Serological detection of antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen in dogs from Costa Rica.

In a study in Costa Rica 314 serum samples from dogs throughout all seven provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis, and of circulating antigen of Dirofilaria immitis. A total of 6.4% (20/314) and 38.2% (120/314) were positive for Anaplasma spp. (An) and E. canis (Ec) antibodies. Overall, 8.0% (25/314) were positive for D. immitis (Di) antigen. One single dog reacted positive with B. burgdorferi s.l. (Bb) antigen (0.3%, 1/314). E. canis positive dogs were detected in all provinces (highest percentages in Guanacaste, Puntarenas [both significantly different compared to the overall] and Limón). Guanacaste and Puntarenas also showed the highest prevalences of Anaplasma spp. (both significantly different compared to the overall). The highest prevalence of D. immitis was detected in Puntarenas (significantly different compared to the overall). Double pathogen exposure (Ec plus An; Ec plus Di; Ec plus Bb) were recorded in 8.9% (28/314). Two dogs showed a triple pathogen exposure (0.6%, 2/314; An, Ec and Di). There was a significant difference between male (11.5%, 18/156) and female (4.4%, 7/158) animals for D. immitis positive results. There was also a significant difference between breed and no breed dogs regarding the characteristics of a general positive test, as well as seropositivity to the single pathogens of Anaplasma spp., E. canis and D. immitis. Finally there was a significant difference in the presence of clinical signs again regarding the characteristics of a general positive test, as well as seropositivity to Anaplasma spp., E. canis and D. immitis. Practitioners in Costa Rica should be aware of the canine vector-borne diseases mentioned as dogs are at risk of becoming infected. Concerning the positive B. burgdorferi s.l. dog, an autochthonous occurrence cannot be confirmed due to a history of adoption and an unusual tattoo number. Veterinary advice to protect dogs and limit transmission of vector-borne pathogens, also to humans, by using prophylactic measures is strongly recommended.

Molecular characterization of human Dirofilaria isolates from Kerala.

Background & objectives: Human dirofilariosis is a well-recognized zoonosis caused by several species of the genus Dirofilaria. The disease is prevalent among canines and human beings in Kerala. The objective of the present study was to confirm the human Dirofilaria isolates by molecular characterization. Methods: The worms or segments obtained from human sources were subjected to diagnostic polymerase chain reaction (PCR) targeting the cytochrome oxidase subunit 1 (COI) and 5S ribosomal RNA (rRNA) genes of Dirofilaria repens. The amplicons were sequenced and analyzed. Results: The filariid nematodes recovered from ocular as well as subcutaneous tissue of human origin were identified as D. repens based on PCR targeting COI as well as 5S rRNA genes. The phylogenetic analysis of the COI gene nucleotide sequence obtained in the present study showed that D. repens shared the closest evolutionary relationship with D. honkongensis. Interpretation & conclusions: Molecular identification of D. repens isolated from human source assumes significance from the point of zoonotic threat of this mosquito-borne nematode. Phylogenetic analysis revealed a close evolutionary relationship with Asian isolate of D. honkongensis. Timely detection and treatment of infection in dogs, together with mosquito control, should be an integral part of the control strategy of this disease.

[(HUMAN DIROFILARIASIS: CLINICAL DIAGNOSTIC FEATURES ASSOCIATED WITH DIFFERENT DEVELOPMENTAL STAGES OF THE PATHOGEN)].

To seek medical advice is due to the time when a person has become infected during the infection transmission season (July-August) and the duration of development of the pathogen Dirofilaria (N.) repens in his/her body Clinical manifes- tations occurred in 61% of the patients 6-10 months after infection, as confirmed by the maximum body sizes (125-160 mm) of removed females which have reached puberty. PCR-based diagnosis in conjunction with microscopic studies improves the efficiency of identifying the patients and the species of the pathogens D.repens and D.immitis. The use of these methods for the first time in 2016 could confirm D.immitis infestation in a 14-month-old infant living in the Solnechnogorsk District, Moscow Region.

Concurrent transcriptional profiling of Dirofilaria immitis and its Wolbachia endosymbiont throughout the nematode life cycle reveals coordinated gene expression.

BACKGROUND: Dirofilaria immitis, or canine heartworm, is a filarial nematode parasite that infects dogs and other mammals worldwide. Current disease control relies on regular administration of anthelmintic preventives, however, relatively poor compliance and evidence of developing drug resistance could warrant alternative measures against D. immitis and related human filarial infections be taken. As with many other filarial nematodes, D. immitis contains Wolbachia, an obligate bacterial endosymbiont thought to be involved in providing certain critical metabolites to the nematode. Correlations between nematode and Wolbachia transcriptomes during development have not been examined. Therefore, we detailed the developmental transcriptome of both D. immitis and its Wolbachia (wDi) in order to gain a better understanding of parasite-endosymbiont interactions throughout the nematode life cycle. RESULTS: Over 215 million single-end 50 bp reads were generated from total RNA from D. immitis adult males and females, microfilariae (mf) and third and fourth-stage larvae (L3 and L4). We critically evaluated the transcriptomes of the various life cycle stages to reveal sex-biased transcriptional patterns, as well as transcriptional differences between larval stages that may be involved in larval maturation. Hierarchical clustering revealed both D. immitis and wDi transcriptional activity in the L3 stage is clearly distinct from other life cycle stages. Interestingly, a large proportion of both D. immitis and wDi genes display microfilarial-biased transcriptional patterns. Concurrent transcriptome sequencing identified potential molecular interactions between parasite and endosymbiont that are more prominent during certain life cycle stages. In support of metabolite provisioning between filarial nematodes and Wolbachia, the synthesis of the critical metabolite, heme, by wDi appears to be synchronized in a stage-specific manner (mf-specific) with the production of heme-binding proteins in D. immitis. CONCLUSIONS: Our integrated transcriptomic study has highlighted interesting correlations between Wolbachia and D. immitis transcription throughout the life cycle and provided a resource that may be used for the development of novel intervention strategies, not only for the treatment and prevention of D. immitis infections, but of other closely related human parasites as well.

