as the pooled whole genomes of four LOE D. immitis populations. Fixation indexes (FST), which allow the genetic structure of each population (isolate) to be compared at the level of single nucleotide polymorphisms (SNP) across the genome, have been calculated. Forty-one previously reported SNP, that appeared to differentiate between susceptible and LOE and confirmed reduced susceptibility isolates, were also investigated in the JYD-34 isolate. RESULTS: The FST analysis, and the analysis of the 41 SNP that appeared to differentiate reduced susceptibility from fully susceptible isolates, confirmed that the JYD-34 isolate has a genome similar to previously investigated LOE isolates, and isolates confirmed to have reduced susceptibility, and to be dissimilar to the susceptible isolates. CONCLUSIONS: These results provide additional evidence for the link between genotype and the reduced susceptibility phenotype observed in such isolates as JYD-34. Further work on other isolates showing reduced susceptibility to ML is required to demonstrate the value of genetic analysis in predicting the response to ML chemoprophylaxis. The authors suggest that genetic analysis may be useful in helping to interpret the results of in vivo efficacy testing of ML heartworm preventives against D. immitis isolates.

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.

Discrimination between six species of canine microfilariae by a single polymerase chain reaction.

Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae (Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.

Circulation of Dirofilaria repens and Dirofilaria immitis in Moldova.

BACKGROUND: Over the last two decades, a significant spread of dirofilariasis has been observed in eastern and central Europe. However, data on the circulation of Dirofilaria spp. in Moldova were absent although direct neighbor states reported high incidence rates of human dirofilariasis. METHODS: Daily mean temperature data were used to calculate Dirofilaria spp. development units, which were used to estimate the potential for complete extrinsic development in the mosquitoes (= sum of potential Dirofilaria spp. transmission days). In addition, 4,481 adult female mosquitoes were collected from 25 trapping sites. From 2010 to 2015, sampling was conducted with Centers for Disease Control miniature light traps, indoor resting mosquito collections as well as human landing catches in urban, rural and natural areas. Mosquitoes were analyzed for the presence of D. repens and D. immitis DNA using a duplex real-time PCR assay targeting nucleotide differences within the cytochrome c oxidase subunit 1 (D. repens) and 16S rRNA gene fragment (D. immitis). RESULTS: The average of the yearly sum of potential Dirofilaria spp. transmission days between 2010 and 2015 ranged from 90 to 140 days with an increasing gradient from the North to the South of Moldova. Positive mosquito pools for D. repens were found countrywide at 13 of the 25 trapping sites and in 17 of the 22 screened mosquito taxa (26.51% of all 347 tested pools), while D. immitis was detected only at 4 of the trapping sites (Center and South) in 4 different mosquito species (8.65% of all 347 tested pools). Highest infection rates (EIR) per 100 specimens for both Dirofilaria species were found in An. maculipennis (s.l.) (D. repens: EIR = 4.91; D. immitis: EIR = 2.01), whereas the most frequent mosquito taxon Cx. pipiens (s.l.)/torrentium had significantly lower infections rates (D. repens: EIR = 0.88; D. immitis: EIR = 0.47). CONCLUSIONS: The temperature conditions in Moldova are suitable for transmission of Dirofilaria spp. within the entire country, which is supported by a wide distribution of Dirofilaria spp.-positive mosquitoes with high infection rates. The low number of reported human cases most likely does not reflect the current epidemiological situation of dirofilariasis in Moldova.

Histochemical differentiation of Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit, Leucognost-SP.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.

Dirofilaria immitis infestation in imported police (K-9) dogs in Iraq: clinicopathological and molecular investigations study / Infecção por Dirofilaria immitis em cães policiais importados no Iraque: estudo clínico-patológico e de investigações moleculares

Dirofilaria immitis, the cause of heartworm infestation (HWI) or dirofilariasis, affects members of the Canidae and remains a worldwide clinical problem. In Iraq, dirofilariasis was believed absent until 2009, when the Karbala Governorate was reported as an endemic area for canine dirofilariasis. Consequently, this study intended to investigate the occurrence of Dirofilaria immitis in police dogs in one police academy in Iraq and to study the gross and histopathological changes in 5 dead dogs, as well as to identify the species of the causative parasite using PCR technique. Thirty-nine police dogs, aged between 6 months and 12 years were included in this study. For the microfilariae investigation, 5 ml blood samples were collected from all dogs in EDTA tubes and examined by Knott's method. The systemic necropsy performed in five dead dogs showed severe clinical signs of dirofilariasis and tissue specimens were sent for routine histopathological processing. For the molecular analysis, adult worms of the detected Dirofilaria spp. were used for DNA extraction and amplification of the cox1 gene. Fifteen of 39 (38.46%) dogs were diagnosed with moderate to severe microfilariasis. The dead dogs revealed typical severe clinical signs of dirofilariasis. Moreover, typical gross and histopathological changes were also seen, accompanied by generalized thromboembolic lesions, suggesting the occurrence of the caval syndrome. The PCR investigation confirmed that D. immitis was the species present in Iraq. In conclusion, this study establishes that Iraq is a newly reported endemic area for dirofilariasis. Moreover, the infestation occurring in these cases most probably happened inside Iraq. The authors recommend doing further epidemiological studies concerning the occurrence of D. immitis in local dogs as well as in the imported dogs in all Iraqi governorates to better understand the epidemiological map of this disease and to introduce an active treatment and preventive program. Awareness and education regarding this disease should be provided to the veterinarians, dog guiders and people in direct contact with dogs, as this disease is one of the important zoonotic diseases.(AU)

