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.

False positive antigen test for Dirofilaria immitis after heat treatment of the blood sample in a microfilaremic dog infected with Acanthocheilonema dracunculoides.

BACKGROUND: Dirofilaria immitis is responsible for heartworm disease in dogs in endemic areas worldwide. Screening for this infection is done by blood tests. Antigen testing is the most sensitive method to detect an infection with adult (female) worms. Microscopic examination of a blood smear or Knott's test can be used to detect circulating microfilariae, the infective larvae. To increase the sensitivity of the antigen test by decreasing the false negative test results, heating of the blood sample has been recommended in recent guidelines. Heating is believed to remove blocking immune-complexes. Circulating microfilariae are not specific findings for heartworm infection, as other nematodes (among others, Acanthocheilonema dracunculoides) can also result in microfilaremia. Although the type of microfilariae cannot be determined by microscopy alone, real-time PCR can reliably identify the infecting nematode species. Correct identification of the parasite is of major importance, as an infection with D. immitis requires antiparasitic therapy, whereas A. dracunculoides is thought to be a clinically irrelevant coincidental finding. The present case report describes a microfilaremic dog where the initial antigen test for D. immitis turned positive after heat treatment, whereas real-time PCR revealed that the microfilariae were A. dracunculoides (syn. Dipetalonema dracunculoides). RESULTS: A circa 5-year old, asymptomatic Spanish mastiff dog was referred for heartworm therapy because microfilariae were found via a screening blood test. The dog was recently imported to the Netherlands from Spain, where it had been a stray dog. Antigen tests on a plasma sample for D. immitis were performed with three different test kits, which all turned out to be negative. However, heat treatment of two of these samples were carried out and both of them led to a positive antigen test result. Real-time PCR showed that the circulating microfilariae belonged to A. dracunculoides species. Three administrations of moxidectin spot-on at monthly intervals resulted in a negative antigen and a negative Knott's tests one month after the last treatment. CONCLUSIONS: We conclude that heat treatment of initially negative blood samples for D. immitis could lead to false positive antigen test results if the dog is infected with A. dracunculoides.

Canine filarial infections in an area of Central Italy (Tuscany-Latium border) historically free from the disease.

This paper reports the presence of various species of filarial nematodes in dogs in an area of central Italy at the border between two regions (Tuscany-Latium), which had previously been free of infection and for which there are no epidemiological data in the literature. Blood and serum samples from a total of 363 adult dogs were collected during in 2016/17 and 2018/19 in the provinces of Grosseto (Tuscany) and Viterbo (Latium). The dogs were of different breeds or mixed breeds, randomly selected, and living in public kennels or privately owned. Of the 363 samples tested, 55 dogs were positive to Knott's test for microfilariae (prevalence 15.1%; 95% confidence interval: 11.5-18.8%). Dirofilaria immitis was found in 25 cases (prevalence 6.9%; 95% CI 4.3-9.5%), D. repens in 27 (7.4%, 95% CI 4.7-10.1%) and Acanthocheilonema reconditum in 3 (0.8%, 95% CI 0.1-1.7%). Out of 55 dogs, only four were infested with both D. immitis and D. repens (1.1%, 95% CI 0.001-2.2%).

Filarial infections in California sea lions vary spatially within the Gulf of California, Mexico.

