Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens of animals and humans.

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.

Camel filariasis (Dipetalonema evansi) and its association with clinical balanoposthitis with reference to prominent changes in clinical findings, serum testosterone, semen analysis, and testicular histopathology.

BACKGROUND: Camel filariasis induced variable clinical syndromes characterized by fever, lethargy, localized dermal lesions, loss of condition, and testicular and scrotal swelling. The objective of the present work focused on clarifying the diagnostic importance of clinical findings, serum testosterone, and semen analysis as well as blood smear and testicular histopathology as a differential tool between only balanoposthitis without filariasis male camels group (OnlyBpgr) and balanoposthitis-filariasis infected male camels group (BpFlgr). The study also monitored the associations between the severity of ticks' infestations in investigated male camels and the occurrence of balanoposthitis only or balanoposthitis with filariasis. RESULTS AND CONCLUSIONS: The study reported significant correlation between serum testosterone, serum cortisol, and sperm vitality and abnormalities percentages. The study included male camels (n = 250) classified into three groups: healthy control group (Contgr; n = 30), OnlyBpgr (n = 210), and BpFlgr (n = 10). These male camels were clinically and laboratory examined, and skin scraping tests and testicular histopathology were conducted. The study confirmed the association of the changes in clinical findings, whole blood picture, serum testosterone, serum cortisol, and semen analysis, with OnlyBpgr and BpFlgr. These changes were more prominent in BpFlgr than in OnlyBpgr. Skin scraping test results revealed a higher severity of live ticks' infestation in BpFlgr than in OnlyBpgr because, unlike OnlyBpgr, all camels in BpFlgr (n = 10) were suffering from live ticks' infestation. It also concluded the higher efficacy of histopathology of testicular tissues in male camels as a diagnostic tool for adult filaria in balanoposthitis-affected male camels than blood smear because all cases of camel filariasis in the current work were negative for microfilaria on microscopic examination of diurnal blood smear as well as testicular histopathology revealed detection of adult filaria in all camel filariasis associated with balanoposthitis. Strong correlation relationships were demonstrated between serum testosterone, serum cortisol, and semen analysis results. Positive correlations were reported between serum testosterone levels and sperm vitality percentages. However, negative correlations were stated between serum testosterone and each of serum cortisol and sperm abnormalities either in Contgr, OnlyBpgr, or BpFlgr.

Cerebral filariasis infection with Litomosoides in Molossus barnesi (Chiroptera: Molossidae) in the Brazilian eastern Amazon, with comments on Molossinema wimsatti Georgi, Georgi, Jiang and Fronguillo, 1987.

During bacterial and viral pathogen investigation of 30 specimens of bats captured in periurban forest areas in the city of Belém, Pará, Brazil, a case of cerebral filariasis was observed. In the course of histopathological examination, adult filariae were found in pseudocystic cavities brain of Molossus barnesi (Molossidae) and classified morphologically as Litomosoides by the shape of the spicules-left spicule with a handle longer than the blade; right spicule curved, with a sclerotized heel supporting a dorsal notch; the area rugosa constituted by a ventral band of small longitudinal crests; tail rounded in males; long esophagus with a slightly glandular distal portion; and a muscular bent vagina. All the specimens lack a stoma (buccal capsule). We compared our filarioids with the description of specimens of Molossinema wimsatti. Morphological characteristics of M. wimsatti resemble the genus Litomosoides. Thus, we believe that M. wimsatti is a synonym of L. molossi Esslinger, 1973, and filarioid specimens from material reported by Lichtenfels et al. (Trans Am Micros Soc 100:216-219, 1981) and from de Souto et al. (J. Helminthol 1195:e65, 2021) most probably correspond to Litomosoides. We suggest that the reduction of the buccal capsule may be attributable to the ectopic location. No evidence of tissue responses by the host was observed. This is the first record of Litomosoides infecting brain tissue of Molossus barnesi from Brazil, representing a record of a new host species. More specimens of bats should be examined in order to find filarioids in the brain and verify its taxonomic position using molecular techniques.