A Dirofilaria immitis, causadora da infestação pelo verme do coração (IVC) ou dirofilariose afeta os membros da família Canidae e ainda é um problema clínico mundial. Até o ano de 2009, acreditava-se que o Iraque fosse livre da dirofilariose, porém nessa ocasião a governadoria de Kerbala foi relatada como uma área endêmica de dirofilariose. Assim, o presente trabalho foi realizado para investigar a ocorrência da Dirofilaria immitis em cães policiais em uma academia de polícia do Iraque, estudar as alterações macroscópicas e histopatológicas em cinco cães mortos, bem como, identificar as espécies do parasita causador com o emprego da técnida de PCR. Trinta e nove cães policiais com 6 meses a 12 anos de idade foram incluídos no estudo. Amostras de sangue de cinco mililitros foram colhidas por animal, em tubos com EDTA e foram examinadas pelo método de Knott. A necropsia sistêmica foi realizada em cinco cães que haviam apresentado sinais clínicos severos de dirofilariose e espécimens dos seus tecidos foram enviados para o processamento histopatológico de rotina. Para a análise molecular dos vermes adultos de Dirofilaria spp, foi empregada a extração do DNA e a amplificação do gene cox1. Quinze de 39(38,46%) cães foram diagnosticados com uma microfilariase variável de moderada para severa. As alterações macroscópicas e histopatológicas foram acompanhadas por lesões generalizadas tromboembólicas sugestivas da ocorrência da síndrome da veia cava. A investigação de PCR confirmou que a D.immitisera a espécie presente no Iraque. A conclusão do estudo estabeleceu que o Iraque deve passar a ser considerado como uma nova área endêmica da dirofilariose. Além da infestação registrada nos casos descritos é provável que ela também esteja presente em outras regiões do Iraque. Os autores recomendam a realização de estudos epidemiológicos para investigar a ocorrência de D.immitis tanto nos cães locais bem como em cães importados em todas as governadorias do Iraque, para ser construído o mapa epidemiológico da distribuição da doença e implantadas as ações de tratamento e de um progrma preventivo. Ações de educação em saúde sobre a doença deverão ser dirigidas para os veterinários, tratadores de cães e para pessoas em geral que tenham contato com os cães, pois esta doença é uma importante zoonose.(AU)

Molecular detection of Dirofilaria immitis, Hepatozoon canis, Babesia spp., Anaplasma platys and Ehrlichia canis in dogs on Costa Rica.

Although vector-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on these conditions in Costa Rica. In PCRs of blood from dogs in Costa Rica, we did not detect DNAs of Rickettsia (R.) felis and Coxiella (C.) burnetii but we did find evidence of infection with Dirofilaria (D.) immitis (9/40, 22.5%), Hepatozoon (H.) canis (15/40, 37.5%), Babesia spp. (10/40, 25%; 2 with B. gibsoni and 8 with B. vogeli), Anaplasma (A.) platys (3/40, 7.5%) and Ehrlichia (E.) canis (20/40, 50%). Nine dogs (22.5%) were free of any vector-borne pathogens while 14 (35%) were infected with a single pathogen, 11 (27.5%) with two, 4 (10%) with three, 1 (2.5%) with four, and 1 (2.5%) with five pathogens. Dogs in Costa Rica are commonly infected with vector-borne agents.

Intraspecific genetic variation and phylogenetic analysis of Dirofilaria immitis samples from Western China using complete ND1 and 16S rDNA gene sequences.

Dirofilaria immitis (heartworm) is the causative agent of an important zoonotic disease that is spread by mosquitoes. In this study, molecular and phylogenetic characterization of D. immitis were performed based on complete ND1 and 16S rDNA gene sequences, which provided the foundation for more advanced molecular diagnosis, prevention, and control of heartworm diseases. The mutation rate and evolutionary divergence in adult heartworm samples from seven dogs in western China were analyzed to obtain information on genetic diversity and variability. Phylogenetic relationships were inferred using both maximum parsimony (MP) and Bayes methods based on the complete gene sequences. The results suggest that D. immitis formed an independent monophyletic group in which the 16S rDNA gene has mutated more rapidly than has ND1.