Human Dirofilaria repens infection in Hungary: a case in the spermatic cord and a review of the literature

Orchiectomy was performed in a 37-year-old Hungarian man exhibiting a swelling in his right testicle. Histology revealed a nodule attached to the spermatic cord, consisting of a granulomatous tissue around sections of a nematode. The worm was identified as Dirofilaria repens, an uncommon parasite in Hungary. As the patient had been abroad only in Italy where cases of dirofilariosis in dogs and humans are relatively frequent, it is assumed that the infection might have been acquired in that country 5 years earlier. This is the fifth case, published so far in the world, of such a localization in a human. The human cases of dirofilariosis reported in Hungary are reviewed.     

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

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

Ocular dirofilariosis by Dirofilaria immitis in a dog: first case report from Europe

A five‐year‐old, entire female mixed‐breed dog was presented with corneal oedema and episcleral hyperaemia in the left eye. The ophthalmological examination revealed the presence of a free‐swimming nematode in the anterior chamber. Circulating microfilariae were not observed by a modified Knott test nor were adult antigens detected in serum by a commercial ELISA. The parasite was surgically removed from the dog's eye, but its anterior end was damaged during the surgery. Based on the morphology of the posterior end, the nematode was preliminarily identified as a male Dirofilaria immitis. The species identification was confirmed by PCR amplification and sequencing of the mitochondrial coxI and 12S rDNA genes, using a DNA barcoding approach. Although other cases of ocular dirofilariosis by D. immitis have been previously recorded in Australia and the United States, the case reported herein is the first in a dog from Europ.     

Serological survey of canine dirofilariosis in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning in Southern China

The present study conducts a serological survey on the presence of canine dirofilariosis in domestic dogs using an enzyme-linked immunosorbent assay (ELISA) kit. A total of 310 household dogs (166 females and 144 males) in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning in Southern China were examined. Of the 310 dogs, 42 (13.5%) were seropositive for dirofilariosis. No statistically significant difference was observed in terms of sex in the seroprevalence of dirofilariosis using the ELISA kit. The positive rates for dirofilariosis were 6.6% in the 0-1-year-old group, 13.8% in the 1-4-year-old group, and 21.6% in the less than 4-year-old group. The statistical analysis revealed that significant differences were observed in the 1-4-year-old group (P=0.037, OR=0.441, 95% CI=0.170-1.144) and less than 4-year-old group (P<0.001, OR=0.256, 95% CI=0.095-0.693). In the regional comparison, the shoreline city Shenzhen (18.8%) had a significantly higher prevalence than urban and mountain areas (P<0.05, OR=0.310, 95% CI=0.066-1.445). In conclusion, Dirofilaria immitis infection in domestic dogs was present in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning. Therefore, heartworm treatment and/or chemoprophylaxis for the captured domestic dogs are necessary in these areas. To the best of our knowledge, the present study is the first using serological methods to examine D. immitis infection in domestic dogs in Mainland China in the recent years.     

Efficacy of a single administration of a spot-on solution containing imidacloprid 10%/moxidectin 2.5% in eliminating Dirofilaria repens microfilariae in naturally infected dogs

In the past decade reports of canine subcutaneous dirofilariosis, caused by the mosquito-transmitted nematode Dirofilaria repens, increased in number in several countries in Europe, along with a rise of human cases. Given the merit to the new approaches for the control and treatment of this infection, the present study evaluated the efficacy of a single application of the spot-on formulation containing imidacloprid 10%/moxidectin 2.5% (Advocate(?), Bayer Animal Health) in the elimination of D. repens microfilariaemia in naturally infected dogs. In September 2009, 18 dogs with a natural infection by D. repens were enrolled in the study. In October 2009 all the dogs were treated once with Advocate(?) and the presence/absence of circulating MF and skin lesions after treatment was evaluated monthly until April 2010. From November 2009 to April 2010 15 dogs scored negative for D. repens while one dog remained negative till March 2010 when it died. Two dogs had a recurrence of microfilariaemia in December 2009 and January 2010 respectively. Nine infected dogs showed skin lesions at the beginning of the trial, which disappeared after treatment in 7 dogs, whereas the other two symptomatic dogs did not show any dermatological improvement until the end of the trial even though they scored negative for D. repens microfilariae. This study demonstrated that a single dermal administration of Advocate(?) is effective in eliminating microfilariae of D. repens and likely has a certain degree of activity in killing subcutaneous adult worms as well. This study demonstrates the efficacy of Advocate(?) in the treatment of dermatitis caused by D. repens. Also, these results are of importance towards further control programs aiming to reduce the number of bites infectious for mosquitoes and the risk of infection for both humans and dogs.     

Preliminary findings on the efficacy of selamectin in the treatment of dogs naturally infected with Dirofilaria repens

Subcutaneous dirofilariosis caused by Dirofilaria repens is common in dogs and it is an emerging helminthozoonosis in Europe, Asia, Africa and also in Hungary. Macrocyclic lactones are used for preventing the infection; however, their activity against the microfilariae and mature stages of this species is questionable. Selamectin is widely used for the prophylaxis of heartworm (D. immitis) infection. The objective of the present study was to test the microfilaricidal efficacy of the topical formulation of selamectin in dogs naturally infected with D. repens . A total of 78 Beagle dogs were examined for the presence of circulating microfilariae by Knott's test. Twenty-three of the microfilaraemic dogs were divided into four groups and included in the trial. The dogs received monthly or biweekly selamectin treatment and were subjected to monthly blood testing for a period of 252 or 336 days. At the end of the study, 65% of the dogs were not microfilaraemic and the rest had low number of microfilariae in their blood. These results indicate that chronic spot-on selamectin treatment may be a useful tool also in the control of canine subcutaneous dirofilariosis.     

Prevalence of Dirofilaria immitis infection in dogs from Dandong, China

The aim of the present study was to estimate the prevalence and risk factors of Dirofilaria immitis infection in dogs from Dandong, China. A total of 886 dogs were examined for D. immitis infection by microscopic examination and PCR, indicating that the prevalence was 16.6% (213/886) and 24.0% (147/886), respectively. The odds of infection were significantly higher in older dogs and dogs sheltered in outdoor, compared to the younger ones and ones sheltered in indoor. No significant difference of infection was observed in different genders, and between pure breed and cross-breed dogs in the same rearing conditions. These results indicated that the risk of exposure to D. immitis in dogs is high in Dandong, China, and prophylaxis against the parasite is advisable to decrease the incidence of canine dirofilariosis.     

Dirofilaria repens infection in a dog imported to Norway

Dirofilaria repens infection was diagnosed in a dog that had been imported to Norway from Hungary three years previously. The dog was a four-year-old castrated male mixed-breed dog and presented for examination of two masses on the right thoracic wall. Fine needle sampling from the subcutaneous nodules and subsequent cytological examination revealed a high number of microfilariae and a pyogranulomatous inflammation. At re-examination approximately 3 weeks later, both masses had apparently disappeared spontaneously, based on both inspection and palpation. However, examination of peripheral blood by a modified Knott’s test revealed a high number of unsheathed microfilariae with mean length of 360 μm and mean width of 6-7 μm, often with the classic umbrella handle appearance of D. repens. Polymerase chain reaction and sequencing confirmed the D. repens diagnosis. Subcutaneous dirofilariosis caused by D. repens is probably the most common cause of human zoonotic dirofilariosis in Europe, but currently is rarely encountered in northern countries such as Norway. However, travelling, import and relocation of dogs have increased, and thus the geographical range of these parasites is likely to increase from traditionally endemic southern regions. Increasing numbers of autochthonous cases of D. repens infections in dogs have been reported in eastern and central Europe. Although infection with D. repens often induces only mild signs or subclinical infections in dogs, they nevertheless represent a reservoir for zoonotic transmission and thus a public health concern, and, in addition, due to the long prepatent period and the high frequency of subclinical infections or infections with unspecific clinical signs, could easily be missed. Lack of experience and expectation of these parasites may mean that infection is underdiagnosed in veterinary clinics in northern countries. Also, predicted climate changes suggest that conditions in some countries where this infection is currently not endemic are likely to become more suitable for development in the intermediate host, and thus the establishment of the infection in new areas.     