Filariasis in dogs brought to the Veterinary Teaching Hospital, University of Peradeniya, Sri Lanka.

Dirofilariasis is the predominant emerging zoonotic filariasis in the world. The two most frequent filarial worms that infect dogs are Dirofilaria repens and Dirofilaria immitis. This study reports filariasis among dogs brought to the Veterinary Teaching Hospital (VTH) at the University of Peradeniya and signifies the first molecular characterization of D. repens, responsible for an emerging zoonotic filarial disease in Sri Lanka. Blood samples were collected and were morphologically analyzed using Modified Knott's Technique, followed by molecular analyses. The difference in filariasis prevalence among gender, breed, and age categories was analyzed using a chi-square test. Infection intensities were analyzed using the Mann-Whitney U test and the Kruskal Wallis test. The dogs were brought to the clinic for either vaccination and/or for a regular checkup, and most were sick having non-specific clinical signs. Among the 87 dogs tested, 27.6% were positive for Dirofilaria. Conventional PCR and bi-directional sequencing of genomic DNA of microscopically tested positive samples revealed that the species in Sri Lanka was D. repens. The infection was significantly higher in males (39.1%) than in females (14.6%; χ2 = 0.447, p = 0.011), though it is not significant between puppies (age < 1 year) and adult dogs. More crossbred dogs were infected compared to older and purebred dogs. There was no difference in intensity of infection based on their gender, age, or breed. Sequences obtained from the current study were unique and were only 63% identical to those of D. repens reported from South India. The high number of Dirofilaria infections in domestic dogs indicates a potential reservoir for emerging human dirofilariasis cases in Sri Lanka. Thus, morphological and molecular diagnosis, along with epidemiological assessment of these zoonoses, is critical for the formulation of effective public health programs and control mechanisms.

Giraffe skin disease: Clinicopathologic characterization of cutaneous filariasis in the critically endangered Nubian giraffe (Giraffa camelopardalis camelopardalis).

Giraffe skin disease (GSD) is an emerging disease of free-ranging giraffe recognized in the last 25 years in several species, including the critically endangered Nubian giraffe (Giraffa camelopardalis camelopardalis) of Uganda. Identifying the cause of GSD and understanding its impact on health were deemed paramount to supporting these vulnerable populations. Sixty-four giraffes were immobilized in Murchison Falls National Park, Uganda, from 2017 to 2019, and GSD lesions were opportunistically biopsied. Fifty-five giraffes (86%) had GSD lesions on the neck, axilla, chest, and cranial trunk. Lesions were categorized into early, intermediary, and dormant stages based on gross and histological characteristics. Early lesions were smaller, crusted nodules with eosinophilic and pyogranulomatous dermatitis and furunculosis. Intermediary lesions were thick plaques of proliferative and fissured hyperkeratosis and acanthosis with dense dermal granulation tissue and severe eosinophilic and granulomatous dermatitis. Lesions appeared to resolve to dormancy, with dormant lesions consisting of hairless plaques of hyperkeratosis with dermal scarring and residual inflammation. The periphery of early and intermediary lesions included follicular granulomas containing adult filarid nematodes, with myriad encysted microfilariae in the superficial dermis. Stage L3 larvae were common in early and intermediary lesions, and dormant lesions had remnant encysted microfilariae with no adult or stage L3 larvae. Nematodes were morphologically and genetically novel with close identity to Stephanofilaria spp. and Brugia malayi, which cause infectious filariasis. Identification of potential insect vectors, long-term monitoring of GSD lesions, and evaluating response to therapy is ongoing in the efforts to help conserve the Nubian giraffe.

Filarial worms: a systematic review and meta-analysis of diversity in animals from Iran with emphasis on human cases.