At least two species of filarial worms, Dirofilaria immitis and Acanthocheilonema (Dipetalonema) odendhali, infect otariid pinnipeds, including the California sea lion (Zalophus californianus). To date, evidence of infection in sea lions has come from dead or captive animals, and little is known about filariasis in free-living populations. We sampled 45 California sea lion adults and 197 pups captured at 12 rookeries from different ecological regions within the Gulf of California and detected and quantified D. immitis and A. odendhali microfilariae in blood smears. We investigated differences in prevalence and parasite load (intensity of infection) among ecological regions. Microfilariae were detected in the blood of 35 of the 45 (77.78%) adult females and in 1 of the 197 (0.51%) pups examined. The average burden of A. odendhali per microlitre of blood was nearly twice that of D. immitis. Prevalence and intensity of infection differed significantly among regions, being highest for colonies within the northern and northcentral regions and lowest in the southern region. Dirofilaria immitis and A. odendhali infections displayed a similar spatial pattern of prevalence. Colony density inversely predicted the prevalence of microfilariae. Based on the clinical parameters typically associated with filarial infections in carnivores and physical examinations, none of the sea lions appeared to have evidence of disease. This is a first approximation to investigate the prevalence of microfilaria infections in free-ranging California sea lions and to explore their relevance to population health.

Massive microfilaremia in a dog subclinically infected with Acanthocheilonema dracunculoides.

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 post-mortem 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.

Filarial infections in dogs in Cyprus, an apparently heartworm free island.

The current study investigated for the first time the occurrence of filarial infections in dogs in Cyprus. Blood samples of dogs from five districts of Cyprus (Lefkosia, Lemessos, Larnaka, Pafos and Ammochostos) were examined by the modified Knott's method and by serology, and the morphological classification of microfilariae was confirmed by molecular methods. A total of 200 dogs, 153 living in shelters and 47 owned dogs, were included in the study. Acanthocheilonema reconditum microfilariae were found in 9 (4.5%) samples and one (0.5%) sample was seropositive for D. immitis. No statistical significance was observed between microfilaraemic samples and the evaluated variables apart from the district (p = .005). The present study showed that dogs in Cyprus can be infected with blood circulating microfilariae and for the first time A. reconditum was reported as autochthonous infection in dogs in the country. No microfilariae of Dirofilaria spp. were detected. However, veterinarians should remain vigilant regarding Dirofilaria infections and should consider preventive protection to the animals, at least in case of travel in enzootic areas.

Filarioid infections in wild carnivores: a multispecies survey in Romania.

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.

Mosquito-borne heartworm Dirofilaria immitis in dogs from Australia.

BACKGROUND: Heartworm (Dirofilaria immitis) in dogs is considered endemic in Australia, but the clinical heartworm disease caused by the heartworm is rare and prevalence is low. The mainstream prevention of the heartworm is based on macrocyclic lactone (ML) administration. The aim of this study was to confirm endemism of the heartworm under current Australian conditions using a cohort of recent microfilaria-positive dogs which were on variable heartworm prevention. METHODS: A hotspot of canine heartworm antigen-positive and microfilaria-positive dogs has been detected recently in Queensland, Australia. Blood samples from 39 dogs from Queensland and two dogs from New South Wales were investigated for canine filarioids. Rapid antigen diagnostic tests capable of detection of D. immitis and real-time PCR for quantification and differentiation between D. immitis from Acanthocheilonema reconditum with quantification of microfilariae in canine blood samples, together with D. immitis specific real-time PCR assay, were applied to microfilaria-positive dogs. The P-glycoprotein genotype was determined to test whether Australian-sourced heartworm shared the same genetic markers as those suspected of ML-resistance in North America. RESULTS: Only D. immitis was detected in the samples from Queensland and New South Wales, Australia. Using high resolution melt real-time PCR and D. immitis specific real-time PCR, the calculated microfilaria concentration ranged from 1 to 44,957 microfilariae/ml and from 7 to 60,526 microfilariae/ml, respectively. DNA sequencing of the PCR products confirmed D. immitis. Fifteen of the examined dogs were on putative, rigorous ML prevention. For the remaining dogs, compliance with heartworm prevention was unknown or reported as inconsistent. Wild-type genotype AA-GG of the P-glycoprotein locus of D. immitis sequence has been obtained for three blood samples. Due to the incomplete history, any suggestion of a loss of efficacy of MLs must be treated as 'remotely possible'. In the immediate future, records of preventative administration and annual antigen testing would be required to determine any problems with the efficacy of preventatives. CONCLUSIONS: The prevalence of canine heartworm in Australia remains poorly understood. It is generally assumed to be low by veterinary practitioners. The localised increase in the study area confirms endemism of canine heartworm and a requirement for ongoing vigilance through annual heartworm testing to better understand the changing distribution of canine heartworm, client compliance, as well as to detect any change in ML-susceptibility.

Dirofilaria infections in dogs in different areas of Greece.

BACKGROUND: The nematodes Dirofilaria immitis and D. repens are enzootic in Greece. In the light of evidence of dirofilariosis spreading to new areas around the world, the aim of the present study was to update and enrich the current knowledge on the prevalence of Dirofilaria infections in dogs in Greece, to assess the risk factors of heartworm infection, and to initiate the drawing of the epizootiological map of canine dirofilariosis, investigating Dirofilaria infections in five locations along the north-south axis of the country, i.e. municipalities of Thessaloniki, Larissa, Achaia, Attica and Heraklion, associated with the five largest urban centres of Greece. METHODS: Blood samples collected from 750 dogs in total, were examined by the modified Knott's method and by serology. A questionnaire including information about each examined dog was filled in and statistical analysis of the results was performed using the Chi-square test and a Binary Multiple Univariate Generalized Linear Model. RESULTS: In total, 31 (4.1 %) out of 750 examined animals were found positive for D. immitis by any of the tests applied (Knott's method and serological examination). Moreover, microfilariae of D. repens and Acanthocheilonema reconditum were detected by the Knott's method in 17 (2.3 %) and 10 (1.3 %) of the animals, respectively. At the municipality level, the prevalence of infection was 14, 7, 5.3, 0.7 and 0 % for D. immitis, 1, 2, 8.7, 0.3 and 0 % for D. repens, and 0, 3, 2.7, 0.7 and 1 % for A. reconditum in Thessaloniki, Larissa, Achaia, Attica and Heraklion, respectively. In addition, in three dogs (one each in Thessaloniki, Achaia and Attica) mixed D. immitis - D. repens infections were detected by the Knott's method. The area of the country, dog's usage and age were determined as risk factors for heartworm infection. CONCLUSIONS: Northern areas of Greece have higher Dirofilaria prevalence and the prevalence in a western province (Achaia) is recorded for the first time. The mosquito population dynamics recorded in the past is likely to play an important role in the distribution of Dirofilaria infections in Greece, and needs further investigation. Similarly, the role of wild reservoirs of filarial parasites in different areas needs to be clarified. Promotion by veterinarians of preventive treatment and compliance by pet owners is essential in all parts of Greece, regardless of the recorded prevalence of infection.

Filarial infections in domestic dogs in Lusaka, Zambia.

Filariae are common parasites of dogs in many parts of the world, but little is known about the status of these infections in sub-Saharan Africa. A study was carried out to determine the occurrence and species of filariae among 272 dogs in Lusaka, Zambia. Giemsa stained blood smear and Knott's concentration methods revealed microfilariae in 16 (5.9%) of the dogs. PCR confirmed that most of these dogs had Acanthocheilonema reconditum infection. Ten (4.0%) of the examined dogs were positive for Dirofilaria immitis circulating antigen (by DiroCHEK(®) test), but D. immitis microfilariae were not identified in any of the dogs and the status of this infection remains unclear. Further studies are needed to explore the occurrence of filariae in Zambian dogs and the zoonotic potential for humans.

Current surveys on the prevalence and distribution of Dirofilaria spp. and Acanthocheilonema reconditum infections in dogs in Romania.

During the last decades, Dirofilaria spp. infection in European dogs has rapidly spread from historically endemic areas towards eastern and northeastern countries, but little or no information is available from these geographical regions. The present study provides a picture of filarial infections in dogs from Romania and compares two tests for the diagnosis of Dirofilaria immitis. From July 2010 to March 2011, blood samples were collected from 390 dogs from nine counties of Romania and serological SNAP tests were performed for the detection of D. immitis antigen. The remaining blood clots were subsequently used for DNA extraction followed by multiplex PCR for assessing filarioid species diversity (i.e. D. immitis, Dirofilaria repens and Acanthocheilonema reconditum). Based on molecular detection, an overall prevalence of 6.92 % (n = 27; 95 % confidence interval (CI) 4.70-10.03 %) for D. repens, 6.15 % (n = 24; 95 % CI 4.07-9.14 %) for D. immitis and 2.05 % (n = 8; 95 % CI 0.96-4.16 %) for A. reconditum was recorded, with significant variations according to sampling areas. Coinfections of D. immitis and D. repens were recorded in 23.91 % (n = 11) positive dogs. A slightly higher prevalence for D. immitis was detected at the SNAP test (n = 28, 7.17 %; 95 % CI 4.91-10.33 %), but this difference was not statistically significant (p = 0.66). However, only 53.57 % (n = 15) of antigen-positive dogs were confirmed by PCR, while other dogs (n = 9) PCR positive for D. immitis were negative at the serology. The present study shows that Dirofilaria species are endemic in the southern and southeastern areas of Romania, This article also provides, for the first time, an epidemiological picture of the distribution of A. reconditum in Romania.

Prevalence of Dirofilaria immitis (Nematoda: Filarioidea) in mosquitoes from northeast Arkansas, the United States.

A mosquito survey was conducted to identify which species of mosquitoes carry Dirofilaria immitis (Leidy) (Nematoda: Filarioidea), dog heartworm, in northeast Arkansas. Using polymerase chain reaction, mosquitoes were analyzed for D. immitis, Dirofilaria repens Railliet & Henry, and Acanthocheilonema dracunculoides Cobbold. Mosquitoes were collected from April to October 2009 using black light ultraviolet traps baited with dry ice. Sixteen mosquito species were identified. D. immitis was identified in nine mosquito species, which included Aedes vexans (Meigen), Anopheles quadrimaculatus Say, Anopheles punctipennis (Say), Culex pipiens quinquefasciatus Say, Culex erraticus (Dyer & Knab), Culiseta inornata (Williston), Psorophora columbiae (Dyer & Knab), Psorophora ferox (Humboldt), and Psorophora howardii Coquillett. No D. repens or A. dracunculoides DNA was amplified. Of the 1,212 mosquito pools tested, 7.3% were positive for D. immitis. Frequency of D. immitis infections from six collection sites ranged from 2.1 to 19.4%. Ae. vexans and An. quadrimaculatus were the two most abundant species, composing 58.7 and 23.7% of the total mosquitoes collected, with 9.6 and 6.9% of pools positive for D. immitis, respectively. To investigate localized vector infection rates of D. immitis, mosquitoes were collected from inside the kennel of a heartworm-positive dog. Of the 114 mosquitoes collected, 84 (73.7%) were positive for D. immitis. The frequency of D. immitis-infected mosquitoes collected near a heartworm-positive dog was considerably higher than in the original six collection sites, suggesting a single heartworm-positive dog potentially increases infection pressure on susceptible animals sharing mosquito exposure.

Potential efficacy of monthly administrations of spot-on moxidectin 2.5%/imidacloprid 10% in the simultaneous prevention of major canine filarioses.