Detection of heartworm infection in dogs via PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples.

OBJECTIVE: To develop and evaluate a rapid and accurate assay involving PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples for the detection of Dirofilaria immitis infection in dogs. SAMPLE: Whole blood nucleic acid extracts from 29 dogs experimentally infected with D immitis (and in which circulating D immitis antigen was detected) and 10 uninfected dogs. PROCEDURES: 16 of the 29 whole blood samples from infected dogs were examined at the time of collection for circulating microfilaria. Nucleic acids were extracted from all whole blood specimens and underwent PCR amplification with 12 PCR primer pairs designed to detect a wide range of pathogens (including the Wolbachia endosymbiont of D immitis) and electrospray ionization mass spectrometry. RESULTS: On the basis of assay results, heartworm infection was detected in 13 of 13 antigen-positive dogs of unknown microfilaria status, 11 of 11 antigen-positive dogs with circulating microfilaria, 0 of 3 antigen-positive dogs tested at 3 months after larval infection, 0 of 2 antigen-positive dogs with occult infections, and 0 of 10 uninfected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: With the assay under investigation, it was possible to identify D immitis infection in dogs with circulating microfilaria via detection of the obligate Wolbachia endosymbiont of D immitis. It was not possible to identify dogs with occult infections, which suggested that circulating microfilaria must be present to detect infection with this assay, although further studies would be required to verify that finding.

Serological update and molecular characterization of Dirofilaria immitis in dogs, South Korea.

Eighty-one dogs in the Chungnam province were tested for heartworm (Dirofilaria immitis) infection by ELISA (SNAP test, IDEXX Laboratories, Maine, USA). Seventeen (20.9%) of the 81 samples were found to be positive and further analyzed by 16S rRNA sequencing. In this study, all dogs tested lived outdoors. Using the chi(2) test and Fisher's exact test, no significant differences in the prevalence of dirofilariasis were observed among different gender and age groups, although the prevalence of this disease among dogs 2-4yrs of age remains highest. Sequence analysis revealed that the species prevalent in Chungnam province were genetically distinct from the type strain of D. immitis based on the nucleotide deletion found at position nt 276 (cytosine) and nucleotide substitution at position 428 (G to A) of the partial 16S rRNA sequence of the type strain. Furthermore, phylogenetic analysis suggests at least 2 groups of D. immitis circulating in the Chungnam area between the year 2007 and 2008.

Pulmonary dirofilariasis with serologic study on familial infection with Dirofilaria immitis.

An asymptomatic patient with a pulmonary coin lesion surgically diagnosed with pulmonary dirofilariasis caused by infection with Dirofilaria immitis (D. immitis) is presented. The preoperative stored serum of the patient was positive for D. immitis by enzyme-linked immunosorbent assay (ELISA). A family study showed that three of five family members were seropositive for D. immitis. These results suggest that family members of a patient with pulmonary dirofilariasis were frequently exposed to D. immitis and serodiagnostic methods are useful for detecting subclinical infection of D. immitis.

Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria immitis, in its mosquito intermediate host.

The mosquito-borne filarial worm, Dirofilaria immitis, causes heartworm disease in dogs. Detection of this parasite in its mosquito intermediate host currently involves dissection and microscopic examination for larval stages. Although this method is used commonly as a screening tool for epidemiological surveys, it lacks both sensitivity and specificity. In this study, a more efficient PCR- and probe-based diagnostic assay was developed. The target selected for this assay is a segment of the 16 S rRNA gene. The assay specifically detects as little as 10 pg of D. immitis genomic DNA, equivalent to DNA derived from one third stage larva (L(3)), but does not detect 100 ng (10 000-fold excess) of the purified DNA from several other filarial nematodes, including Dirofilaria striata, Dirofilaria tenuis, Dipetalonema reconditum, Wuchereria bancroftii, Brugia pahangi, B. malayi, Onchocerca volvulus or Loa loa. This assay also detects one L(3)of D. immitis, the minimal biological unit of infection, in a pool of 200 mosquito heads. This assay can serve as a highly specific and sensitive tool for efficiently screening the large numbers of mosquitoes to determine, with statistical validity the seasonal transmission pattern of D. immitis in a locality prior to designing a rational preventive medication program for that parasite.

Identification of Dirofilaria in man by multilocus electrophoretic analysis.

Biochemical keys based on the electrophoretic patterns of nine enzymatic loci diagnostic between Dirofilaria repens and D. immitis were used to examine an immature female of Dirofilaria removed from a 32-year-old woman resident in Caserta, Italy. The worm, tentatively assigned on a morphological basis to D. conjunctivae-D. repens, showed at all the loci examined the same electrophoretic pattern as an Italian dog strain of D. repens. This genetic evidence is in substantial agreement with the hypothesis, already supported by morphological and epidemiological data, that D. conjunctivae (Addario, 1885) Desportes, 1939-1940 and D. repens Railliet and Henry, 1911 should be considered as synonyms. According to the law of priority D. conjunctivae should be the valid name for the species; however, it is proposed to preserve the name repens, well-established for the dog subcutaneous filaria. Multilocus electrophoretic analysis appears to be a valuable tool for the identification of aetiological agents of human zoonotic filariae, particularly in areas where more than one species of the subgenus Nochtiella are present.