Current systematic review and meta-analysis demonstrate the prevalence reports of filariasis in animals in Iran along with human cases. Studies were screened, relevant papers were selected and the random-effect model was used by forest plot with 95% confidence interval (CI). Of 17 records of human case-reports, particularly from Khuzestan province (5 cases), Dirofilaria repens was the most detected parasite (10 cases) with higher involvement of the right eye (7 cases) than other organs. Eleven animal species were reported to be parasitised by filarioids in Iran. The prevalence of Dirofilaria immitis in canids was 14.69% (95% CI: 10.33-19.67), with highest rates (20.92%; 95% CI: 13.84-29.03) in free-ranging dogs. Male (10.07%; 95% CI: 5.10-16.47) and more than 1-year old (20.77%; 95% CI: 8.66-36.42) dogs were more likely to be found infected. The frequency of other filarioids of zoonotic interest was: Acanthocheilonema reconditum in dogs 2.15% (95% CI: 0.71-4.33), Dipetalonema evansi in camels 10.16% (95% CI: 4.73-17.34), Onchocerca cervicalis in horses 3.63% (95% CI: 1.44-6.75%) and Onchocerca fasciata 16.57% (95% CI: 10.12-24.24%) in camels. Still, our knowledge on parasitic filariae in Iran is limited and more investigation is needed in both human and animal populations.

Cercopithifilaria species in dogs and ticks from Greece.

Filarioids of the genus Cercopithifilaria (Spirurida, Onchocercidae) are parasites of wild and domestic animals in tropical and subtropical regions being transmitted by ixodid ticks. Though this filarioid species have been studied in canine and tick populations in Europe, data on their species diversity and geographical distribution in Greece is scant. Thus, the aims of this study were to investigate the presence of Cercopithifilaria spp. in dogs and ticks across Greece and to assess the possible risk factors. A total of 500 skin biopsies were collected from dogs, while 508 ticks were collected from 180 infested animals and examined. Sediments from skin biopsies were microscopically screened for detection of dermal microfilaria (mfs). Skin samples (n = 115) and tick specimens (n = 153) were molecularly subjected by PCR. Overall, 70 samples (14%) scored positive for mfs. Specifically, 68 samples (13.6%) were positive for Cercopithifilaria bainae and two (0.4%) were co-infected with C. bainae and Cercopithifilaria sp. II. Molecular analyses revealed that all sequences obtained belong to C. bainae. Haplotype I was the most frequent (92.6%), followed by haplotype XVIII (3%) and haplotypes II and IX (1.5%). Three new haplotypes of C. bainae, named XIX, XX, and XXI, were also identified. Among the risk factors examined, habitat, dog use, body weight, tick infestation history, and the use of acaricides were associated with the presence of C. bainae. The estimated prevalence of Cercopithifilaria spp. demonstrates that these filarioids are common in dogs and ticks in Greece. Finally, the identification of 7 haplotypes for C. bainae confirms their genetic variability.

Effect of Brugia pahangi co-infection with Plasmodium berghei ANKA in gerbils (Meriones unguiculatus).

Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.

New host and geographical records for Parafilaroides normani (Nematoda: Filaroididae) Dailey, 2009 in South American fur seal, Arctocephalus australis, from southern Brazil.

Lungworms are a common finding in seals and fur seals around the world. However, from existing records, the biogeographical distribution of filaroid helminths appears to be restricted, and these parasites are endemic in only certain areas and species, mainly in the Northern Hemisphere. The occurrence of infection in pinniped species in the Southern Hemisphere is scarce. The objective of this work is to verify the prevalence of lungworms in Arctocephalus australis in waters off the southern coast of Brazil. Twenty subadult specimens of A. australis found recently dead on the southern coast of Brazil were necropsied and their lungs were examined. Parasitic cysts were found in only one specimen (prevalence of 5%). The helminths were morphologically identified as Parafilaroides normani (Metastrongyloidea: Filaroididae). This helminth species has been reported in pinnipeds from Australia, New Zealand and South Africa. This is the first record of P. normani in A. australis and for the western South Atlantic, providing additional data regarding the biogeographic distribution of the parasite.

Therapeutic efficacy of doxycycline in domestic cats naturally infected with Brugia malayi in field condition.

