Molecular genetic comparison of Onchocerca sp. infecting dogs in Europe with other spirurid nematodes including Onchocerca lienalis

In the past 15 years, subconjunctival onchocercosis has been reported from 63 dogs in south-western United States (Arizona, California, Utah) and Southern and Central Europe (Germany, Greece, Hungary, Portugal, Switzerland). To reveal the taxonomic status of the parasite responsible for these infections, fragments of the mitochondrial cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes of three European strains of canine Onchocerca sp. and the 16S ribosomal RNA (16S rRNA) gene of their Wolbachia endosymbionts were sequenced and compared to the homologous sequences of other spirurid nematodes. The evolutionary divergence between COI and ND5 gene sequences of Greek, Hungarian and Portuguese strains of canine Onchocerca sp. were similar in magnitude to that seen within Thelazia callipaeda or Onchocerca lienalis. The evolutionary divergence between the sequences of canine Onchocerca sp. and other Onchocerca spp. including O. lienalis were similar or higher in magnitude to that seen between other Onchocerca spp. The results of the current and earlier phylogenetic analyses indicate that canine Onchocerca sp. separated from other Onchocerca spp. early in the evolution. Based on the similar clinical pictures, the identical morphology of nematodes and the sequence analyses of COI and ND5 genes of the worms and 16S rRNA gene of their wolbachiae, the Onchocerca worms isolated from European dogs appear to belong to the same species. The results support the earlier biological and morphological arguments that a distinct species, most likely O. lupi originally described from the subconjunctival tissues of a Caucasian wolf is responsible for canine ocular onchocercosis in Europe.     

Ocular onchocercosis in dogs: aberrant infection in an accidental host or lupi onchocercosis?

Four adult dogs that had spent their entire life in Hungary, were found to be infected with filaroid nematodes of the genus Onchocerca. The morphology and location of the parasites as well as pathological lesions were similar to those described earlier in the one Hungarian and five US dogs. Only moderate morphological differences were noted between the adults of Onchocerca sp. infecting dogs and O. volvulus of man or O. lienalis of cattle. Nevertheless, the morphology of microfilariae of Onchocerca from dogs is unique within the genus. Their length was less than half the length of microfilariae of other Onchocerca spp. known so far. In addition to size differences, several characteristic morphological features were observed. The unsuccessful attempt to infect dogs with O. lienalis, the absence of O. volvulus and O. lienalis in endemic regions of canine onchocercosis, the different size, morphology, and location of the adults in dogs and cattle, the exceptionally small size and unique morphology of microfilariae of Onchocerca of canids indicate that a distinct species might be responsible for canine onchocercosis. Since the larval concentration in the skin was high (50-3600 microfilariae g(-1)) in all affected dogs, the diagnosis prior to surgical removal of worm nodules can be based on the examination of a small skin snip collected from the head or abdominal region. Infections in dogs may provide a model to study human onchocercosis, therefore, further studies are encouraged on the feasibility of experimental infection of dogs with this Onchocerca species.     

Electron microscopic and molecular identification of Wolbachia endosymbionts from Onchocerca lupi: implications for therapy

It was recently demonstrated that Wolbachia intracellular bacteria (alpha 2 proteobacteria, Rickettsiales) living in filarial nematodes are obligatory symbionts of their hosts. Herein, we report the electron microscopic and 16S ribosomal DNA-based (16S rDNA) identification of the endobacteria harboring in Onchocerca lupi. The worm nodules containing the nematodes were removed from three Hungarian dogs naturally infected with O. lupi. Wolbachia-like endobacteria were detected by electron microscopy in the lateral chords of both adult worms and microfilariae. The endosymbionts in O. lupi resemble in location, size, and morphology the wolbachiae found in other filariae. The presence of wolbachiae in O. lupi was also confirmed by PCR amplification of the 16S rDNA of the bacteria. The 16S rDNA-based phylogenetic analysis revealed that the endosymbionts of O. lupi infecting dogs belong to the supergroup C of Wolbachia pipientis and are not identical with those of other Onchocerca spp. sequenced so far. Since intermittent treatment with oxytetracycline has adulticid and microfilaricid activity by depletion of Wolbachia endobacteria, this antibiotic treatment regimen may offer an alternative of ivermectin or diethylcarbamazine in the suppression of postoperative microfilaridermia in Onchocerca-infected dogs and may prevent relapse.     

