Identity of Microfilariae Circulating in Dogs from Western and South-Western Romania in the Last Decade

Dirofilaria infections in dogs are recognized as widespread mosquito-borne diseases with zoonotic potential, that are caused by the filarioid nematode (Onchocercidae) species Dirofilaria immitis and Dirofilaria repens. The long-term survey conducted in western and south-western Romania was undertaken in order to provide valuable data on the occurrence of Dirofilaria infections in dogs. Blood samples from 1088 dogs, originating from 73 localities of 11 western and south-western counties in Romania, were collected and examined using the modified Knott test. Subsequently, all of the microscopically positive samples were subjected to molecular analysis for confirmation. Altogether, the data obtained showed the percentage of dogs with circulating microfilariae to be 21.42% (233/1088) of dogs tested in the investigated region. The identified species, in cases of monoinfections, were D. immitis, D. repens, and Acanthocheilonema reconditum in 106 (9.74%) samples, 102 (9.38%) samples, and 1 (0.09%) sample, respectively. Twenty-four (2.21%) samples were simultaneously positive for D. immitis and D. repens. There was no association (p > 0.05) between infection status and breed; however, sex and lifestyle were positively associated (p < 0.05) with the percentage of dogs with circulating microfilariae and might be regarded as risk factors for infection. The results of the present investigation indicate potential zoonotic risks for humans living in the screened area and support the imperative to increase awareness among both veterinarians and physicians, regarding the continuous spread of these zoonotic filariae.

Multiple vector-borne pathogens of domestic animals in Egypt

Vector Borne Diseases (VBDs) are considered emerging and re-emerging diseases that represent a global burden. The aim of this study was to explore and characterize vector-borne pathogens in different domestic animal hosts in Egypt. A total of 557 blood samples were collected from different animals using a convenience sampling strategy (203 dogs, 149 camels, 88 cattle, 26 buffaloes, 58 sheep and 33 goats). All samples were tested for multiple pathogens using quantitative PCR and standard PCR coupled with sequencing. We identified Theileria annulata and Babesia bigemina in cattle (15.9 and 1.1%, respectively), T. ovis in sheep and buffaloes (8.6 and 7.7%, respectively) and Ba. canis in dogs (0.5%) as well as Anaplasma marginale in cattle, sheep and camels (20.4, 3.4 and 0.7%, respectively) and Coxiella burnetii in sheep and goats (1.7 and 3%; respectively). New genotypes of An. centrale, An. ovis, An. platys-like and Borrelia theileri were found in cattle (1.1,3.4, 3.4 and 3.4%, respectively), An. platys-like in buffaloes (7.7%), An. marginale, An. ovis, An. platys-like and Bo. theileri in sheep (3.4, 1.7, 1.7 and 3.4%, respectively), An. platys, An. platys-like and Setaria digitata in camels (0.7, 5.4 and 0.7%, respectively) and Rickettsia africae-like, An. platys, Dirofilaria repens and Acanthocheilonema reconditum in dogs (1.5, 3.4, 1 and 0.5%, respectively). Co-infections were found in cattle, sheep and dogs (5.7, 1.7, 0.5%, respectively). For the first time, we have demonstrated the presence of several vector-borne zoonoses in the blood of domestic animals in Egypt. Dogs and ruminants seem to play a significant role in the epidemiological cycle of VBDs.

An epidemiological survey of Dirofilaria spp. and Acanthocheilonema spp. in dogs from the Republic of Moldova

Background During the last decades, filarial infections caused by Dirofilaria spp. have spread rapidly within dog populations of several European countries. Increasing scientific interest in filariasis, and the availability of new diagnostic tools, has led to improved knowledge of the biology, morphology, and epidemiology of different species of filarial worms. However, data are still scarce for a number of countries, including the Republic of Moldova. Thus, we assessed the epidemiological status of canine filariasis in the Republic of Moldova to address part of this knowledge gap. Methods A total of 120 blood samples were collected between June 2018 and July 2019 from dogs originating from the cities of Cahul and Chişinău. The samples were examined microscopically, and multiplex polymerase chain reaction was performed to evaluate filarioid species diversity. Results Microscopic examination revealed that 12 dogs (10.0%) were positive for circulating microfilariae. The molecular test showed that one dog was positive for Acanthocheilonema reconditum (0.8%), one for Dirofilariaimmitis (0.8%), six for Dirofilariarepens (5.0%), and four (3.3%) harboured a co-infection with D. immitis and D. repens. Prevalence was significantly higher in dogs aged ≥ 2 years. Conclusions The epidemiological survey presented here for the Republic of Moldova confirmed the presence D. immitis, D. repens and A. reconditum in dogs that had not received any heartworm preventive. Graphical abstract

Ocorrência de microfilaremia em cães (Canis familiaris) em municípios do Rio de Janeiro

Esse trabalho teve como objetivo realizar o levantamento epidemiológico de cães com microfilaremia em Centro Diagnóstico Veterinário localizado no município de Niterói, estado do Rio de Janeiro. A microfilaremia pode ser causada por várias espécies de filarídeos, como a Dirofilaria immitis, Dirofilaria repens e Acanthocheilonema reconditum. Essas espécies liberam microfilárias na corrente sanguínea de seu hospedeiro infectado, sendo a espécie que mais acomete os cães, Dirofilaria immitis, causadora da parasitose denominada dirofilariose canina. Para a realização deste estudo foram analisados 10.996 exames hematológicos, no período de janeiro a setembro de 2020, dos quais 292 foram positivos (2,66%) para microfilaremia, sendo 284 (97,26%) pela técnica do esfregaço sanguíneo, dois (0,70%) pela técnica da gota espessa e seis (2,05%) por ambos os métodos. Os dados foram coletados dos municípios de Niterói (36%), São Gonçalo (30,5%), Maricá (29,5%) e Itaboraí (4,1%). O mês de janeiro teve 42 casos positivos (3,77%), fevereiro 34 (3,48%), março 9 (0,86%), abril 41 (4,15%), maio 42 (3,54%), junho 40 (2,45%), julho 48 (3,45%), agosto 17 (1,12%) e setembro 19 (1,37%). Dentre os animais positivos para microfilaremia 147 eram machos (50,3%), 144 fêmeas (49,3%) e 1 não foi informado (0,3%), com idade média de 6 anos, não tendo sido encontradas diferenças estatísticas entre os gêneros e raças dos animais. Face ao exposto a média de casos positivos para microfilaremia canina foi de 2,65% do total de exames analisados, ressaltando a importância dos achados hematológicos e da solicitação de métodos mais sensíveis na rotina clínica.

Epidemiological Survey of Dirofilaria Spp. and Acanthocheilonema Spp. in Dogs from the Republic of Moldova

Background: During the last decades, the spread of the infection with filarial nematodes Dirofilaria spp. in dog’s population in various European countries has been dynamic and rapid. The increased interest of the scientific community as well as the availability of new diagnosis tools, allowed a better knowledge on the biology, morphology and epidemiology of different species of filarial worms. However, in some countries, data are still scarce on this topic. To address some knowledge gaps, we assessed the epidemiological status of different canine filariasis in the Republic of Moldova. Methods: A total of 120 blood samples were collected between June 2018 and July 2019 from dogs originating from Cahul and Chișinău cities. The samples were microscopically examinated and multiplex PCR was performed to evaluate filarioid species diversity. Results: Microscopic examination revealed that 12 dogs (10%) were positive for circulating microfilariae. Molecular test showed that 1 dog was positive for Acanthocheilonema reconditum (0.83%), 1 for D. immitis (0.83%), 6 for D. repens (5%), and 4 (3.33%) harboured a coinfection with D. immitis and D. repens. Conclusions: This study is the first epidemiological survey of filaroid worms in dogs from the Republic of Moldova and the first to show the presence of D. repens and A. reconditum in this species.

Haematological and biochemical abnormalities in hunting dogs infected with Acanthocheilonema reconditum, associated risk factors, and a European overview

Acanthocheilonema reconditum is a filarial parasite transmitted by arthropods (fleas, lice, and ticks) that infect dogs. There is minimal published data available to date on potential haematological and biochemical changes associated with this parasitic infection. Study aims were (i) provide an overview of A. reconditum in Europe, (ii) define A. reconditum prevalence and risk factors in a specific dog population (hunting) from southern Italy, and (iii) assess the frequency of haemato-biochemical abnormalities associated with infection. Blood samples collected from 3020 dogs were tested by a modified Knott’s technique to count and identify microfilariae. Eighty-four dogs were infected by A. reconditum (2.78%; 95% CI 2.19–3.37%). Microfilariae ranged from 1 to 212/ml. Based on clinical examination, all but six dogs with non-specific symptoms were healthy. Haematological abnormalities included leucocytosis (n = 15), with eosinophilia (n = 14) and monocytosis (n = 13). Serum biochemical abnormalities included increased total serum proteins (n = 19), albumins (n = 7), total globulins (n = 14), ALT (n = 1), and ALP (n = 1); one dog was hypoalbuminemic, and BUN was mildly increased in 2 dogs. Risk factors included the province origin (Napoli, OR=5.4, 95%CI: 2.1–14.0; Caserta, OR=5.1, 95%CI: 2.5–10.6), hunting wild mammals (OR=2.8, 95% 95%CI: 1.6–4.8), and ectoparasite infestation (OR=1.9, 95%CI: 1.1–3.1). There was a negative correlation between microfilaraemic load and decreased albumin level (−0.37; p=0.021). Our results showed that A. reconditum circulates within the hunting dog population of southern Italy, with seemingly low pathogenic potential.

Surveys of Canine Filarioids In Nigeria: The Path Travelled and the Way Forward

Filarioid worms infecting dogs have recently received increased attention globally because of their zoonotic potential. In Africa and, particularly, in Nigeria, however, where there is preponderance of the risk factors for vector-borne diseases transmission, there are few reports of the disease in the canid and felid definitive hosts, the wild/domestic reservoirs and humans. Thus, the epidemiology of the disease in Nigeria remains sketchy and needs to be investigated. A retrospective analysis of reported canine filarioids in Nigeria was undertaken with the view to highlight what has been done and reported, existing gaps in knowledge, what needs to be done to bridge the gap and possibly how it could be done. Thirteen published works on canine filarioids using classical laboratory methods in Nigeria, reported the finding of Dirofilaria immitis (0.4–15.1%), Dirofilaria repens (0.1–9.4%), Acanthocheilonema reconditum (0.4–9.2%) and a case of A. dracunculoides in an unspecified dog population. In most instances, the species identification of the filarioids reported was not conclusive due to limitation of the diagnostic methods employed. No human infection due to any zoonotic canine filarioid has been reported in Nigeria. Suggestions for prospective filarioids research in Nigeria were made. Keywords: Filarioids, dog, zoonosis, diagnosis, Nigeria

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.

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

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

Molecular Approach for the Diagnosis of Blood and Skin Canine Filarioids

The zoonotic Onchocerca lupi and tick-transmitted filarioids of the genus Cercopithifilaria remain less well known due to the difficulties in accessing to skin samples as target tissues. Here, we proposed a molecular approach reliying on multiplex qPCR assays that allow the rapid identification of filarioids from canine blood, skin, and tick samples. This includes two newly developed duplex qPCR tests, the first one targeting filarial and C. grassii DNA (CanFil-C. grassii). and the second qPCR assay designed for the detection of Cercopithifilaria bainae and Cercopithifilaria sp. II DNAs (C. bainae-C.spII). The third one is a triplex TaqMan cox 1 assay targeting DNA of blood microfilariae (e.g., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum). The novel duplex qPCRs developed were validated in silico and by screening of known DNA collection. The qPCR assays were also used for screening the blood and tick samples of 72 dogs from Algeria. This allowed the identification of canine filariasis infection with 100% of specificity and 89.47% and 100% of sensitivity from naturally infected blood and tick samples, respectively. The prevalences of 26.39% for D. immitis and 5.56% for both D. repens and A. reconditum were reported in blood and tick samples. Cercopithifilaria DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for C. bainae and Cercopithifilaria sp. II, respectively. Co-infections were diagnosed in 6.94% and 13.89% of blood and tick samples, respectively. Whereas all samples were negative for C. grassii DNA. The use of engorged ticks instead of blood and skin samples could be an easier option for the surveillance of all canine filarioids herein investigated. The multiplex qPCR assays herein validated were shown to be useful in the detection of filarial co-infections by overcoming sequencing of positive samples.