OBJECTIVE: The aim of this study was to evaluate the efficacy of oral doxycycline treatment for Brugia malayi as measured by microfilarial and filarial DNA clearance in naturally infected domestic cats. METHODS: This study included 8 domestic cats that lived with families that resided in Tak Bai District of Narathiwat Province, which is located in Southern Thailand. The study area is a known B. malayi endemic area. All study cats received doxycycline treatment doses by their respective owners according to a previously described protocol. Briefly, doxycycline (VibraVet@) was given orally once a day during weeks 1-4, 10-11, and 16-17. Blood collections were performed at baseline before treatment, and then every month for 12 months after the initial dose of doxycycline to assess microfilaraemia by Giemsa stain, and filarial DNA detection by high-resolution melt (HRM) real-time polymerase chain reaction (PCR). RESULTS: One month after the start of doxycycline treatment, five of eight cats were negative for microfilaraemia, and 4 of those were negative for filarial DNA. All cats receiving doxycycline treatment were negative for microfilaria by Giemsa stain, and for filarial DNA by HRM real-time PCR within 8 months after receiving the initial dose of doxycycline treatment. CONCLUSION: Administration of oral doxycycline to domestic cats naturally infected with B. malayi in disease endemic areas can significantly reduce microfilaraemia at 1 month and filarial DNA was undetectable by 8 months after the initial dose of doxycycline treatment. No recurrence of microfilaraemia or filarial DNA was observed in study cats at 1 year after the start of doxycycline. Included cats appeared to tolerate doxycycline (VibraVet@) well, with no adverse drug reactions reported by any study cat owner.

Dermatitis caused by autochthonous Cercopithifilaria bainae from a dog in Florida, USA: clinical, histological and parasitological diagnosis and treatment.

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.

New insights into the clinico-histopathological and molecular features of Pelecitus (Filarioidea: Onchocercidae) from a raptor bird.

Some Onchocercidae nematodes such as Pelecitus are parasites of medical and veterinary importance. The adult stage of Pelecitus has been reported infecting birds, and the microfilaria has been associated to human blindness. However, in some of these cases, the nematode was incompletely identified at the species level due to the scarcity of morphological taxonomic keys and, also, to the lack of molecular diagnostic analysis. Here, we report a new Pelecitus species in a crested caracara (Caracara cheriway) producing a severe tenosynovitis and microfilarial dermatitis. It is also the first record of Pelecitus in an American bird of prey. Clinical and histopathological features are described, contributing towards our understanding of the pathogenesis of Pelecitus and the health and conservation of wild bird populations. Our study also provides new information on the molecular diagnosis of this parasite and highlights the potential role of wild birds as Pelecitus reservoirs, and health risk for humans and wildlife.

GRANULOMATOUS FILARIAL ENCEPHALOMYELITIS CAUSED BY CHANDLERELLA QUISCALI IN A NORTHERN CRESTED CARACARA (CARACARA CHERIWAY).

A northern crested caracara (Caracara cheriway) was presented after being found nonambulatory in a field. On physical examination, the bird had severe hind-limb paresis. The bird did not improve after 10 days of hospitalization and was euthanized. Histologic examination of the cerebrum and spinal cord revealed multiple adult filarial nematodes surrounded by granulomatous inflammation with several multinucleated giant cells. These parasites were confirmed to be Chandlerella quiscali with polymerase chain reaction. This is the first report of C. quiscali in a bird of prey.

Co-infections and environmental conditions drive the distributions of blood parasites in wild birds.

Experimental work increasingly suggests that non-random pathogen associations can affect the spread or severity of disease. Yet due to difficulties distinguishing and interpreting co-infections, evidence for the presence and directionality of pathogen co-occurrences in wildlife is rudimentary. We provide empirical evidence for pathogen co-occurrences by analysing infection matrices for avian malaria (Haemoproteus and Plasmodium spp.) and parasitic filarial nematodes (microfilariae) in wild birds (New Caledonian Zosterops spp.). Using visual and genus-specific molecular parasite screening, we identified high levels of co-infections that would have been missed using PCR alone. Avian malaria lineages were assigned to species level using morphological descriptions. We estimated parasite co-occurrence probabilities, while accounting for environmental predictors, in a hierarchical multivariate logistic regression. Co-infections occurred in 36% of infected birds. We identified both positively and negatively correlated parasite co-occurrence probabilities when accounting for host, habitat and island effects. Two of three pairwise avian malaria co-occurrences were strongly negative, despite each malaria parasite occurring across all islands and habitats. Birds with microfilariae had elevated heterophil to lymphocyte ratios and were all co-infected with avian malaria, consistent with evidence that host immune modulation by parasitic nematodes facilitates malaria co-infections. Importantly, co-occurrence patterns with microfilariae varied in direction among avian malaria species; two malaria parasites correlated positively but a third correlated negatively with microfilariae. We show that wildlife co-infections are frequent, possibly affecting infection rates through competition or facilitation. We argue that combining multiple diagnostic screening methods with multivariate logistic regression offers a platform to disentangle impacts of environmental factors and parasite co-occurrences on wildlife disease.

First report of Filaria martis Gmelin, 1790 in the European mink, Mustela lutreola (Linnaeus, 1761).

The riparian European mink (Mustela lutreola), currently surviving in only three unconnected sites in Europe, is now listed as a critically endangered species according to the IUCN. Habitat loss and degradation, anthropic mortality, interaction with the feral American mink (Neovison vison), and infectious diseases are among the principal causes of its decline. Surveys of helminth parasites of this host that also include focus on subcutaneous potentially pathogenic helminths such as those belonging to the genus Filaria are very scarce. We report here the presence of specimens of Filaria martis in the subcutaneous connective tissues of three M. lutreola individuals from Spain. This is the first finding of a subcutaneous nematode in a representative of the genus Mustela. The report also enlarges the known range of the definitive hosts of this nematode. These worms were mainly located in the dorsal region of mink and more rarely in the knees, elbows, and hips. Skin sloughing was only observed in one M. lutreola with both septicaemia and an associated high burden of F. martis. Therefore, more attention should be paid to potentially pathogenic helminths when designing conservation programs dedicated to M. lutreola.

Canine filarial infections in Liguria, north-west Italy.

This paper reports the findings of a study on the presence of various species of filarial nematodes in dogs in Liguria, north-west Italy, a region traditionally considered free from the disease. Between 2009 and 2012 blood samples were taken from 365 dogs in rural areas in Liguria. The blood samples were then submitted to Knott's test, histochemical staining, polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis antigens. Overall, 35 of the 365 dogs were positive using Knott's test for microfilariae (prevalence 9.6%; 95% confidence interval (CI): 6.6-12.6%). Acanthocheilonema reconditum was the most prevalent species (8.0%), while Dirofilaria repens (1.4%) and Dirofilaria immitis (0.6%) were less common. One co-infection by D. repens and A. reconditum was observed. All morphological identifications were confirmed by histochemical staining and PCR. In addition, a retrospective analysis of data on D. immitis antigens in 11,363 samples of canine sera was carried out. Sera were collected and analysed for D. immitis antigens by the Istituto Zooprofilattico Sperimentale (IZS) of Piedmont, Liguria and Aosta Valley (Imperia section) between 2004 and 2013 during annual tests for leishmaniasis on autochthonous dogs throughout Liguria. Serological data from IZS showed an overall seroprevalence of 0.65% (95% CI: 0.50-0.80%) for D. immitis throughout the region. The present study updates the epidemiological map of canine filarial infections in Italy and suggests the need for surveillance and prophylaxis in Liguria.

Molecular and phylogenetic analysis of the filarial nematode Micipsella numidica from the hare Lepus europaeus in Italy.

The genus Micipsella comprises three species of filariae to date identified in lagomorphs only, whereas the other genera belonging to the subfamily Splendidofilariinae are described as parasites of birds, reptiles and mammals. In the present study seven specimens of Micipsella numidica (Seurat, 1917), collected from the hare Lepus europaeus in Italy, were characterized genetically by molecular amplification of the mitochondrial genes (12S rDNA; cox1) and the 5S rDNA gene spacer region. Phylogenetic trees inferred using available sequences from filariae and those identified in this study evidenced a close relationship between M. numidica and Splendidofilariinae of other mammals and reptiles (Rumenfilaria andersoni and Madathamugadia hiepei). The present findings, apart from adding new data about the hosts in Italy, support the taxonomic position of M. numidica and highlight the substantial biological and molecular differences existing between Splendidofilariinae and other Onchocercidae. The study also contributes to our knowledge of the molecular/genetic diagnosis of filarial parasites of veterinary and medical concern in any vertebrate or invertebrate host.

Comparative Studies on Nucleic Acid Based Biosensors for Identification of Filarial Nematode.

Background: The microfilarial nematodes, found mainly in blood circulation, form a special group of human and veterinary parasitic diseases. Recently, the rapid nucleic acid based biosensors (NABs) have been established in our laboratory for the employment of a point-of-care diagnostic test. Herein, NABs involved in the exploitation of lateral flow dipstick (LFD), gold nanoparticle (AuNP) and turbidity in combination with PCR and LAMP amplification for detection of filarial nematodes. The validation of each NABs was investigated in comparison to that of standard detection methods using the same unknown blood specimens. Objective: To compare the sensitivity, specificity and accuracy of NABs with standard detection methods. Material and Method: In this study, the microfilariae of Dirofilaria immitis was used as the representative model for filarial nematode. The PCR and LAMP primers were designed and synthesized according to the specific nucleotide regions of a small subunit gene of the parasite as well as the DNA probes. The fifty unknown blood samples were sent as a gift from Prasu Arthorn Animal Hospital, Faculty of Veterinary Science, Mahidol University. The samples were tested by using PCR, PCRLFD, PCR-AuNP, LAMP, LAMP-LFD, LAMP-AuNP, LAMP-turbidity and film blood smear. The efficacy of NABs was compared to a standard film blood smear in terms of sensitivity, specificity and accuracy. Results: Upon detection of fifty unknown blood samples, LAMP-LFD assay presented 100% of sensitivity, specificity and accuracy. The data revealed that sensitivity, specificity and accuracy of NABs varied from 66.67-100.00% when compared to a film blood smear stained with Giemsa dye. The data clearly indicated that LAMP-LFD was a preferred choice for the use as a point-of-care NABs. Conclusion: NABs were highly sensitive performing as a selective diagnostic tool that could be applied particularly as the rapid screening tests for filarial nematodes hence as a recommended epidemiological survey.

Intraperitoneal development of the filarial nematode Brugia malayi in the Mongolian jird (Meriones unguiculatus).

In the present study, we describe intraperitoneal development of the FR3 strain of Brugia malayi in Mongolian jirds (Meriones unguiculatus). The third molt for male worms occurred between 4 and 7 days postinfection (dpi) and between 4 and 8 dpi for females. The fourth and final molt occurred between days 21 and 29 for males and 25 and 34 for females, considerably earlier than the times reported for subcutaneous infection models using cats and jirds. The timing of the third molt coincided largely with reports for subcutaneous Brugia pahangi infections of cats and jirds, but the final molt occurred considerably later and lasted longer than those reported for subcutaneous B. pahangi models. Spermatogenesis occurred by at least 50 dpi in adult males, and insemination of females likely occurred between 50 and 60 dpi. Microfilariae were observed in the uteri and ovejectors of adult females at 65 dpi.

First report of Cercopithifilaria spp. in dogs from Eastern Europe with an overview of their geographic distribution in Europe.

Dogs in Europe may be parasitized by a variety of species of filarioids. While some species have been studied for decades, others have been only recently reported, and their range of distribution is still unknown. The present study was aimed to investigate the occurrence of Cercopithifilaria spp., whose microfilariae reside in the dermis and are transmitted by Rhipicephalus sanguineus s.l. ticks, in a dog population from Romania and to present the current knowledge of their distribution in Europe. Sampling was performed in a rural locality from Danube Delta region of Romania, location chosen due to the presence of the only known vector for these species. Skin samples were collected from 39 dogs. The samples were tested for the presence of Cercopithifilaria spp. by PCR methods. One animal (2.56%) was positive, and the amplified sequence showed a 100% similarity to Cercopithifilaria bainae. This study reports C. bainae for the first time in Eastern Europe, extending the known range of the genus in Europe.