Subconjunctival filariasis due to Onchocerca sp. in dogs: report of 23 cases in Greece

In the present study, we describe a series of 23 cases of ocular subconjunctival parasitic granulomas in dogs, admitted to the Clinic of Surgery, Faculty of Veterinary Medicine of the Aristotle University of Thessaloniki, Greece, between 1997 and 2000. The ophthalmic manifestations in all animals were periorbital swelling, discomfort, photophobia, conjunctival congestion, and discharge. A more detailed examination revealed the presence of periocular masses (nodules) on the subconjunctival bulbar space. Granulomatous or cyst-like formations were extracted surgically, and were found to contain thread-like nematode parasites. A histologic and parasitologic examination of tissues and parasites was carried out. Diagnosis of parasitic granulomas was made and the parasite was identified as Onchocerca sp. This is the largest series of cases reported of aberrant Onchocerca infections in dogs coming from one geographic location.     

Canine ocular onchocercosis in Hungary

An adult male mongrel dog that had spent its entire life in Hungary, was found to have infection with filaroid nematodes of the genus Onchocerca. The gravid male and female parasites were embedded in bean-sized granulomatous masses on the conjunctiva and the sclera of both eyes. The cuticle of females consisted of two separated layers in longitudinal sections, the external layer bearing ridges and the internal layer showing striations. The ridges were marked, rounded in shape, and the ratio of body diameter to the distance between ridges varied between 7:1 and 10:1. At midbody of the worms, two striations could be seen between each pair of ridges: one under every ridge and one between neighbouring ridges. Numerous exceptionally small (96.4 microm x 6.4 microm) microfilariae were seen in the uteri of females and the surrounding tissues and isolated from skin biopsy materials. The morphology and location of the parasite and histopathological lesions of the Hungarian case were similar to that described in dogs in the United States. This case is the first documented ocular Onchocerca infection in dogs outside the western United States. Thus, onchocercosis should be considered in the differential diagnosis of ocular and periocular nodules in dogs also in Europe.     

Canine ocular onchocerciasis in the United States: two new cases and a review of the literature

Since 1991, 53 cases of canine ocular onchocerciasis have been reported in the literature worldwide, 43 of these were from Greece, five from Hungary, and five from the western United States. Information on the histopathologic features of canine ocular onchocerciasis is limited. We describe the histopathologic features of canine ocular onchocerciasis in two dogs from California that presented clinically with firm episcleral nodules and uveitis unilaterally. Pertinent literature and pathogenesis are reviewed; recognizable clinical features and treatment are discussed. The cases presented were diagnosed via histopathology of the enucleated globes and episcleral granulomas at the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW). Positive identification of adult Onchocerca within episcleral granulomas was made based on light-microscopy features. Histopathologic examination of both globes revealed episcleral parasites surrounded by granulomas containing few to moderate numbers of eosinophils. Other sequelae, in both cases, included lymphoplasmacytic uveitis, preiridal fibrovascular membranes, peripheral anterior synechiae, retinal degeneration, and optic nerve head cupping. Both male and female worms were present, as were in utero microfilariae in both cases. Worms in both cases were tentatively identified as Onchocerca lienalis. Ocular onchocerciasis should be a differential consideration in cases of canine conjunctival nodules or periorbital swelling, particularly in dogs from the western United States.     

Molecular phylogenetic analysis of Onchocerca lupi and its Wolbachia endosymbiont

The morphology of Onchocerca lupi, responsible for canine ocular onchocercosis, is unique within the genus. Earlier analyses of the 5S ribosomal RNA gene spacer region sequence of the parasite and the 16S ribosomal RNA gene sequence of its Wolbachia endosymbiotic bacteria (Rickettsiales) supported the morphological and biological arguments that O. lupi is a distinct species. However, the exact phylogenetic position of O. lupi and its endosymbiont could not be unambiguously determined. Herein we report analyses based on the mitochondrial cytochrome oxidase I (COI) gene of the filarial species and the Wolbachia surface protein (wsp) and the bacterial cell-cycle ftsZ genes of their wolbachiae. Our results indicate that O. lupi separated from other Onchocerca spp. early in evolution. This is in line with the previous morphological analysis demonstrating that O. lupi is an atypical Onchocerca species showing both primitive and evolved characters. The phylogenetic trees generated for the COI sequences of filariae and the wsp and ftsZ sequences of their wolbachiae were congruent with each other, which supports the hypothesis that nematodes and their Wolbachia endobacteria share a long co-evolutionary history.     

