Molecular survey of Dirofilaria immitis and Dirofilaria repens by direct PCR for wild caught mosquitoes in the Republic of Korea

Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted to determine the potential vectors and the relative mosquito infection rates of Dirofilaria immitis and Dirofilaria repens in Gyeonggi and Gangwon Provinces, Republic of Korea, 2005. Dirofilaria spp. were confirmed by polymerase chain reaction (PCR) using species-specific primers for D. immitis and D. repens. Minimum field infection rates (MFIR) [MFIR = (number infected pools/total number of mosquitoes) x 1000] of 12 pools/2059 total number mosquitoes (5.8) and 21 pools (10.1) of the wild caught mosquitoes were positive by PCR for D. immitis and D. repens, respectively. Dual infections of both D. immitis and D. repens were detected by PCR in eight pools (3.9). Anopheles sinensis sensu lato (includes An. sinensis s.s., An. pullus, An. kleini, An. belenrae, and An. lesteri), An. sineroides, Aedes vexans nipponi, Culex pipiens and Armigeres subalbatus were found to be infected and are potential vectors of D. immitis and D. repens in Korea. Infections observed in mosquitoes represent potential health risks for domestic animal and human populations.     

Prevalence and epidemiological aspects of Dirofilaria immitis in dogs from Kayseri Province, Turkey

This study was conducted to determine the prevalence of Dirofilaria immitis infection and to investigate the risk factors related to heartworm disease in dogs from Kayseri, Turkey. Blood samples were collected from 280 dogs from May 2005 to March 2006 and were examined by membrane filtration-acid phosphatase histochemical staining and antigen Elisa techniques to detect circulating microfilariae and antigens of D. immitis, respectively. Of the total of 280 dogs, 27 were positive for D. immitis with a prevalence value of 9.6%. In addition 29.6% of positive dogs determined to have occult D. immitis infections. D. immitis was the only canine filarial parasite present in the study area. The mean number of microfilariae in infected dogs was 4730+/-5479 per ml of blood. The highest heartworm prevalence were observed in 7-10 age group (28.6%) followed by 4-6 (17.1%) and 0.5-3 (4.8%) age groups. The differences between 0.5-3 and other age groups were found significant, whereas no statistically significant difference was observed between 4-6 and 7-10 age groups. The infection was more prevalent in males, larger breeds and the dogs not on prophylaxis. No statistically significant difference was observed between stray and owned dogs. Our results suggest that heartworm treatment and prophylaxis should be considered in Kayseri Province.     

Old problems on a new playing field: Helminth zoonoses transmitted among dogs, wildlife, and people in a changing northern climate

Although surveillance is limited, indigenous residents at latitudes ranging from 53 to 73°N in Canada appear to have a higher occurrence of infection with some zoonotic parasites than the general population. Conversely, they are relatively naïve to other zoonotic parasites that have previously been unable to establish at northern latitudes. For those parasites that circulate among dogs, wildlife, and people, potential risk factors in the North include limited availability of veterinary services, presence of free-roaming dog populations, and consumption of locally harvested fish and wildlife. These regions are also experiencing some of the greatest impacts of climate change in North America, including increased temperature, precipitation, and frequency and severity of extreme weather. We review the current taxonomy, genetic diversity, host and geographic distributions, epidemiology and risk factors for 3 genera of helminths (Diphyllobothrium spp., Echinococcus spp., and Toxocara sp.) in Canada's North in order to identify climate-sensitive aspects of their ecology. Free-living stages of parasitic zoonoses endemic in the Arctic (such as Diphyllobothrium dendriticum, the cervid strain of Echinococcus granulosus, and Arctic strains of Echinococcus multilocularis) will experience trade-offs between enhanced survival under wetter conditions and increased mortality under warmer conditions. Climate change might also lead to the introduction and establishment in the Arctic of parasitic zoonoses previously restricted to the sub-Arctic, such as Diphyllobothrium latum, Toxocara canis, and the prairie strain of E. multilocularis. Molecular techniques applied in broad geographic surveys are needed to address critical knowledge gaps in the geographic distribution, genetic diversity, and public health significance of zoonotic helminths already in the circumpolar North, and to determine the current barriers to range expansion of temperate-adapted parasites into the North. Dogs will continue to play important roles in the North, including that of a “bridging” host between sylvatic cycles and human communities. In a warming north, increased opportunities for business, agriculture, and tourism favor importation of dogs and their parasites into a newly suitable environment. Collaborations among veterinarians, public health personnel, and policy-makers are needed to enhance surveillance and mitigate for dog-transmitted parasitic zoonoses in a changing North.     

Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection

Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0-6, 10-12, 16-18, 22-26, and 28-34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.     

Climate and the epidemiology of gastrointestinal nematode infections of sheep in Europe

The free-living stages of gastrointestinal nematode parasites of sheep are strongly affected by climate. Thus, extreme heat and cold are detrimental to development and survival, while, within tolerable limits, increasing temperatures generally accelerate development but increase mortality. Moisture is needed for development and translation of larvae from faeces to pasture, and so rainfall is a limiting factor for transmission. Together, these factors underpin seasonal patterns of infection in sheep, as well as geographic variation in the epidemiology and relative importance of different species within Europe. Local knowledge and experience enable treatment to be targeted appropriately to prevent dangerous levels of infection. This traditional know-how can be supplemented by predictive epidemiological models, built on thorough understanding of the influence of climate on larval availability. However, management also has a dominant role in determining patterns of infection, and is itself influenced by climate. Current geographic variation in nematode epidemiology across Europe, and knowledge of systems from outside Europe, can provide only limited perspectives on the likely effects of climate change on disease in future. This is because disease arises from complex interaction between host and parasite factors, and the implementation of optimal control strategies to meet new challenges will be slowed by the inertia of current systems. Approaches to nematode control must therefore take account not only of parasite biology, but also the forces that shape sheep farming systems and management decisions.     

Genetic polymorphism in Dirofilaria immitis

Dirofilaria immitis is the causative agent of heartworm disease. Reports of macrocyclic lactone inefficacy prompted an investigation of genetic polymorphism in D. immitis. Currently, there is a lack of genetic information for this parasite. Information on baseline levels of genetic heterogeneity in the dog heartworm would have important implications for the possible development of macrocyclic lactone resistance in D. immitis. Genetic variability was investigated to assess the extent of genetic polymorphism in D. immitis populations from the USA (field and laboratory samples) and Japan (field samples). Single nucleotide polymorphisms (SNPs) were investigated in a full-length β-tubulin gene and segments of genes encoding heat shock protein 60 (Hsp60), a P-glycoprotein (Pgp), sarco-endoplasmic reticulum Ca(2+)ATPase (Serca) and phosphofructokinase (PFK). Significant differences in SNP frequencies were found in all genes except PFK. A combined genotype built from two SNPs from β-tubulin, Hsp60, Pgp and Serca was analyzed in the US lab, US field and Japan field samples. Some combined genotypes were unique to each sample group while others were shared between groups. F coefficients calculated for 15 SNPs from β-tubulin, Hsp60, Pgp and Serca were not in equilibrium. Differences in F coefficients were observed between the groups. D. immitis was found to be genetically heterogeneous. This genetic heterogeneity has implication for the development of genetically selected strains of the parasite.     

Identification and characterization of the cofactor-independent phosphoglycerate mutases of Dirofilaria immitis and its Wolbachia endosymbiont

Drug treatments for heartworm disease have not changed significantly in the last decade. Due to concerns about possible drug resistance and their lower efficacy against adult worms, there is a need for the development of new antifilarial drug therapies. The recent availability of genomic sequences for the related filarial parasite Brugia malayi and its Wolbachia endosymbiont enables genome-wide searching for new drug targets. Phosphoglycerate mutase (PGM) enzymes catalyze the critical isomerization of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG) in glycolytic and gluconeogenic metabolic pathways. There are two unrelated PGM enzymes, which are structurally distinct and possess different mechanisms of action. The mammalian enzyme requires 2,3-bisphosphoglycerate as a cofactor (dependent PGM or dPGM), while the other type of PGM does not (independent PGM or iPGM). In the present study, we have determined that Dirofilaria immitis and its Wolbachia endosymbiont both possess active iPGM. We describe the molecular characterization and catalytic properties of each enzyme. Our results will facilitate the discovery of selective inhibitors of these iPGMs as potentially novel drug treatments for heartworm disease.     

Effects of doxycycline on early infections of Dirofilaria immitis in dogs

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.     

Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis

The close emotional tie between people and companion animals is a beneficial relation known as the human-animal bond. However, pet dogs and cats can play an important role in the transmission of helminthic zoonotic agents such as the tapeworms Echinococcus and the roundworms Toxocara which are directly transmitted from pets to the human environment without the involvement of vectors or intermediate hosts. In humans, echinococcosis has emerged in Europe and toxocarosis is still persisting in large endemic areas despite the availability of highly efficient anthelminthics for dogs and cats. Ecological changes significantly contributed to these trends: the high wild fox populations and the high density of freely roaming dogs and cats maintain a permanent infection pressure of these and other parasites. Further, the establishment of urban recreational environments closer to natural ecological systems boosted vole populations that represent urban reservoirs for zoonotic helminths. A good understanding of the parasites’ biology and epidemiology including the transmission to humans is required for planning and implementing effective prevention strategies. The continuous education of veterinarians and the information of the pet owners by providing uniform recommendations are of priority importance. A close collaboration between veterinary and public health professionals in a ‘One Health’ concept is required.     

Dirofilaria immitis and Wolbachia-derived antigens: its effect on endothelial mammal cells

Antigens of both Dirofilaria immitis and Wolbachia symbiont bacteria are implicated in the inflammatory pathology of heartworm infection. The aim of the present study was to compare the stimulatory capacity of in vitro cultures of vascular endothelial cells by the adult somatic antigens of D. immitis (DiSA) and the recombinant form of the Wolbachia surface protein (rWSP), during the first 24h of stimulation. Our results indicate a different stimulatory activity of the two antigens. Both the DiSA and rWSP stimulate the production of the enzymes responsible of the arachidonic acid metabolism, cyclooxygenase-2, 5-lipoxygenase (5-LO), and leukotriene B4. Only DiSA stimulates the production of prostaglandin E2. Related to the adhesion molecules, the DiSA stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas rWSP stimulates ICAM-1, PECAM-1, and vascular cell adhesion molecule-1 (VCAM-1). Expression of E-cadherin and vascular endothelial growth factor also were stimulated by rWSP. Neither of the two antigens altered the basic physiological mechanisms of endothelial cells, such as cell proliferation, cell cycle, or apoptosis. The biological and pathological significance of these finding are discussed.     

A review of parasitic zoonoses in a changing Southeast Asia

Parasitic zoonoses are common and widely distributed in the Southeast Asian region. However, the interactions between parasites, hosts and vectors are influenced by environmental, socio-cultural and livestock production changes that impact on the distribution, prevalence and severity of disease. In this review we provide an update on new knowledge in the context of ongoing changes for the food-borne pig associated zoonoses Taenia solium and Trichinella spp., the food-borne trematodes Opisthorchis viverrini and Clonorchis sinensis, the water-borne trematodes Schistosoma spp., the vector-borne zoonotic protozoa Plasmodium knowlesi and Leishmania spp. and the soil-borne zoonotic hookworm Ancylostoma ceylanicum. These various changes need to be considered when assessing or developing regional control programs or devising new research initiatives in a changing SE Asia.     

Prevalence of the Dirofilaria immitis infection in dogs from Merida, Yucatan, Mexico

The aim of the present study was to estimate the prevalence of Dirofilaria immitis infection, and to examine the relationship between host factors (gender, age and breed) and D. immitis infection in dogs. The study was designed as a cross-sectional study. A total of 676 dogs were examined for D. immitis infection. Adult worms (necropsy) and blood samples were collected from all animals for diagnosis of D. immitis. Blood samples were examined using a modified Knott's and thick drop techniques. Fifty-six of 676 dogs were diagnosed infected with D. immitis. The odds of infection were 2.85 times higher in female dogs, compared to male dogs (OR = 2.85, 95% CI = 1.5-5.5, P = 0.01). In addition, the odds of infection were 2.11 times higher in > or =3 years old dogs, compared with younger dogs (OR = 2.11, 95% CI = 1.1-4.2, P = 0.03). This information is important for a better understanding of the epidemiology of D. immitis in dogs in Yucatan, Mexico.     

Dirofilaria immitis infection in dogs: cardiopulmonary biomarker levels

Cardiopulmonary biomarkers are biological parameters that can be objectively measured and quantified as indicators of pathogenic processes (heartworm disease) or as indicators of response to therapeutic intervention. To determine levels of cardiopulmonary biomarkers in canine dirofilariasis, measurements of cardiac troponin T, cardiac troponin I, myoglobin, and D-dimer concentrations were performed for dogs with and without evidence of adult heartworm infection. The results showed that levels of cardiac troponin T were undetectable in all dogs studied while levels of cardiac troponin I were higher in dogs infected with Dirofilaria immitis. In healthy dogs, levels of myoglobin and D-dimer were below detection limits of the instrument and were significantly higher in heartworm-infected dogs, notably in microfilaremic dogs. The results suggest the possibility of using troponin I and myoglobin as markers for cardiac damage and the D-dimer as a supportive tool for a diagnosis of pulmonary thromboembolism in dogs with cardiopulmonary dirofilariasis.     

Bluetongue in Europe and the Mediterranean Basin: history of occurrence prior to 2006

Bluetongue virus (BTV) exists around the world in a broad band covering much of the Americas, Africa, southern Asia and northern Australia. Historically, it also occasionally occurred in the southern fringes of Europe. It is considered to be one of the most important diseases of domestic livestock. Recently BTV has extended its range northwards into areas of Europe never before affected and has persisted in many of these locations causing the greatest epizootic of bluetongue (BT), the disease caused by BTV, on record. Indeed, the most recent outbreaks of BT in Europe are further north than this virus has ever previously occurred anywhere in the world. The reasons for this dramatic change in BT epidemiology are complex but are linked to recent extensions in the distribution of its major vector, Culicoides imicola, to the involvement of novel Culicoides vector(s) and to on-going climate-change. This paper investigates these recent outbreaks in the European theatre, up to the beginning of 2006, highlights prospects for the future and sets the scene for the following papers in this special issue.     

Impact of climate change in the epidemiology of vector-borne diseases in domestic carnivores

Vector-borne diseases are medically important in humans and animals but were long considered tropical and known to first affect production animals. This is no longer true and we can see today that they are common in domestic animals and that they are also present in temperate countries, especially in Europe. In recent years, an increase in the diagnosis of vector borne diseases among humans and animals has been observed, which may partly due to the development of diagnostic tools. Their study requires exchanges and collaborations between the many actors involved, especially since the epidemiology seems to be constantly evolving. The veterinary practitioner is the first one to notice the emergence of cases and to implement prevention measures. He also acts as a sentinel to alert epidemiologists. Many factors can explain the epidemiological changes, i.e. all human factors, such as the increase in commercial transportation, but also owners traveling with their pet during the holidays, the development of “outdoor” activities, the increase of individual housings with gardens; to these human factors must be added the ignorance of the risks, linked to animals in general and to wildlife in particular; then the environmental changes: forest fragmentation, establishment of parks; the increase of wild mammal populations (deer, carnivores, rodents, etc.); finally, climate changes. Climate change is a reality which may explain the increase of density of arthropod vectors, but also of their hosts, changes in periods of activity and variations in geographical distribution. The authors show the proof of the climate modifications and then explain how it has an impact in Europe on ticks, mosquitoes, sandflies and even fleas. They conclude on the practical consequences for veterinary practitioners, especially with the diagnosis of parasitic diseases or diseases in areas where they usually do not occur. However, not any epidemiological modification should be linked to climate change, since many other factors are involved and often even overriding.     

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.     

Feline heartworm (Dirofilaria immitis) infection: a statistical elaboration of the duration of the infection and life expectancy in asymptomatic cats

A study was conducted to assess the duration and the outcome (self-cure or death) of feline heartworm infection and the life expectancy of infected cats. To be included in the study, cats had to be positive for heartworm antibody (Ab) and heartworm antigen (Ag) and had to demonstrate the presence of worms by echocardiography. Self-cure was defined as (1) negative for heartworm Ag and (2) no further visualization of worms by echocardiography. Of the 1962 eligible cats, 364 (18.5%) were positive for heartworm Ag and 131 were positive for heartworm Ag and for echocardiography diagnosis (prevalence 6.7%). None of the cats was microfilaremic. Of 43 asymptomatic cats included into the follow-up study with owners' consent, 34 (79%) self-cured and nine (21%) died. Eleven (26%) cats remained asymptomatic and self cured within 21-48 months, 23 (53%) showed symptoms but self-cured within 18-49 months, 6 (14%) died within 8-41 months of follow-up and 3 (7%) suddenly died after 38-40 months, which was related to heartworm infection. The probability for death or sudden death increased significantly with age at diagnosis, but no difference was detected by gender, survival time after diagnosis, or the presence or absence of symptoms. The presence/absence of symptoms showed significant interaction with the age at diagnosis (i.e., symptomatic cats showed increasing duration of heartworm infection along with age at diagnosis compared to that for asymptomatic cats. Heartworm-infected cats survived significantly longer than heartworm-negative cats affected by hypertrophic cardiomyopathy, chronic renal failure, or neoplastic diseases.     

Experimental infection of dogs with various Bartonella species or subspecies isolated from their natural reservoir

Dogs can be infected by a wide variety of Bartonella species. However, limited data is available on experimental infection of dogs with Bartonella strains isolated from domestic animals or wildlife. We report the inoculation of six dogs with Bartonella henselae (feline strain 94022, 16S rRNA type II) in three sets of two dogs, each receiving a different inoculum dose), four dogs inoculated with B. vinsonii subsp. berkhoffii type I (ATCC strain, one mongrel dog) or type II (coyote strain, two beagles and one mongrel) and B. rochalimae (coyote strain, two beagles). None of the dogs inoculated with B. henselae became bacteremic, as detected by classical blood culture. However, several dogs developed severe necrotic lesions at the inoculation site and all six dogs seroconverted within one to two weeks. All dogs inoculated with the B. v. berkhoffii and B. rochalimae strains became bacteremic at levels comparable to previous experimental infections with either a dog isolate or a human isolate. Our data support that dogs are likely accidental hosts for B. henselae, just like humans, and are efficient reservoirs for both B. v. berkhoffii and B. rochalimae.     

Efficacy of danofloxacin 18% injectable solution in the treatment of Escherichia coli diarrhoea in young calves in Europe

The efficacy of danofloxacin 18% against naturally occurring Escherichia coli diarrhoea was investigated in calves at seven European sites. Treatment commenced on day 0, with either a single subcutaneous injection of danofloxacin 18% (n=267) at 6 mg/kg repeated on day 2 if required, or reference product containing baquiloprim/sulphadimidine (n=37) or gentamicin (n=98) administered as recommended. E. coli was isolated from 90% to 100% of calves pre-treatment, and the prevalence of serotypes K99 and F41 was 8-46% and 46-92%, respectively. In both treatments, the majority of calves (93.2-93.9%) showed clinical improvement and completed the studies. There were significant reductions for both treatments, in severity of clinical signs on days 4 and 10 compared to day 0 (P<0.0001), and between days 4 and 10 (P<0.05), but no significant differences between treatments (P>0.05). Danofloxacin 18% was clinically safe, and as effective as the reference products in the treatment of E. coli diarrhoea in calves.