Intron 1 of the interferon gamma gene: Its role in nematode resistance in Suffolk and Texel sheep breeds

Genetic variation at intron 1 of the interferon gamma gene has recently been associated with variation in faecal egg count in Romney, Merino and Soay breeds of sheep. The Texel breed is more resistant to gastrointestinal nematode infection than the Suffolk breed, based on faecal egg count. Hence, the objective of this experiment was to characterise the polymorphisms of intron 1 of the interferon gammagene in Suffolk and Texel sheep, and to determine if the characterised haplotype variants were associated with faecal egg count variation in these breeds. Intron 1 haplotypes and faecal egg measurements were determined for Suffolks (n=113) and Texels (n=135). Four haplotypes were identified in Suffolks (A, B, C and D), two haplotypes as previously described, and two previously unknown haplotypes. However, there was no association between interferon gamma intron 1 haplotype and faecal egg count in Suffolks. In contrast, two previously identified haplotypes (A and B) were present in the Texel breed, and the B haplotype was associated with resistance to nematode infection (P=0.02). These results suggest that intron 1 of the interferon gamma gene has an important role in resistance to nematode infection in the Texel breed.     

Intron 1 of the interferon γ gene: Its role in nematode resistance in Suffolk and Texel sheep breeds

Genetic variation at intron 1 of the interferon gamma gene has recently been associated with variation in faecal egg count in Romney, Merino and Soay breeds of sheep. The Texel breed is more resistant to gastrointestinal nematode infection than the Suffolk breed, based on faecal egg count. Hence, the objective of this experiment was to characterise the polymorphisms of intron 1 of the interferon gammagene in Suffolk and Texel sheep, and to determine if the characterised haplotype variants were associated with faecal egg count variation in these breeds. Intron 1 haplotypes and faecal egg measurements were determined for Suffolks (n=113) and Texels (n=135). Four haplotypes were identified in Suffolks (A, B, C and D), two haplotypes as previously described, and two previously unknown haplotypes. However, there was no association between interferon gamma intron 1 haplotype and faecal egg count in Suffolks. In contrast, two previously identified haplotypes (A and B) were present in the Texel breed, and the B haplotype was associated with resistance to nematode infection (P=0.02). These results suggest that intron 1 of the interferon gamma gene has an important role in resistance to nematode infection in the Texel breed.     

First molecular detection of Dirofilaria immitis (Spirurida: Onchocercidae) infection in dogs from Northern Algeria

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioid nematodes that affect dogs and other domestic and wild carnivores, causing heartworm disease and subcutaneous dirofilariosis, respectively. In Algeria, the data about the epidemiology of these infections is largely unknown. The present study was designed to establish the occurrence of D. immitis and D. repens in dogs in Algeria using molecular tools.In 2014 and 2015, a total of 209 dogs over one year of age of different breed and sex, living in Northern Algeria, were examined and blood samples were collected from each dog. The presence of D. immitis and D. repens in these samples was detected by real-time PCR followed by standard PCR and sequencing. Overall, the blood of 209 dogs from two departments was collected and only 3 (1.4%) of the blood samples were found positive for D. immitis DNA. Sequencing of the corresponding amplicon displayed a 99.8% identity to D. immitis, confirming the presence of this mosquito-borne nematode in Algeria. Furthermore, all tested samples were negative for D. repens.     

Serological and molecular survey of Dirofilaria immitis infection in stray cats in Gyunggi province, South Korea

The aim of this study was to carry out a serological and molecular survey for the presence of Dirofilaria immitis infection in stray cats using an ELISA kit and PCR assay. One hundred and fifty-five stray cats (77 females and 78 males) in Gyunggi province in South Korea, were used in this study. Four (2.6%) tested with the ELISA kit showed a positive reaction, and all positive samples by the ELISA kit showed a positive reaction by PCR analysis. No significant difference was observed between the male (2.6%) and female (2.6%) cat groups by ELISA kit. The positive rates for dirofilariosis were 2.8% in the 4-6-year-old group, and 18.2% in the > 6-year-old group by ELISA kit. With regard to the age element, older cats showed a higher prevalence of D. immitis infection in this study. A statistical analysis revealed that significant difference was observed in > 6-year-old group (p < 0.01). In conclusion, D. immitis infection in stray cats was present in Gyunggi province, although its incidence was low. Therefore, heartworm treatment and/or prophylaxis for stray cats captured are required in this area.     

Effect of nematophagous fungus Duddingtonia flagrans and energy supplementation on the epidemiology of naturally infected kids

Gastrointestinal (GI) nematode infection is a major constraint for grazing livestock production. The increasing prevalence and severity of anthelmintic-resistant nematodes in many parts of the world has led to a search for non-chemical control options. Under experimental conditions, the nematophagous fungus Duddingtonia flagrans is emerging as an alternative to chemotherapy for the control of GI nematode infection in biological production systems. Also, recent information points to the role of energy nutrition to increase the immune response against GI nematode infection. In this study the effect of D. flagrans and energy supplementation on the epidemiology of GI nematode infections is explored on grazing kids. Four groups of 10, 4-month old goats were turned out on infected pasture in the early spring and allocated to four separate paddocks where they were rotationally grazed for 16 weeks. One of these groups (F) received 0.5 x 10(6) D. flagrans spores/kg BW/d. Another group (S) was supplemented with 100 g barley grain per day. A third group (F+S) received both nematophagous fungi and barley supplement treatments simultaneously while the fourth group (C) was used as a non-treated control. Both nematophagous fungi and barley supplement had a significant effect (P<0.01) on reducing pasture infectivity, faecal egg excretion and worm burdens at slaughter that was particularly evident for Trichostrongylus colubriformis. The combination of both treatments showed a synergistic effect on the control of gastrointestinal nematode infections. At slaughter, the average total post-mortem worm count of the F+S group was reduced by 65% compared with the non-treated control. The results herein show that D. flagrans can act as an efficient biological control agent against kid GI nematode infections on pasture, which could further improve carcass characteristics. While small amounts of energy supplement can also reduce kid infection, the effect of D. flagrans as a biological control agent appeared clearly enhanced both in magnitude and duration by energy supplementation. This has clear implications for grazing animals and provides an efficient method for the practical control of parasitic nematodes in biological production systems.     

Oxidant/antioxidant balance, minerals status and apoptosis in peripheral blood of dogs naturally infected with Dirofilaria immitis

The present study was aimed to examine lipid peroxides, antioxidants, zinc, copper, iron panels and apoptosis in peripheral blood of dogs with clinical dirofilariosis. In comparison to healthy control, erythrocytic lipid peroxides level and superoxide dismutase activity were significantly (P<0.01) higher in diseased dogs. However, level of reduced glutathione and activities of glutathione peroxidase, glutathione-S-transferase and catalase were significantly (P<0.01) lower in diseased dogs. Blood zinc, copper and iron levels in dogs with dirofilariosis were significantly (P<0.01) lower than the healthy control. The percent apoptotic peripheral leukocytes were significantly higher in dogs with dirofilariosis in comparison with healthy control. From the present study, it is concluded that significant alterations in oxidant/antioxidant balance, mineral status and rate of apoptosis in peripheral blood may be implicated in the pathogenesis of clinical canine dirofilariosis.     

Canine dirofilariasis in the canton of Ticino and in the neighboring areas of northern Italy

The distribution of canine dirofilariosis in Southern Ticino (Switzerland) and in the neighbouring provinces of Varese and Como (Italy) was investigated. Blood samples were collected from 308 dogs which had remained in the local area and were outdoor-housed, older than 1.5 years and had not been treated previously with preventive or microfilaricidal drugs. Microfilariae of Dirofilaria immitis and D. repens were found in 33 (10.7%) and 17 (5.5%) dogs, respectively. Ten more dogs (3.2%) tested positive for circulating antigens. Four infected dogs lived in Southern Ticino: two harboured D. immitis, one D. repens and one had a mixed infection. In addition, 3887 mosquitoes were captured in five sample sites by means of dog-baited traps. Culex pipiens, Aedes geniculatus and Ae. vexans were the most abundant species. Infective stages of D. immitis were observed in local strains of Ae. geniculatus and Cx. pipiens, following engorgement on a microfilaraemic dog and the successive rearing in laboratory conditions. A chemo-prophylactic scheme in four administrations between July and October is recommended for Southern Switzerland.     

Autochthonous canine Dirofilaria repens in the vicinity of Warsaw

In 2009 microfilaremia was recognized in ten dogs living in five distinct districts located near Warsaw, the capital of Poland. Based on PCR results, all the animals were found to be infected with Dirofilaria repens. Nine of the infected dogs have never travelled outside the country and it was assumed that the cases were native. Monitoring the infection in the European countries should be introduced to establish the actual geographic range of dirofilariosis.     

Geographical information systems as a tool in the control of heartworm infections in dogs and cats

Geospatial tools (e.g., geographical information systems, remote sensing, global positioning systems, and virtual globes) are very useful for the simultaneous visualization of health data with environmental data, which holds promise to understand environmental-health linkages and to generate new hypotheses to be tested in future research. Current epidemiological studies clearly show that the distribution patterns of vector-borne infections are changing; for example, in Europe, heartworm infection and subcutaneous dirofilariosis are spreading throughout areas that previously had little to no incidence of heartworm. In view of the changes of the distribution patterns of Dirofilaria immitis and Dirofilaria repens, geospatial tools are now more useful for mapping (including territorial sampling), monitoring, ecological analysis, risk assessment, forecasting (including the choose of the timing of treatment), early warning, and surveillance of both heartworm and subcutaneous dirofilariosis. All these issues have control of these infections as the ultimate goal.     

Feline dirofilariosis: antibody response to antigenic fractions containing specific 20 to 30 kDa polypeptides from the adult Dirofilaria immitis somatic antigen.

Fractions from the adult somatic antigen (SA) Dirofilaria immitis complex, containing polypeptides from 20 to 30kDa, previously identified as molecular markers of feline dirofilariosis are isolated by sequential application of gel filtration and anion exchange chromatography. Indirect enzyme-linked immunosorbent assays, employing these fractions (20-26kDa/ELISAF1 and 30kDa/ELISAF7) show multivalent diagnostic capacities: they were able to detect pre-patent infections 2 months after infection, infections in clinical phase, and the fall of antibodies after the worms were removed from the heart, or the application of a ivermectin treatment. The results obtained by the two tests correlated well, in spite of the fact that ELISAF1 was most useful to detect antibodies in sera from cats in the clinical phase, while ELISAF7 has more sensitivity for the early detection of the infections. Both ELISAs were useful in the detection of the decrease of antibodies after the worms were removed by surgery or pharmacological treatment.     

Dispharynx nasuta (Nematoda:Acuariidae) infection causing proventricular lesions and death in three captive rosellas (Psittaciformes:Psittacidae)

Two adult male Crimson rosellas (Platycercus elegans) and an adult female eastern rosella (Platycercus eximius) were found dead in their cages in a private aviary in March 2009, April 2010, and February 2010, respectively, without premonitory signs. Their postmortem examination showed pectoral muscle atrophy and a distended proventriculus. The proventricular mucosal surface was covered with a gelatinous, whitish material, where a large number of nematode parasites identified as Dispharynx nasuta were present. A histologic examination revealed a hyperplastic mucosa, spirurid nematodes in the mucosal layer and inflammatory mononuclear cells in the lamina propria. This is the first report of a D. nasuta infection associated with proliferative proventriculitis and subsequent death in psittacine birds.