Dirofilaria immitis, Dirofilaria repens, and Acanthocheilonema reconditum are the most important canine filariae. These species are expanding their distribution in both endemic regions and in previously free areas, thus enhancing the risk for single and mixed infestations in both dogs and humans. The present study evaluated the potential use of moxidectin in a spot-on formulation in preventing these dog filarioses in a confined area where the three major canine filariae live in sympatry. The trial was performed in a private shelter with a history of mixed infestations. Twenty-nine negative dogs were monthly treated with a spot-on formulation containing moxidectin 2.5%/imidacloprid 10% in spring and summer of 2010. The blood of the dogs was examined monthly with classical and molecular assays until December 2011. Twenty-six dogs completed the study, and they scored constantly negative to all diagnostic assays. Three further dogs completed the study in February 2011 until when they were filariae-negative. These results show that monthly treatment with moxidectin may potentially contribute in reducing the spreading of canine filarial diseases. The permanent negativity of treated dogs for the study period is discussed in relation to the potential use of this spot-on formulation in the prevention of single and mixed infestations of major vector-borne canine filariae.

Ecological, morphological, and molecular studies of Acanthocheilonema odendhali (Nematoda: Filarioidea) in northern fur seals (Callorhinus ursinus) on St. Paul Island, Alaska.

Studies of northern fur seal (Callorhinus ursinus Linnaeus, 1758) infection by the filariid nematode Acanthocheilonema odendhali were carried out in 2011-2012 on St. Paul Island, Pribilof Archipelago, Alaska. Skins of 502 humanely harvested northern fur seals from haul-out areas of five rookeries, Polovina (n = 122), Morjovi (n = 54), Zapadni (n = 72), Lukanin (n = 109), and Gorbatch (n = 145), were examined. A. odendhali was found in 18% of northern fur seals. The prevalence of infection ranged from 12.5% up to 22.9% on different haul-out areas on the island. The mean intensity of infection was 1.3 (range 1-7). Detailed morphological examination of collected specimens was performed using light microscopy. Several characters were added to the morphological description of the species, among them lateral thickening of the body cuticle, especially prominent in males, variations in number and position of genital papillae in males, transverse striation of the cuticle, and terminal dilation on tail end in microfilariae. The adult specimens studied had a shorter esophagus than type specimens from the California sea lion described by Perry (1967). Comparison of partial sequences of the mitochondrial cox1 gene from specimens collected from five sampling sites on St. Paul Island and a specimen from the type host and territory in California showed no significant differences and strongly supported conspecificity of the material from Alaska with A. odendhali.

Morphometric analyses of canine blood microfilariae isolated by the Knott's test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis.

BACKGROUND: Considering the increasing importance of small animals travel medicine and the spread of filariae with zoonotic potential to non-endemic European areas, routine filarial diagnosis in dogs is becoming important. Dirofilaria immitis, D. repens, Acanthocheilonema dracunculoides and A. reconditum are the most common canine filarial nematodes presenting blood circulating microfilariae (mf) which can be differentiated to species level by the acid phosphatase activity patterns or by PCR. Available data on the size of the mf vary considerably in the literature. The aim of this study was to validate morphometric criteria for filarial identification in blood samples of dogs after concentration of mf with the modified Knott's technique. METHODS: Morphometric analysis of 10 mf from samples identified to species level by acid phosphatase activity and partially confirmed by PCR were performed with specimens from 377 dogs. RESULTS: The mean length and width of D. immitis mf from 60 dogs were 301.77 ± 6.29 µm and 6.30 ± 0.26 µm, of D. repens mf from 171 dogs 369.44 ± 10.76 µm 8.87 ± 0.58 µm, of A. dracunculoides mf from 133 dogs 259.43 ± 6.69 µm and 5.09 ± 0.47 µm and of A. reconditum mf from 13 dogs 264.83 ± 5.47 µm and 4.63 ± 0.52 µm.For a subset of 30 samples, morphometric analysis was repeated with identical results in two laboratories. Furthermore, the size of mf concentrated and fixed by the Knott's technique was shown to be stable over 105 days. CONCLUSIONS: The Knott's test enables to clearly distinguish between D. immitis, D. repens and Acanthocheilonema spp. However, due to the overlapping size ranges of A. dracunculoides and A. reconditum, biochemical or molecular methods are required to distinguish these two species.