Teat onchocercosis in cows with reference to prevalence, species involved and pathology

The present study reports teat onchocercosis in cows in the province of Kars in north eastern Turkey with reference to the prevalence, species involved and pathological findings. In the study, 600 cows of various breeds and ages slaughtered in the local abattoir were examined and 145 (24%) were found to have various lesions on their teats, of which 45 (31%) were infected with three Onchocerca species (Onchocerca gutturosa, O. lienalis and an unidentified Onchocerca spp.), identified according to their morphological characteristics. Gross pathological examination of the teats with lesions revealed scabies and chaps of varying sizes, healed sores and small nodules. Histopathologically, large numbers of microfilariae were commonly observed within the collagenous fibre bundles of the dermis, with only a few microfilariae in the dermis in some cases. The microfilariae were also often found to have accumulated around the perivascular spaces and were frequently associated with infiltration by inflammatory cells, predominantly eosinophil leukocytes and mononuclear cells. In two cases, microfilariae were discovered exclusively in the lumen of the capillaries of the dermal papillaries. In one case, in which microfilarae were not encountered, sections of adult worms surrounded by neutrophil leukocytes were detected free in the teat canal. The presence of extensive teat lesions along with microfilariae of the Onchocerca species as found in this study warrants further assessment of the impact of onchocercosis on dairy farming.     

Onchocercosis of an intervertebral joint capsule causing cervical vertebral stenotic myelopathy in a horse.

A novel case where onchocercosis was identified as a cause of cervical myelopathy in the horse is described. A 15-year-old Connemara mare was euthanized due to progressive locomotion disturbance. Postmortem examination revealed soft-tissue swelling in the intervertebral joint capsule of C6-7 with narrowing of the vertebral canal. On light microscopy, axonopathy was pronounced in the corresponding segment of the spinal cord. Fibrous tissue and eosinophilic granulomas were found in the joint capsule, together with parasites identified histologically as Onchocerca sp.     

Ocular onchocercosis in dogs: a review

In recent decades, sporadic cases of ocular Onchocerca species infection have been reported in dogs in the USA and Europe. In the acute stage of the disease severe inflammation of the ocular and periocular tissues was observed. In chronic cases, the strongly coiled, gravid nematodes were incorporated in pea- to bean-sized granulomatous nodules in various parts of the eye, including the retrobulbar space, orbital fascia, eyelid, third palpebra, conjunctiva and sclera. Apart from the ophthalmological significance of the disease, the large number of microfilariae in the skin may be responsible for acute and chronic dermatological problems. The geographical distribution and prevalence of the infection may be greater than currently thought, because the lesions may have been erroneously regarded as other ocular diseases. Onchocerciasis is the world's second most prevalent infectious cause of blindness in human beings and parasitologists have long searched for an experimental model of human onchocerciasis; ocular onchocercosis infections in dogs may provide a useful experimental system.     

Introduction to the alpha-proteobacteria: Wolbachia and Bartonella, Rickettsia, Brucella, Ehrlichia, and Anaplasma

Wolbachia is an obligate intracellular endosymbiont and likely mutualist living within the heartworm Dirofilaria immitis and a number of other filarial nematodes in the family Onchocercidae. The bacterial infection is passed from worm to worm transovarially; the organisms are in ovarian cells, the developing microfilariae, and multiply and persist in all later developmental stages through the mosquito and into the next host. Besides being present in the ovaries of the adult worms, they also are present in large numbers within the hypodermal tissues of the nematode. It is now know that these bacteria that were first observed in heartworms more than 30 years ago are actually related to similar Wolbachia bacteria that are found in arthropods. Wolbachia is an alpha-proteobacteria, and this group includes a number of important arthropod-transmitted bacterial agents of dogs and cats: Rickettsia rickettsii, R. felis, Anaplasma platys, Ehrlichia canis, E. chaffeensis, and E. ewingii. Alpha-proteobacteria are also important as obligate intracellular mutualists in plants in which they are responsible for nitrogen fixation. Recent work on the treatment of heartworms in dogs with doxycycline stems from related work with the human filarial nematode Onchocerca volvulus that causes river blindness in people.     

Prevalence and features of canine atopic dermatitis in Hungary

Medical records of 600 dogs diagnosed with atopic dermatitis were reviewed and evaluated with reference to history, geographical distribution, breed predilection, clinical signs and positive reactions to allergens as determined by intradermal skin testing (IDT) manufactured by Artuvetrin Laboratories. In 66.6% of dogs, the age of onset of atopic dermatitis was between 4 months and 3 years. Dogs living in the garden suburb of Budapest were more sensitive to house dust mites, fleas and moulds, and dogs from the western part of Hungary were more sensitive to weeds than to other allergens (p < 0.01). Positive reactions were most common to Dermatophagoides farinae followed by human dander. The breed distribution found in the present study was consistent with that reported in the literature, except for the breeds Hungarian Vizsla, Pumi, French bulldog, Doberman Pinscher and Bobtail which were over-represented among atopic dogs compared to the breed distribution of the general dog population of a large city in Hungary. Breeds with verified adverse reaction to food were Cocker spaniels, French bulldogs, Bullmastiffs, Bull terriers, St. Bernards, Tervurens, West Highland White terriers and American Staffordshire terriers (p < 0.05). The clinical signs of atopic dermatitis and their occurrence are in accordance with the data described in the literature.     

