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Veterinary Diagnosis of Filarial Infection
Abstract
Filarial worms are a unique group of parasites with importance in both human and veterinary medicine. These parasites are typically long‐lived and difficult to detect, often causing chronic disease states over a period of years and, for these reasons, effective diagnostic testing is crucial for their control. Adult filarial worms tend to occupy inaccessible anatomical sites within the host, but microfilariae disperse widely in the blood or skin to allow uptake and transmission by the hematophagous insects necessary to complete the life cycle, and the detection of this microscopic stage represents a fundamental form of diagnostic testing. Immunodiagnostic and DNA‐based tests have since been developed for several filarial species, as well as methods for visualizing adult parasites in situ . All these techniques carry their own distinct strengths and weaknesses, so reliable diagnosis often requires a strategic combination of tests. Accurate diagnosis is important for potentially fatal infections like canine heartworm and is also essential for identifying emergent zoonoses, like Onchocerca lupi , and potential animal reservoirs, as with Brugia malayi . Accurate parasite detection and identification is useful not only in clinical settings but also greatly assists research efforts. This chapter will review the diagnostic methods available for some of the most common species of filarial nematodes in small animal veterinary medicine.
... Accurate diagnosis of filarial worm infections is challenging, particularly when using microscopic methods as morphological identification can be difficult between closely related species. Moreover, microfilaremia for some filarioids is periodic meaning that microfilaria only appear in the blood at certain times in the day, whilst at other points they may fluctuate down to diagnostically undetectable levels [20][21][22]. Concentration techniques such as the modified Knott's test can increase the sensitivity of diagnosis by microscopy, although the challenges of morphological identification remain [20]. Serodiagnostic methods have also been widely utilised in the context of filarial worm diagnosis, nonetheless such methods may not be able to distinguish between active infections and historical ones, or have poor specificity and an inability to provide an exact, species-level diagnosis [22][23][24]. ...
... Moreover, microfilaremia for some filarioids is periodic meaning that microfilaria only appear in the blood at certain times in the day, whilst at other points they may fluctuate down to diagnostically undetectable levels [20][21][22]. Concentration techniques such as the modified Knott's test can increase the sensitivity of diagnosis by microscopy, although the challenges of morphological identification remain [20]. Serodiagnostic methods have also been widely utilised in the context of filarial worm diagnosis, nonetheless such methods may not be able to distinguish between active infections and historical ones, or have poor specificity and an inability to provide an exact, species-level diagnosis [22][23][24]. ...
Background
Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. ‘hongkongensis’. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies’ (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott’s test (MKT).
Results
We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. ‘hongkongensis’. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections.
Conclusions
Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT’ small and portable MinION™ means that such methods could be deployed for field use.
... In dogs, the pathogenic importance of these infections has been considered minimal, but in some cases, lymphadenomegaly and lymphedema may be observed (158). Other canine species reported in dogs include B. ceylonensis in India and Sri Lanka, B. patei in Kenya (Pate Island), and Brugia species presenting high similarity to B. malayi and B. pahangi in Chad (159,160). ...
... Somatic staining patterns for Brugia spp. infecting dogs have been previously discussed elsewhere (13,14,159). ...
Filarioid nematodes, which are vector-borne parasites of cosmopolitan distribution, of dogs are medically important. They are represented by species in which microfilariae were found to be circulating in the bloodstream (e.g., Dirofilaria sp., Acanthocheilonema sp., and Brugia sp.) or skin-dwelling (e.g., Cercopithifilaria sp. and Onchocerca sp.). Those species whose microfilariae are detected in blood have been extensively studied, especially Dirofilaria immitis, due to their clinical importance. In recent decades, there has been an increased interest by the scientific community in filarioid nematodes whose microfilariae are detected in the skin because of the zoonotic aspect of Onchocerca lupi. In the United States (US), although D. immitis has been considered the main filarioid infecting dogs, the intense animal movement and global canine filarioid diversity may indicate that the likely presence of cutaneous filarioid nematodes is more common than previously expected. Hence, a question remains: Are these canine filarioid nematodes emerging, neglected, or simply underdiagnosed in the US? In this review, we provide an overview of pertinent information that briefly summarizes the biology of the different canine filarioid nematode species, clinical signs associated with infections, and currently available diagnostic tools using molecular and microscopy-based methods and highlight knowledge gaps where research and surveillance efforts remain necessary. The data herein presented serve as an alert to the scientific community about the importance of filarioid nematodes infecting dogs other than D. immitis. Additionally, the zoonotic potential of several filarioid species reinforces the necessity of a proper diagnosis and the need for broader surveillance to understand their diversity and distribution, to highlight the potential introduction of certain species, and mitigate their establishment in the country and new animal and human cases.
Abstract
Background: Diroflaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of
zoonotic canine flariosis.
Methods: We developed a combined multiplex approach for flaria and Wolbachia detection using the 28S-based
pan-flarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples
using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D.
immitis and D. repens. The approach was complemented by a duplex qPCR for the diferential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-flarial cox1 and pan-Wolbachia ftsZ PCRs to identify other
flarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate
the approach. Spearmanʼs correlation was used to assess the association between flarial species and the strain of
Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for
D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens
DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections.
Results: The detection limit for Wolbachia and flariae qPCRs ranged from 5×10−1
to 1.5×10−4 mf/ml of blood.
When tested on clinical samples, 29.2% (49/168) tested positive for flariae or Wolbachia DNA. Filarial species and
Wolbachia genotypes were identifed by the combined multiplex approach from all positive samples. Each species
of Diroflaria was signifcantly associated with a specifc genotype of Wolbachia. Compared to the true positives, the
approach showed excellent agreement (k=0.98–1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the
duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k=0.6) and a weak
(k=0.15) agreements before and after thermal pre-treatment of sera, respectively.
Conclusions: The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult
canine flariosis. The current diagnosis of heartworm disease based on antigen detection should always be confrmed
by qPCR essays. Sera heat pre-treatment is not efective and strongly discouraged.
Keywords: Canine flariosis, Multiplex qPCRs, Diferential diagnosis, Heartworm disease, Diroflaria immitis
Background:
Cercopithifilaria bainae is a filarioid nematode of dogs. Infection with the parasite was not reported in the USA until 2017, when a dog with skin lesions in Florida was diagnosed. Brown dog ticks, Rhipicephalus sanguineus (sensu lato), are the purported tick vectors, and are widespread in the USA. Therefore, C. bainae is likely present in additional states. Here, we tested dogs and ticks in Oklahoma for evidence of C. bainae infection.
Methods:
Dermal punch biopsies were opportunistically collected from municipal shelter and client-owned dogs. Multiple skin samples collected from interscapular and head regions were tested by saline sedimentation to recover live microfilariae for morphometric identification and by PCR to amplify a 330 bp region of the filarioid 12S rRNA gene. Also, ticks observed on surveyed dogs were collected, identified to species level, and tested for filarioid DNA.
Results:
A total of 496 saline sedimentations were performed on 230 shelter and 20 client-owned dogs. Cercopithifilaria bainae infections were identified in 2.6% (6/230) of shelter dogs by morphometry of microfilariae in sedimentations and/or amplification of DNA from skin. DNA sequences amplified from PCR positive skin samples were 99-100% identical to C. bainae reported in Italy. All skin samples from client-owned dogs were negative for filarioid infection by saline sedimentation and PCR. A total of 112 ticks, comprised of four species, were collected. Two of 72 R. sanguineus (s.l.), both engorged females found attached to a C. bainae infected dog, harbored C. bainae DNA (99-100% identity). One attached R. sanguineus (s.l.) male on the same dog harbored filarioid DNA sequence which was difficult to interpret at numerous base-pair locations, but was closest in identity (~80%) to C. bainae.
Conclusions:
The distribution of C. bainae is more widespread than previously known. To our knowledge, we document C. bainae infections in dogs and DNA in brown dog ticks in Oklahoma for the first time. As brown dog ticks are commonly found throughout the USA, veterinarians in this region should consider C. bainae infection as a differential diagnosis in canine patients with dermatitis or polyarthritis.
The suitability of acetic acid as a safer alternative to formalin in the modified Knott test was evaluated for the diagnosis of canine heartworm (Dirofilaria immitis). Microfilaria concentration was measured by both methods and found to agree within reasonable limits (-5.84 % bias; -88.1-76.4 % limits of agreement). The level of agreement was lower when samples were prepared with a 24 h delay, but this was due to the formalin method tending to yield lower counts (-20.1 % bias; -90.5-50.2 % limits of agreement). Clearing the sample of hemoglobin improves readability and is a key feature of the modified Knott test. Hemolysis was significantly lower in the acetic acid method than the formalin method as measured by red blood cell count (6.83 × 106 and 8.79 × 106 cells/ml, respectively; p = 0.015) and absorbance at 415 nm (33.20 and 34.75, respectively; p < 0.001). Visual assessment, however, revealed little practical difference in readability. Finally, lengths of microfilariae were measured to ensure the validity of species identification by the acetic acid method; mean length was significantly shorter after acetic acid treatment (273 μm) than formalin treatment (316 μm; p < 0.001). Length reduction was also observed in acetic acid-treated Acanthocheilonema reconditum (254 μm versus 262 μm; p = 0.035), though these samples were stored prior to testing and are not directly comparable. We conclude that, while the readability of samples is similar for both methods, species differentiation must still be accomplished by other means. For most clinical purposes in determining the presence or absence of blood circulating microfilariae, however, acetic acid appears to be a suitable alternative to formalin in the modified Knott test.
Vector-borne diseases (VBDs) are among the leading causes of morbidity and mortality in humans and animals. The scale of VBDs is increasing worldwide, including in the Mediterranean Basin, a region exposed to climate changes. Indeed, weather conditions may influence the abundance and distribution of vectors. The vector-borne nematode diseases of dogs and cats, such as dirofilariosis, onchocercosis, thelaziosis, Cercopithifilaria, and Acanthocheilonema infections, are some of these vectorized diseases, several of which are zoonoses. They are all caused by parasitic nematodes transmitted by arthropods, including mosquitoes (Dirofilaria spp.), black flies (Onchocerca lupi), drosophilids (Thelazia callipaeda), ticks (Acanthocheilonema dracunculoides and Cercopithifilaria bainae), and fleas and lice (Acanthocheilonema reconditum). The control and prevention of these infections and diseases require a multidisciplinary approach based on strengthening collaboration between the different actors in the fields of health, research, sociology, economics, governments and citizens, to improve human, animal, and ecosystem health. This is the concept of "one health." The review aimed to provide a general update on the spatial and temporal distribution of vector-borne nematodes diseases affecting companion animals and humans, as well as the vectors involved in the Mediterranean area. Simultaneously, certain epidemiological parameters, diagnosis, treatment, and control of these diseases based on the "one health" concept will also be discussed.
The balance of expense and ease of use vs. specificity and sensitivity in diagnostic assays for helminth disease is an important consideration, with expense and ease often winning out in endemic areas where funds and sophisticated equipment may be scarce. In this review, we argue that molecular diagnostics, specifically new assays that have been developed with the aid of next-generation sequence data and robust bioinformatic tools, more than make up for their expense with the benefit of a clear and precise assessment of the situation on the ground. Elimination efforts associated with the London Declaration and the World Health Organization (WHO) 2020 Roadmap have resulted in areas of low disease incidence and reduced infection burdens. An accurate assessment of infection levels is critical for determining where and when the programs can be successfully ended. Thus, more sensitive assays are needed in locations where elimination efforts are approaching a successful conclusion. Although microscopy or more general PCR targets have a role to play, they can mislead and cause study results to be confounded. Hyper-specific qPCR assays enable a more definitive assessment of the situation in the field, as well as of shifting dynamics and emerging diseases.
Canine filariasis is caused by several nematode species, such as Dirofilaria immitis, Dirofilaria repens, Brugia pahangi, Brugia malayi, and Acanthocheilonema reconditum. Zoonotic filariasis is one of the world’s neglected tropical diseases. Since 2000, the World Health Organization (WHO) has promoted a global filarial eradication program to eliminate filariasis by 2020. Apart from vector control strategies, the infection control of reservoir hosts is necessary for more effective filariasis control. In addition, many studies have reported that Wolbachia is necessary for the development, reproduction, and survival of the filarial nematode. Consequently, the use of antibiotics to kill Wolbachia in nematodes has now become an alternative strategy to control filariasis. Previously, a case of subconjunctival dirofilariasis caused by Dirofilaria spp. has been reported in a woman who resides in the center of Bangkok, Thailand. Therefore, our study aimed to principally demonstrate the presence of filarial nematodes and Wolbachia bacteria in blood collected from domestic dogs from the Bangkok Metropolitan Region, Thailand. A total of 57 blood samples from dogs with suspected dirofilariasis who had visited veterinary clinics in Bangkok were collected. The investigations for the presence of microfilaria were carried out by using both microscopic and molecular examinations. PCR was used as the molecular detection method for the filarial nematodes based on the COI and ITS1 regions. The demonstration of Wolbachia was performed using PCR to amplify the FtsZ gene. All positive samples by PCR were then cloned and sequenced. The results showed that the filarial nematodes were detected in 16 samples (28.07%) using microscopic examinations. The molecular detection of filarial species using COI-PCR revealed that 50 samples (87.72%) were positive; these consisted of 33 (57.89%), 13 (22.81%), and 4 (7.02%) samples for D. immitis, B. pahangi, and B. malayi, respectively. While the ITS1-PCR showed that 41 samples (71.93%) were positive—30 samples (52.63%) were identified as containing D. immitis and 11 samples (19.30%) were identified to have B. pahangi, whereas B. malayi was not detected. Forty-seven samples (82.45%) were positive for Wolbachia DNA and the phylogenetic tree of all positive Wolbachia was classified into the supergroup C clade. This study has established fundamental data on filariasis associated with Wolbachia infection in domestic dogs in the Bangkok Metropolitan Region. An extensive survey of dog blood samples would provide valuable epidemiologic data on potential zoonotic filariasis in Thailand. In addition, this information could be used for the future development of more effective prevention and control strategies for canine filariasis in Thailand.
Dirofilaria repens is a nematode affecting domestic and wild canids, transmitted by several species of mosquitoes. It usually causes a non-pathogenic subcutaneous infection in dogs and is the principal agent of human dirofilariosis in the Old World. In the last decades, D. repens has increased in prevalence in areas where it has already been reported and its distribution range has expanded into new areas of Europe, representing a paradigmatic example of an emergent pathogen. Despite its emergence and zoonotic impact, D. repens has received less attention by scientists compared to Dirofilaria immitis. In this review we report the recent advances of D. repens infection in dogs and humans, and transmission by vectors, and discuss possible factors that influence the spread and increase of this zoonotic parasite in Europe. There is evidence that D. repens has spread faster than D. immitis from the endemic areas of southern Europe to northern Europe. Climate change affecting mosquito vectors and the facilitation of pet travel seem to have contributed to this expansion; however, in the authors’ opinion, the major factor is likely the rate of undiagnosed dogs continuing to perpetuate the life-cycle of D. repens. Many infected dogs remain undetected due to the subclinical nature of the disease, the lack of rapid and reliable diagnostic tools and the poor knowledge and still low awareness of D. repens in non-endemic areas. Improved diagnostic tools are warranted to bring D. repens diagnosis to the state of D. immitis diagnosis, as well as improved screening of imported dogs and promotion of preventative measures among veterinarians and dog owners. For vector-borne diseases involving pets, veterinarians play a significant role in prevention and should be more aware of their responsibility in reducing the impact of the zoonotic agents. In addition, they should enhance multisectorial collaboration with medical entomologists and the public health experts, under the concept and the actions of One Health-One Medicine.
Electronic supplementary material
The online version of this article (10.1186/s13071-018-3205-x) contains supplementary material, which is available to authorized users.
Background
Cercopithifilaria bainae is a tick‐vectored filarioid nematode associated with erythematous dermatitis in dogs. It has not been reported previously in the United States.
Hypothesis/Objective
To describe clinical, histological and parasitological diagnosis and treatment of C. bainae in a dog.
Animals
An 11‐month‐old golden retriever/standard poodle mixed breed dog from Florida (USA).
Methods and materials
The dog had no travel history within or outside the United States, was presented with a one month history of annular erythematous plaques on the head and ulcers on the medial canthi. Lesions were unresponsive to antibiotic treatment.
Results
Histopathological evaluation of skin biopsies revealed an eosinophilic to lymphohistiocytic perivascular dermatitis with multiple microgranulomas and rare 5–10 μm diameter microfilariae within microgranulomas. Microfilarial morphology was consistent with C. bainae. PCR and sequencing of 18S rRNA and mitochondrial cytochrome oxidase subunit I genes confirmed the nematodes as C. bainae. The dog was treated with a commercial spot‐on containing imidacloprid and moxidectin, and clinical resolution occurred.
Conclusions and clinical importance
To the best of the authors’ knowledge, this is the first report of C. bainae in a dog in the United States and the first description of dermatological lesions caused primarily by C. bainae.
Background
Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens.
Methodology/Principal findings
Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack.
Conclusions/Significance
Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.
Acanthocheilonema reconditum (A. reconditum) is a nematode with low pathogenicity that causes canine filariasis, without clinical signs. This filarid produces microfilariae that circulate in the blood of their natural hosts. Microfilariae of A. reconditum can be easily confused with microfilariae of Dirofilaria immitis (D. immitis) which causes a severe disease in dogs. The aim of this article is to describe a case of A. reconditum infection in a dog, diagnosed by fine needle aspiration cytology (FNAC) of two cutaneous nodules, blood smear examination, and further confirmed by molecular assay (PCR). This report alerts veterinarians to the presence of A. reconditum in the region of Santa Maria, Rio Grande do Sul, Brazil, and highlights the importance of performing the differentiation of microfilaria species. In addition, it demonstrates the importance of fine needle aspiration cytology as a diagnostic tool.
Human brugian filariasis has re-emerged in Sri Lanka after a quiescent period of four decades. This study investigated the prevalence of canine and feline filarial parasites in three localities with human sub-periodic brugian filariasis, in order to determine their potential reservoir status. All reachable dogs and cats, both stray and domestic, within a 350m radius of an index case of brugian filariasis in three locations (Madampe, Wattala and Weliweriya) were screened for microfilariae using Giemsa stained thick blood smears. A representative sample of canine and feline blood samples positive for Brugia spp. microfilariae by microscopy, from each of the three locations, were further analyzed by PCR with specific primers for internal transcribed spacer region 2 (ITS2) of the ribosomal DNA. A total of 250 dogs and 134 cats were screened. The overall microfilaraemia rates were high among both dogs (68.8%) and cats (47.8%). The prevalence of microfilaraemia was significantly higher among dogs than cats (p<0.05). Two filarial species were identified based on morphology of microfilariae: Dirofilaria (Nochtiella) repens (dogs, 54.4% and cats, 34.3%) and Brugia spp. (dogs, 51.6% and cats, 30.6%). PCR analysis of canine (n = 53) and feline (n = 24) samples elicited bands in the region of 615bp, which confirmed Brugia malayi infection. Co-infection with D.(N.) repens was detected by PCR with an additional band at 484bp, in 36 canine and 17 feline samples. Overall microfilaraemia rates of dogs (81.8%) and cats (75%) in Madampe (rural) were significantly higher than in urbanized Wattala (dogs, 62.4% and cats, 26.0%) (p<0.05). High rates of zoonotic filarial infections strongly implicate dogs and cats as potential reservoirs for human dirofilariasis and brugian filariasis in Sri Lanka.
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
Background:
Dirofilariosis is an emerging vector-borne parasitic disease in Europe. Monitoring of wild and domestic carnivores demonstrated circulation of Dirofilaria spp. in Romania in the past. For the implementation of control measures, knowledge on the native mosquito community responsible for Dirofilaria spp. transmission is required.
Methods:
Mosquito samples originated from a longitudinal study previously performed in the Danube Delta Biosphere Reserve. Mosquito pools were screened for Dirofilaria immitis and Dirofilaria repens. The samples comprised 240,572 female mosquito specimens collected every ten days between April and September in 2014 at four different trapping sites. In addition, blood samples of 36 randomly selected dogs were collected in 2016 in each of the four mosquito sampling sites. A duplex real-time assay was used to detect the presence of one or both Dirofilaria species for each sample. This assay targets the cytochrome c oxidase subunit 1 and the 16S rRNA gene fragments to differentiate both parasites.
Results:
Dirofilaria immitis and D. repens were detected in mosquito pools at all four trapping sites. In the 2118 mosquito pools tested, D. immitis was identified for eight and D. repens for six of the 14 screened mosquito taxa, with a higher prevalence of D. immitis (4.53% of analysed pools) compared to D. repens (1.09%). Dirofilaria spp. were also identified in dogs from the same sampling sites with a prevalence of 30.56%. For both Dirofilaria species, the highest estimated infection rates (EIRs) were found in Anopheles maculipennis (s.l.) (D. immitis: EIR = 0.206 per 100 specimens, D. repens: EIR = 0.066 per 100 specimens). In contrast, Coquillettidia richiardii and Anopheles hyrcanus as the most frequent taxa had infection rates which were significantly lower: Cq. richiardii (D. immitis: EIR = 0.021; D. repens: EIR = 0.004); An. hyrcanus (D. immitis: EIR = 0.028; D. repens: EIR = 0.006). The number of positive pools per calendar week was positively correlated with the number of screened pools per calendar week, suggesting constant Dirofilaria spp. transmission during the observation period.
Conclusions:
This study further confirms significant circulation of Dirofilaria spp. in eastern Europe, with high parasite prevalence in domestic canids and mosquitoes. Therefore, systematic monitoring studies are required to better understand the environmental risk factors for Dirofilaria transmission, allowing the implementation of effective surveillance and control measures.
Identification of filarial species in dogs is clinically important because of zoonotic concerns and therapeutic implications. The present study was carried out to identify the filarial parasites causing microfilaraemia in dogs in Thrissur District, Kerala- an endemic area for human Brugian filariasis. Out of the 1600 dogs screened by wet blood film examination, 130 were positive for microfilariasis. Giemsa staining of blood smears revealed that 90 out of 130 dogs had unsheathed microfilariae, 24 had sheathed microfilariae and 16 had combined infection of sheathed and unsheathed microfilariae. Results of micrometry and histochemical staining of the sheathed microfilariae were in conformity with that of Brugia malayi. The DNA isolated from the sheathed microfilariae amplified the primers specific for the Hha 1 repeats of the B. malayi. Cloning and sequencing revealed that the amplified fragment corresponded to the 140-292 base pairs of the 320 base pair Hha1 repeat of Brugia malayi. The amplified DNA fragment also contained restriction sites for Alu 1 and Rsa 1which confirmed that the present isolate is Brugia malayi. The present study confirmed the presence of B. malayi in dogs in Kerala, India. © 2017, Malaysian Society for Parasitology. All rights reserved.
The ocular onchocercosis is caused is by the zoonotic parasite Onchocerca lupi (Spirurida: Onchocercidae). A major hindrance to scientific progress is the absence of a reliable diagnostic test in affected individuals. Microscopic examination of skin snip sediments and the identification of adults embedded in ocular nodules are seldom performed and labour-intensive. A quantitative real-time PCR (qPCR) assay was herein standardized for the detection of O. lupi DNA and the results compared with microscopic examination and conventional PCR (cPCR). The specificity of qPCR and cPCR was assessed by processing the most common filarial nematodes infecting dogs, skin samples from O. lupi infected (n = 35 dogs) or uninfected animals (n = 21 dogs; n = 152 cats) and specimens of potential insect vector (n = 93 blackflies; n = 59 mosquitoes/midges). The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of DNA from adult specimen and from a pool of microfilariae. The qPCR on skin samples revealed an analytical specificity of 100% and a sensitivity up to 8 x 10-1 fg/2μl O. lupi adult-DNA and up to 3.6 x 10-1 pg/2μl of mfs-DNA (corresponding to 1 x 10-2 mfs/2μl). Only 9.5% O. lupi-infected skin samples were positive for cPCR with a sensitivity of 8 x 10-1 pg/2μl of DNA. Out of 152 blackflies and mosquitoes/midges, eight specimens experimentally infected (n = 1 S. erythrocephalum; n = 1 S. ornatum; n = 6 Simulium sp.) were positive by qPCR. The qPCR assay herein standardized represents an important step forward in the diagnosis of zoonotic onchocercosis caused by O. lupi, especially for the detection and quantification of low number of mfs. This assay provides a fundamental contribution for the establishment of surveillance strategies aiming at assessing the presence of O. lupi in carnivores and in insect species acting as potential intermediate hosts. The O. lupi qPCR assay will enable disease progress monitoring as well as the diagnosis of apparently clinical healthy dogs and cats.
Detection of circulating antigen of Dirofilaria immitis has been a mainstay of identifying heartworm infection in clinical practice for the past three decades. Several validated commercial antigen tests have very good sensitivity, specificity, and positive predictive values, especially when used in patients for which heartworm infection is likely. In some dogs and cats infected with heartworm, antigen may not be available for detection although present in the patient sample; heat pretreatment of these samples reveals the antigen, changing the false negative to positive. This phenomenon was documented in the literature in the 1980s but subsequently overlooked by the heartworm research community for many years. In this review, we provide a summary of the current understanding of the role of heat reversal in diagnosing heartworm infection. This additional diagnostic step is most important for patients in which heartworm infection is likely, such as dogs or cats in an endemic area with an inconsistent history of heartworm preventive use, or dogs with a prior diagnosis of heartworm infection that were recently treated. To illustrate the concept, we share a summary of results from canine samples tested at the state veterinary diagnostic laboratory in Oklahoma, USA in 2017 by modified Knott test and by commercial antigen test before and after heat treatment of samples; in this sample set, heat treatment changed all D. immitis microfilaria-positive but antigen-negative samples to antigen-positive. Pet dogs with a history of consistent preventive use are unlikely to become positive with heat pretreatment; for that reason, routine pretreatment of all samples tested in a veterinary practice is not recommended. We also review known causes of false negative and false positive results on heartworm antigen tests that, although uncommon, can complicate accurate diagnosis in individual patients. Together, this review provides a primer to aid understanding of strategies that can enhance accurate diagnosis of heartworm infection in veterinary practice and clinical research.
Background
The emergence of macrocyclic lactone resistance in canine heartworm poses a substantial threat to what is currently the only effective, FDA-approved available method of prevention. Further study of the biotypes is necessary to understand the mechanism of resistance and evaluate novel prevention options. Identifying cases of drug-resistant infection remains problematic, however, especially when poor compliance and insufficient testing are concerns. Furthermore, a definitive demonstration of resistance requires experimental infection and treatment, which is prohibitively costly.
Methods
With the aim of identifying likely cases of macrocyclic lactone-resistant heartworm and preventing their continued spread, we describe an algorithm for determining the likelihood of drug resistance and appropriate treatment strategies for each case.
Results
This algorithm relies on the microfilarial suppression test (MFST), which has been used previously as an efficient and discrete measure of suspected resistance. By standardizing this method in a format that is readily available to practitioners, it could become possible to preliminarily survey the emergence and spread of resistance.
Conclusion
Heartworm isolates identified through this method can be used in research to better understand macrocyclic lactone resistance so prevention strategies can be adapted.
Background
Two main Dirofilaria species infect dogs: D. immitis and D. repens. While D. immitis has a worldwide distribution, D. repens is currently found only in Europe, Asia, and Africa. Adult D. repens are located in subcutaneous tissues of natural hosts where they survive for long periods of time. First-stage larvae, microfilariae, circulate in the peripheral bloodstream, where they are taken up by the mosquito intermediate hosts. Infected mosquitoes then transmit infective third-stage (L3) larvae to new hosts through the blood meal. In dogs, most infections are asymptomatic, although cutaneous disorders such as pruritus, dermal swelling, subcutaneous nodules, and ocular conjunctivitis can be observed. Currently, two factors have increased the concerns about this parasitic infection 1) its spread throughout the European countries and to other continents and its prevalence in dog populations, where in some cases it has overcome D. immitis; and 2) its zoonotic potential, which is much greater than that of D. immitis.
Results
Different hypotheses can be put forward to explain these concerns. First, climate change has allowed more favorable conditions for survival of culicid vectors. Second, accidental hosts such as humans may have a less efficient immune reaction against a parasite that is located in subcutaneous tissues, and thus less exposed to the host’s immune response than, for instance, D. immitis. Furthermore, the absence of clinical signs in the majority of canine infections and the difficulty in diagnosing the infection, due to the lack of serologic tests and thus the reliance on the identification of microfilariae and differentiation from D. immitis to confirm the presence of the parasite, favor the further spread of this species. Finally, among the macrocyclic lactones currently used to prevent heartworm infection, only moxidectin has been found to be fully effective against the infective larvae transmitted by mosquitoes and partially effective (efficacy 96%) against adult D. repens in experimental studies.
Conclusions
Dirofilaria repens infection is much more difficult than D. immitis to diagnose and control in the reservoir population (microfilaremic dogs). In addition, lack of familiarity with D. repens infection could lead to lack of vigilance underestimation for this parasite. The number of human cases in Europe and Asia is currently a serious public health concern. Medical doctors and veterinarians must collaborate closely for better control and surveillance of D. repens infection.
Background
Heartworm antigen testing is considered sensitive and specific. Currently available tests are reported as detecting a glycoprotein found predominantly in the reproductive tract of the female worm and can reach specificity close to 100%. Main concerns regard sensitivity in the case of light infections, the presence of immature females or cases of all-male infections. Research and development have been aimed at increasing sensitivity. Recently, heat treatment of serum prior to antigen testing has been shown to result in an increase in positive antigen test results, presumably due to disruption of natural antigen–antibody complexes. Cross-reactions in dogs with both natural and experimental infections with Angiostrongylus vasorum and Spirocerca lupi have been reported, but cross-reactions with other helminths have not been extensively studied. In order to evaluate potential cross-reactivity with other canine and feline parasites, two studies were performed. Study 1: Live adults of Dirofilaria immitis, Dirofilaria repens, Toxocara canis, Toxocara cati, Dipylidium caninum, Taenia taeniaeformis and Mesocestoides spp. larvae were washed and incubated in tubes with saline solution. All worms were alive at the time of removal from the saline. Saline solutions containing excretory/secretory antigens were then tested for heartworm with six different, commercially available antigen tests. All results were evaluated blind by three of the authors. Study 2: Sera from dogs with natural infections by A. vasorum or D. repens, living in areas free of heartworm disease, were tested with the same tests before and after heat treatment (103 °C for 10 min).
Results
Results suggest that antigens detected by currently available tests are not specific for D. immitis. They may give positive results through detection of different parasites’ antigens that are normally not released into the bloodstream or released in a low amount and/or bound to antibodies. Tests may even detect antigens released by male D. immitis adult worms. D. repens appears to release more detectable antigens than the other worms studied.
Conclusions
Cross-reaction with A. vasorum and D. repens does occur in the field and could potentially occur with other helminths. Heat treatment decreases specificity by enhancing cross-reactivity.
Background
Dirofilaria immitis, a filarial nematode, is the most important parasite-affecting dogs, causing cardiopulmonary dirofilariasis. Current diagnostic tools for detecting D. immitis include morphological assays, antigen detection, and X-ray. Herein, we developed a method for the molecular detection of D. immitis in blood using polymerase chain reaction (PCR).
Methods
The study was conducted at Eulji University, Republic of Korea in 2016. To detect D. immitis-specific gene regions, we aligned the cytochrome c oxidase subunit I (COI) genes of seven filarial nematodes and designed primers targeting the unique region. We used dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-targeted primers as the internal control. We conducted PCR-amplified genomic DNA from canine blood samples. The products were confirmed by sequencing.
Results
Gene alignment revealed a D. immitis COI-specific gene region, and the activity of designed primers was confirmed by PCR and sequencing. Plasmid DNA made from the PCR products was a positive control. The limit of detection for our method was 50 copies. The D. immitis COI and dog GAPDH genes could be discriminated from blood samples simultaneously.
Conclusion
This study provides a method for highly specific and sensitive molecular diagnosis of D. immitis used as a diagnostic and therapeutic tool from the early stage of infection.
Background
Filarioids are vector-borne parasitic nematodes of vertebrates. In Europe, eight species of filarioids, including zoonotic species, have been reported mainly in domestic dogs, and occasionally in wild carnivores. In Romania, infections with Dirofilaria spp. and Acanthocheilonema reconditum are endemic in domestic dogs. Despite the abundant populations of wild carnivores in the country, their role in the epidemiology of filarioid parasites remains largely unknown. The aim of the present study was to assess the host range, prevalence and distribution of filarioid infections in wild carnivores present in Romania.
Methods
Between May 2014 and February 2016, 432 spleen samples originating from 14 species of wild carnivores have been tested for the presence of DNA of three species of filarioids (D. immitis, D. repens and A. reconditum).
Results
Overall 14 samples (3.24%) were molecularly positive. The most prevalent species was D. immitis (1.62%), accounting for 50% (n = 7) of the positive animals. The prevalence of D. repens was 1.39%, while that of A. reconditum was 0.23%. No co-infections were detected. Dirofilaria immitis DNA was detected in five golden jackals, Canis aureus (7.58%), one red fox, Vulpes vulpes (0.33%), and one wildcat, Felis silvestris (10%). The presence of D. repens DNA was detected in two red foxes (0.66%), two golden jackals (3.03%), one grey wolf (7.14%), and one least weasel, Mustela nivalis (33.33%). Acanthocheilonema reconditum DNA was found only in one red fox (0.33%).
Conclusion
The present study provides molecular evidence of filarial infections in wild carnivore species in Romania, suggesting their potential epidemiological role and reports a new host species for D. repens.
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. ResultsThe 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.
The Onchocerca lupi nematode is an emerging helminth capable of infecting pets and humans. We detected this parasite in 2 dogs that were imported into Canada from the southwestern United States, a region to which this nematode is endemic. We discuss risk for establishment of O. lupi in Canada.
Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propi-dium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic. Author Summary Dirofilaria repens is a filarial nematode which mainly infests the dog, but humans may be occasionally infested, too. The spread of the parasite is mediated by a number of mosquitoes species, which are well recognized as vectors of D. repens. The majority of reports of the disease come from the European Countries, especially those along the Mediterranean basin, but in the last decade several cases have been recorded also from Asian and African PLOS Neglected Tropical Diseases |
Background
Onchocerca lupi causes ocular pathology of varying severity in dogs from south-western United States, western Europe and northern Asia. This filarioid has also been recognized as a zoonotic agent in Tunisia, Turkey, Iran and the USA, though the information about the biology and epidemiology of this infection is largely unknown. In Europe, O. lupi has been reported in dogs from Germany, Greece, Hungary, Portugal and Romania and in a cat from Portugal. The present study was designed to establish the occurrence of O. lupi in dogs in southwestern Spain.
In the present study a total of 104 dogs of different breed, sex, and age living in a shelter in Huelva (SW Spain) were examined. Skin snip samples were collected using a disposable scalpel in the forehead and inter-scapular regions and stored as aliquots in saline solution (0.5 ml) before light microscopy observation of individual sediments (20 μl) and molecular examination.
Results
Of the 104 dogs examined, 5 (4.8 %) were skin snip-positive for O. lupi: two by microscopy and three by PCR. One of the O. lupi infected dogs showed neurological signs but ocular ultrasonography and/or MRI detected no abnormalities.
Conclusions
This first report of O. lupi infection in dogs in southern Spain expands the range of geographical distribution of this parasite and sounds an alarm bell for practitioners and physicians working in that area.
During the last two decades, Belarus faces an increase of human cases of Dirofilaria (Nematoda, Spirurida, Onchocercidae) infections. However, comprehensive analyses explaining this development and the identification of mosquito vector species are missing. Here, we present results using temperature data from Belarus and show that the annual number of human Dirofilaria cases is significantly correlated with the yearly average temperatures (Spearman’s rho = 0.49, p < 0.05) and the average sum of potential Dirofilaria transmission days (Spearman’s rho = 0.46, p < 0.05), suggesting that autochthonous transmission is at least in part responsible for the increasing number of clinical Dirofilaria cases in the country. In addition, 467 female mosquitoes were collected from different sampling sites in the regions of Brest and Minsk, which were analyzed by molecular methods for the presence of Dirofilaria repens and Dirofilaria immitis DNA, respectively. Two pools (5.56 %) were tested positive for Dirofilaria (estimated infection rate per 100 specimens = 0.44, 95 % confidence interval = 0.08–1.43), comprising one Anopheles claviger s.l. pool that was positive for D. repens and one Culex pipiens s.l./Culex torrentium pool positive for D. immitis DNA. This, to our knowledge, is the first molecular evidence for the presence of Dirofilaria in mosquitoes from Belarus, suggesting a high probability of autochthonous Dirofilaria transmission in the country.
This study establishes a relationship between positive canine heartworm (Dirofilaria immitis) test results frequently observed in California sea lions ( Zalophus californianus ) and infection with the filarid nematode Acanthocheilonema odendhali. Four commercially available canine heartworm antigen tests were evaluated for cross-reaction with A. odendhali in California sea lions. Sera were tested from fifteen California sea lions with A. odendhali-associated microfilaremia, confirmed by blood smear, and with no evidence of D. immitis infection at necropsy. Ninety-five percent of tests were falsely positive for D. immitis. This study also determined that the prevalence of A. odendhali infection in stranded California sea lions from central California is approximately 23% by comparing the number of findings of mircofilaremia to the total number of California sea lions sampled at The Marine Mammal Center between 2005 and 2011, inclusive. Acanthocheilonema odenhali microfilaremia in California sea lions is likely to cross-react with canine heartworm antigen tests, and clinicians should interpret results with caution.
This case-series describes the 6 human infections with Onchocerca lupi, a parasite known to infect cats and dogs, that have been identified in the United States since 2013. Unlike cases reported
outside the country, the American patients have not had subconjunctival nodules but have manifested more invasive disease
(eg, spinal, orbital, and subdermal nodules). Diagnosis remains challenging in the absence of a serologic test. Treatment
should be guided by what is done for Onchocerca volvulus as there are no data for O. lupi. Available evidence suggests that there may be transmission in southwestern United States, but the risk of transmission to
humans is not known. Research is needed to better define the burden of disease in the United States and develop appropriately-targeted
prevention strategies.
In this work a new species of the genus Cercopithifilaria Ebehard, 1980 is proposed considering the differential charecteres of the microfilarias Dipetalonema grassii (Noé, 1907) and C. bainae n. sp.
Dirofilaria repens is a parasite of animals and humans, transferred by mosquitoes. The assessment of the presence of D. repens-infected vertebrate hosts in the investigated area can be performed by xenomonitoring—detection of the parasite in blood-feeding arthropods. Our study aimed to evaluate PCR xenomonitoring of mosquitoes as a tool for dirofilariosis surveillance in Poland. We were also interested whether inter-study comparisons at the international level would be possible. Mosquitoes were collected in a single locality in Mazowsze province in Poland, in which between 12 and 20 % of dogs were infected with D. repens and autochthonous human dirofilariosis was confirmed. All captured female mosquitoes were divided into pools; alternatively, single mosquitoes were analyzed; DNA was isolated and subjected to PCR and real-time PCR for detection of D. repens. The estimations of infection rates of mosquitoes with D. repens, based on PCR results, varied from 0 to 1.57 % even between assays for detection of distinct fragments of the same marker—cytochrome oxidase subunit one gene. Polymorphisms of the DNA sequence within binding sites of the primers used in D. repens xenomonitoring assays, applied in European studies, were identified. Non-specific amplification of Setaria tundra (Nematoda: Onchocercidae) DNA occurred. Surveillance of dirofilariosis by PCR mosquito xenomonitoring is possible; however, the efficiency of the approach on territories where the prevalence of the disease among definitive hosts is lower than 12 % remains unknown. Furthermore, mosquito infection rate estimations can be PCR assay dependent, which makes inter-study comparisons difficult. The results obtained in independent European xenomonitoring studies were contradictory. International collaboration would be required to establish a standardized set of assays for sensitive and specific xenomonitoring-based dirofilariosis surveillance.
This study aimed to evaluate the efficacy of imidacloprid 10 %/moxidectin 2.5 % (w/v) spot-on (Advocate®/Advantage® Multi, Bayer) against adult Dirofilaria repens in a blinded, placebocontrolled randomised laboratory study. Twentyfour Beagle dogs were experimentally infected with approximately 75 infective D. repens larvae each on study day (SD) 0. Treatment was initiated on SD 228 after patency had been confirmed in 21 dogs, using a modified Knott Test. Eleven dogs received monthly treatments with imidacloprid/moxidectin at the minimum therapeutic dose (10 mg/kg imidacloprid and 2.5 mg/kg moxidectin) for six consecutive months and 12 control dogs were treated with a placebo formulation. Approximately one month after the last treatment, all dogs were euthanised and necropsied for the detection of D. repens worms. Eleven control dogs harboured live adult D. repens (range 2-11, geometric mean 5.44). Eight of 11 imidacloprid/moxidectin-treated dogs were free of live worms. The live worm count was reduced by 96.2 % (range 0-1, geometric mean 0.21). The majority of dead worms were encapsulated and degenerated. After the first treatment, Knott Tests were negative in all imidacloprid/moxidectin-treated dogs and this status was maintained in 10 dogs until study end. One dog showed a low microfilariae count (1 and 4/mL) on four occasions but was also negative before necropsy. The treatment was well tolerated by all study animals. It is concluded that six consecutive monthly treatments with imidacloprid/moxidectin spot-on are effective and safe against adult D. repens and provide an option for preventing the further spread of this zoonotic parasite.
In southern California, ocular infections caused by Onchocerca lupi were diagnosed in 3 dogs (1 in 2006, 2 in 2012). The infectious agent was confirmed through morphologic analysis of fixed parasites in tissues and by PCR and sequencing of amplicons derived from 2 mitochondrially encoded genes and 1 nuclear-encoded gene. A nested PCR based on the sequence of the cytochrome oxidase subunit 1 gene of the parasite was developed and used to screen Simulium black flies collected from southern California for O. lupi DNA. Six (2.8%; 95% CI 0.6%-5.0%) of 213 black flies contained O. lupi DNA. Partial mitochondrial16S rRNA gene sequences from the infected flies matched sequences derived from black fly larvae cytotaxonomically identified as Simulium tribulatum. These data implicate S. tribulatum flies as a putative vector for O. lupi in southern California.
Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.
Canine filarioids are worldwide distributed nematodes transmitted by arthropods with variable virulence depending on the species. Dirofilaria immitis is the most virulent and serological antigen tests are commonly employed to detect it. This study reports on the heaviest cavity filariasis recorded so far in a dog, which showed no apparent clinical signs of infection. The 6-year-old male was positive to a D. immitis antigen test. Blood samples collected and analyzed with the modified Knott's test for microfilariae revealed 264,367 microfilariae/ml. In a postmortem examination 791 adult filarial nematodes were found in the dog's thoracic and peritoneal cavities. Morphological and molecular analysis identified the nematode as Acanthocheilonema dracunculoides and no other species were present. This is evidence that massive A. dracunculoides infections in dogs may not be clinically evident, they may cause serologic cross-reaction with D. immitis infection and become a life-threatening condition if dogs are treated with a microfilaricidal treatment without previously performing an adequate diagnosis.
Dirofilaria immitis and D. repens are endemic throughout Europe and southern eastern regions of Asia and reported with increasing frequency in Africa. Nevertheless, the increased awareness of veterinary practitioners, even in countries where the prevalence is low, has led to a decrease D. immitis prevalence in dogs, especially in previously endemic/hyper-endemic areas. Prevalence has significantly increased, however, in areas where heartworm has apparently spread more recently, such as Central and North Eastern Europe. Furthermore, autochthonous cases have been observed in Siberia. Low seroprevalence has been reported in Croatia, while in Romania it has reached 14%. In Greece, the prevalence ranges between 0.7% and 25% whilst in Turkey is 0-18%. Data for canine dirofilariosis in Africa is scarce, and most are case reports. Overall, the dominant species is Achanthocheilonema dracunculoides, although both D. immitis and D. repens have been reported from some countries. In the Far East, the prevalence ranges from 2% to 15% in northeastern of China. In Hong Kong a novel species has been found in dogs and humans (Candidatus Dirofilaria hongkongensis). In India, the prevalence ranges from 4.7%-29.5% in Northeastern states.
The main factors that have influenced the spreading of Dirofilaria infections are the climate changes and the introduction of new, invasive, competent mosquito species such as Aedes albopictus and Ae. koreicus. Other factors include relocation and insufficient prevention in dogs, manly in the new areas of colonization.
Feline heartworm infection has been diagnosed in every European country when diagnosed either by the Knott test or by serology for circulating antibodies and antigens of the parasite. However, prevalence is much lower than in dogs.
In spite of the continuing spreading of heartworm infection, D. repens is the main concern in Europe, mostly for physicians, while the infection is nearly always asymptomatic in dogs. The infection is spreading from Portugal to the Southeastern regions of Finland and Siberia, and in some areas its prevalence overlaps that of D. immitis. Many reasons make more difficult the control of D. repens than D. immitis: the frequent lack of clinical symptoms of suspicion, the specific diagnosis being possible only by blood examination and the inefficacy of some macrocyclic lactones.
Filariae of animals, especially those of mammals, often infect humans and typically produce cryptic infections. These “zoonotic” infections have been reported from virtually all parts of the world including temperate zones. Infections may be symptomatic or not, and the parasites are found in surgical tissue biopsy specimens or, more rarely, are removed intact from superficial sites such as the orbit or conjuctivae. Typically, these worms tend to occupy tissue sites similar to those occupied in the natural animal host, with the exception of the eyes. Many kinds of filariae have been isolated from humans, including species of Dirofilaria, Brugia, Onchocerca, Dipetalonema, Loaina and Meningonema. Worms have been found in subcutaneous tissues, the heart and lungs, lymphatics, the eye, and the central nervous system. Specific identification of these filariae is based on their morphological features in histologic sections. Unfortunately, some of these worms cannot be identified even at the generic level. There are other species of filariae, presumed to be zoonotic, which produce patent infections in humans but are poorly and incompletely known. These include Microfilaria semiclarum and Microfilaria bolivarensis. It is probable that almost any filaria parasitizing animals can, under proper circumstances, infect humans and undergo some degree of development. Undoubtedly, additional species of filariae will continue to be isolated from humans in the future.
Dirofilaria immitis and D. repens are filarioid nematodes of animals and humans, transmitted by the bite of infected mosquitoes. Domestic and wild canids are a major natural host and reservoir for these parasites. In this study, we designed a duplex real-time PCR protocol targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene, detecting both D. immitis and D. repens using two primer pairs and two Dirofilaria-specific hydrolysable probes. The sensitivity and specificity of the primers and probes were tested in both experimental and naturally infected samples. The detection limits of this assay were evaluated using plasmid DNA from D. immitis and D. repens. No cross-reaction was observed when testing this system against DNA from other filarial nematodes. The detection limit of the real-time PCR system was one copy per reaction mixture containing 5 I of template DNA. Field application of the new duplex real-time assay was conducted in Corsica. The prevalence rate of D. immitis was 21.3% (20/94) in dogs. In a locality where most dogs with Dirofilaria spp. infection were found, D. immitis and D. repens were detected in 5% (20/389) and 1.5% (6/389) of the Aedes albopictus population, respectively. These results suggest that this sensitive assay is a powerful tool for monitoring dirofilariosis in endemic or high risk areas.
To compare the usefulness of two commercial tests for detecting the antigens of Dirofilaria immitis in dogs, one based on whole-blood agglutination (WBA) and the other on ELISA, 100 stray dogs from North Taiwan were tested before necropsy. Of the 53 dogs found to contain D. immitis at necropsy, which had a mean (S.D.) burden of 8.2 (10.6) worms/dog, 45 were found to be positive by WBA and 47 by ELISA. All the false negatives were dogs with very low worm burdens. Although the ELISA was more sensitive (83.9% v. 71.7%) and specific (100% v. 85.1%) than the WBA, the latter is simpler to use and less time-consuming. In terms of their general use for diagnosis of canine heartworm, there seems little to choose between the two tests. The false negatives observed with both tests are not likely to be a problem as they represent dogs with worm burdens which are probably too low to cause significant clinical manifestations or pathology. As the positive predictive value of the WBA test declines dramatically with prevalence of infection, this test may not be suitable for detecting D. immitis in canine populations in which heartworm infection is rare.
Brugia pahangi and sub periodic Brugia malayi coexist in many areas of Malaysia where they are found in many of the same animal hosts (Laing et al., 1960; Mullin et al., 1972). The latter, however, infects man as well as animals while the former is not thought to be a parasite of humans. These two species are difficult to differentiate morphologically, particularly in the microfilarial stage. Since the diagnosis of most human cases of filariasis is based on the detection of microfilariae in the peripheral blood, it is possible that a significant number of infections in Malaysia diagnosed as due to B. malayi may, in fact, have been due to B. pahangi. Experimental infection of B. pahangi in man has been reported (Edeson et al., 1960a). Also, clinical manifestations similar to tropical eosinophilia syndrome were reported in a human volunteer experimentally exposed to B. pahangi (Buckley, 1958a, 1958b). Consequently, a relatively simple and reliable method of differentiating these two parasite species would be advantageous in any field or laboratory study. Chalifoux and Hunt (1971) introduced a histochemical method for demonstrating differences between the microfilariae of two common canine filarial worms, Dirofilaria immitis and Dipetalonema reconditum, based on the specific distribution of acid phosphatase activity in these organisms. Later, Chalifoux et al. (1973) using the same technique, were able to differentiate 11 types of circulating microfilariae isolated from seven species of New World monkeys. The present study was designed to determine whether microfilariae of B. malayi and those of B. pahangi could be accurately and consistently differentiated on the basis of the specific distribution of their acid phosphatase activity.
The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have shown that parasite-derived cell-free DNA (cfDNA) can be detected in host blood and may be a promising diagnostic marker for parasitic infections. Thus, our aim was to detect D. immitis-derived cfDNA in host serum by nested PCR. Sera were collected from 12 dogs with natural D. immitis infections; eight were microfilaria (mf)-positive, and the remaining four were mf-negative. Culture fluids derived from single-sex adult D. immitis worms (mf-producing females and males) were also tested for cfDNA. All mf-positive sera were positive by nested PCR, whereas no amplification products were detected in mf-negative sera. The culture fluid of mf-producing females was positive by nested PCR but that of males was negative. All products amplified by nested PCR were sequenced to confirm that the amplicons were those of D. immitis. These results indicate that D. immitis DNA circulates freely in dog serum, except in mf-negative dogs. Additionally, D. immitis cfDNA may primarily be derived from the mf, and adult worms appeared to be minor contributors of cfDNA concentrations in serum; however, the contribution of D. immitis cfDNA derived from larvae of other developmental stages is unclear. An evaluation of the kinetics of D. immitis cfDNA in host serum throughout the parasite life cycle could facilitate the development of early molecular diagnostic techniques. To the best of our knowledge, this is the first report of the detection of mitochondrial DNA from a filarial parasite in host serum.
Editor,—Human ocular invasion by non-human filarial parasites has been reported for more than 200 years.1 2However, only just over a handful have actually been removed, described and identified in detail.3-6 Furthermore, the Dipetalonema species that have been described in three cases were thought to be from the body cavity of the natural hosts—the porcupine and the beaver.4 5 7 8
This report describes a case of Dipetalonema reconditum (usually associated with canine filariasis) in the human eye. It is noteworthy that this worm has morphological similarities to the canine heartworm Dirofilaria imitis , which in the past has been described in the human eye5 9 but not satisfactorily identified.10
### CASE REPORT
A 62 year old white resident of suburban Victoria, Australia, presented with a red and irritated right eye of 2 weeks' duration. This was exacerbated after a rural walking trip and did not improve with topical lubrication. He also noted mild diplopia on extreme right gaze. On examination, the visual acuity was 6/6 in the right eye and 6/4 in the left. There was mild limitation of right eye abduction. Localised bulbar conjunctival …