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.     

Aortic onchocercosis and elaeophorosis in traditional TSZ-cattle in Tabora (Tanzania): prevalence and pathology

Onchocerca armillata was found in 3838 (95.4%) and Elaeophora poeli in 70 (1.7%) out of 4025 samples of aortas collected from cattle slaughtered at Tabora in Tanzania during the calendar year 1988. Gross lesions of the affected aortas varied from mild to severe, characterised by parasitic tunnels, nodules and corrugated calcified ridges on the aortic wall. Histological sections revealed changes as a result of tissue reaction against the parasites which were embedded into the intima of affected aortas. Calcification and hyaline degeneration were common features. The high prevalence of O. armillata and the extensive pathological lesions observed would seem to warrant assessment of the importance of onchocercosis in animal production in the tropics. Meanwhile, further studies are required to elucidate the epizootiology of aortic onchocercosis and elaeophorosis in order to devise practicable diagnosis, treatment and control methods.     

Orbital fat prolapse in the dog

Five dogs presented with varying degrees of subconjunctival swelling and enophthalmos. Biopsy revealed the subconjunctival tissue to be fat. Surgical excision in two cases resulted in resolution of the clinical signs and demonstrated that the fat came from the retrobulbar region.     

Genetic diversity of Hungarian canine distemper virus strains

To achieve proper diagnosis of dogs based on acute clinical symptoms and poorly preserved field samples taken from animals that died due to canine distemper (CD), a new differential diagnostic test has been developed based on polymerase chain reaction (PCR). In this study, more than 150 samples collected from dogs showing respiratory, gastrointestinal and neurological signs suggesting canine distemper virus (CDV) infection were examined. The samples consisted of urine, blood and nasal swabs collected from clinically ill patients, sent to our laboratory by clinicians from various veterinary clinics throughout Hungary. Various organs collected during the necropsy of dogs with pathological changes that suggested CDV infection were also included. Three distinct PCRs were designed. For diagnostic purposes, a primer pair specific to a 409 bases-long segment within the conservative part of the large polymerase region (L) of the CDV genome was designed. Using this test, out of the 150 analyzed samples, 46 (30.66%) proved to be positive for CDV, indicating that CDV still represents a high risk to the canine population in Hungary. For the phylogenetical analysis, a primer pair that completely encompasses the hemagglutinin (H) gene of the CDV genome was designed. The amplicons of this region were sequenced in both directions using the appropriate primers. Our results indicate that several different CDV genotypes are currently present in Hungary. Nine of the analyzed Hungarian strains turned out to belong to the so-called Arctic group of CDVs, and were most closely related to non-European strains from North America, China and Greenland, as well as to the phocine distemper virus 2 (PDV-2) isolated from Baikal seals (Phoca sibirica). One of the Hungarian strains showed high similarity to other European isolates from Denmark, Germany, Italy and Turkey, as well as to other isolates from geographically more distant regions, such as the USA. Three Hungarian strains seem to join a new cluster that is formed by only a couple of strains, one isolated from a mink in Denmark, and another from a dog in North America. Using a third set of primers, a restriction fragment length polymorphism (RFLP) assay has also been designed for the fast and reliable differentiation of the wild-type CDVs from the vaccine strains.     

Nodular onchocercosis of red deer in central Spain

The presence of nodular onchocercosis was investigated post-mortem in 142 red deer (11 calves, 35 yearlings and 96 adults) shot from February 1998 to January 1999, and July-November 1999 in "Quintos de Mora" (Toledo, central Spain), a game property belonging to the National Wildlife Reserves. Between 6 and 13 animals were analysed monthly by inspection for subcutaneous nodules of Onchocerca spp. Subcutaneous nodules of Onchocerca sp. were detected in 24% of the animals. Anatomical locations for nodules were the back and flanks. Infection ranged from 1 to 29 nodules per animal. Adult worms collected from nodules were identified as O. flexuosa. No apparent seasonal trend was observed either in prevalence or in mean intensity of infection, which fluctuated between 48% (5+/-8 nodules) in winter 1998 and 5% (1+/-0 nodules) in summer 1999. Prevalence of infection was significantly higher in adult (30%) than in young animals (9% in calves, 11% in yearlings), although no age effect on intensity was observed. The size of the nodules was measured to evaluate the age of infection. Small (recent) nodules (5-6mm of diameter) were collected in late spring, summer and fall; medium-sized nodules (12-15 mm) were in second half of summer, fall and winter, and large (mature) nodules (20-25 mm) in fall, winter and part of spring. Significant differences were found among host age groups.     

Sequence and phylogenetic analysis of the 16S rRNA gene of Ehrlichia canis strains in dogs with clinical monocytic ehrlichiosis

The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype.