Thelazia callipaeda (Spirurida, Thelaziidae) of carnivores and humans: morphological study by light and scanning electron microscopy

Thelazia nematodes cause ocular infection in several mammals and are transmitted by dipteran flies. Thelazia callipaeda Railliet and Henry 1910 (Spirurida, Thelaziidae) is common in the Far East, where it causes eyeworm infection in carnivores and humans. In the past few years, it has also been reported in dogs, cats and foxes in Northern and Southern Italy. Although a great deal of work has been carried out in China and other Eastern countries, knowledge of the morphology of T. callipaeda is scanty. Eighty-three nematodes collected from the eyes of naturally infected dogs from the Basilicata region (southern Italy) were examined by light and scanning electron microscopy, and the most important features of the nematodes were described. The morphological characteristics useful for the identification of T. callipaeda are discussed.     

Enzooticity of the dogs,the reservoir host of Thelazia callipaeda,in Korea

The reservoir hosts of Thelazia callipaeda were examined. The eyes of the 76 dogs raised at farm. 78 military dogs (shepherds), 96 cattle, and 105 pigs were investigated for the presence of eyeworm. Among them, six worms of T. callipaeda were collected from two dogs raised at farm (2.7%), and 188 worms from 26 shepherds (33.5%). No worms were recovered from the cattle or pigs. These results suggest that the dogs, especially the military dogs are serving as a reservoir host of T. callipaeda in Korea.     

Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: evidence for pathogen transmission by a male arthropod vector

Knowledge about Phortica variegata (Drosophilidae, Steganinae), the intermediate host of the eyeworm Thelazia callipaeda (Spirurida, Thelaziidae), is confined to experimental studies. To investigate the role P. variegata plays in the transmission of T. callipaeda under natural conditions, the population dynamics of these flies in the natural environment and their feeding preferences (on vegetables and/or animal lachrymal secretions) were examined. From April to November 2005, a total number of 969 (557 males and 412 females) P. variegata flies were collected weekly in a region of southern Italy with a history of canine thelaziosis. The flies were identified and dissected or subjected to a PCR assay specific for a region within the ribosomal ITS-1 DNA of T. callipaeda. The zoophilic preferences of P. variegata were assessed by collecting flies around the eyes of a person or around a fruit bait. Seven hundred and twenty flies (398 males and 322 females) were dissected under a stereomicroscope; 249 flies (158 males and 91 females) that died prior to the dissection were subjected to molecular investigation. Only P. variegata males were infected with larval T. callipaeda both at dissection (six, 0.83%) and with the specific PCR (seven, 2.81%), representing a total percentage of 1.34% flies infected. Interestingly, only males were collected around the eyes, compared with a male/female ratio of 1:4 around the fruit. This survey indicated that P. variegata males act as intermediate hosts of T. callipaeda under natural conditions in Europe. Both the zoophilic behaviour of P. variegata males on lachrymal secretions and their role as vector of T. callipaeda have been discussed as they represent a peculiarity in medical and veterinary entomology. The synchrony between the fly population dynamics and the biology of the nematode in the definitive host provides an interesting model for exploring the co-evolution of Thelazia spp. with their hosts.     

Helminths in the wolf, Canis lupus, from north-western Spain

Fifteen helminth species were collected from 47 wolves (Canis lupus ) which were surveyed from 1993 to 1999 in northwestern Spain. These included the trematode Alaria alata (2.1%); the cestodes Taenia hydatigena (44.7%), T. multiceps (29.8%), T. serialis (2.1%), Dipylidium caninum (6.4%) and Mesocestoides sp. aff. litteratus (4.2%); and the nematodes Pearsonema plica (7.4%), Trichuris vulpis (10.6%), Trichinella britovi (12.8%), Ancylostoma caninum (8.5%), Uncinaria stenocephala (51.1%), Toxocara canis (6.4%) Toxascaris leonina (4.2%), Angiostrongylus vasorum (2.1%) and Dirofilaria immitis (2.1%). Only two wolves were not infected. A single infection occurred in 28.9% of the cases, but the commonest infracommunity (31.1%) involved three species. The helminths Alaria alata, Taenia hydatigena, Mesocestoides sp. aff. litteratus, P. plica, Trichuris vulpis, and Ancylostoma caninum parasitizing C. lupus are reported for the first time in Spain. Taenia serialis and D. immitis are reported for the first time in wolves in Europe. Angiostrongylus vasorum represents a new host record for wolves. The helminth fauna of Spanish wolves is compared with that of other European wolf populations. Some epidemiological considerations of the helminth fauna of wolves in Spain and the health risk to humans are also discussed.     

What Drives Wolves: Fear or Hunger? Humans,Diet, Climate and Wolf Activity Patterns

Activity patterns of animals depend on environmental and intrinsic factors. Studies undertaken across the current wolf ( Canis lupus ) range suggested a number of variables that may be correlated with activity patterns of wolves. These factors vary locally and there has been no attempt so far at defining those that ubiquitously impact wolf behaviour. I compared 11 studies (from Alaska to Israel) to assess the influence of (1) public road density, (2) human population density, (3) human-caused mortality, (4) proportion of domestic animals in wolf diet, (5) proportion of forest, (6) latitude and (7) mean annual temperature on nocturnal wolf activity and movements. Nocturnal activity was mainly correlated to the proportion of domestic animals in the diet and the density of public roads, whereas nocturnal movements were mainly correlated to latitude. The importance of latitude indicates that sun periodicity might represent an important signal (`Zeitgeber') for circadian rhythms in wolves. Environmental constraints such as high temperatures during the day and a higher hunting success in crepuscular periods probably limit the ability of wolves to avoid humans by nocturnal behaviour. I therefore suggest that in regions where wolves hunt wild prey, they experience a trade-off between predation risk by humans and increased hunting success during twilight hours.     

Molecular characterization of the first internal transcribed spacer of ribosomal DNA of the most common species of eyeworms (Thelazioidea: Thelazia)

Spirurid nematodes of the genus Thelazia cause ocular infection in several mammals and are transmitted by dipteran flies. Of the 10 described species, T. gulosa, T. rhodesi, T. skrjabini (affecting cattle), T. lacrymalis (affecting horses), and T. callipaeda (affecting carnivores and humans) are the most common. The aim of this study was to characterize the first internal transcribed spacer (ITS1) ribosomal DNA sequences of these 5 species. The length of the ITS1 sequences ranged from 357 bp (T. lacrymalis) to 905 bp (T. callipaeda). Interspecific differences ranged from 35 to 77%. Polymorphic sites were detected in each species. Intraspecific variation varied from 0.3 to 2.5%. The characteristics of the ITS1 of Thelazia spp. show similarities to those of other Spirurida. The findings of this present study show that the ITS1 represent a powerful genetic marker for the molecular identification of eyeworms and a useful tool for developing molecular epidemiological techniques to study Thelazia spp. transmission patterns and prevalence in definitive and intermediate hosts.     

Viral gut metagenomics of sympatric wild and domestic canids,and monitoring of viruses: Insights from an endangered wolf population

Animal host–microbe interactions are a relevant concern for wildlife conservation, particularly regarding generalist pathogens, where domestic host species can play a role in the transmission of infectious agents, such as viruses, to wild animals. Knowledge on viral circulation in wild host species is still scarce and can be improved by the recent advent of modern molecular approaches. We aimed to characterize the fecal virome and identify viruses of potential conservation relevance of diarrheic free‐ranging wolves and sympatric domestic dogs from Central Portugal, where a small and threatened wolf population persists in a highly anthropogenically modified landscape. Using viral metagenomics, we screened diarrheic stools collected from wolves (n = 8), feral dogs (n = 4), and pet dogs (n = 6), all collected within wolf range. We detected novel highly divergent viruses as well as known viral pathogens with established effects on population dynamics, including canine distemper virus, a novel bocavirus, and canine minute virus. Furthermore, we performed a 4‐year survey for the six wolf packs comprising this endangered wolf population, screening 93 fecal samples from 36 genetically identified wolves for canine distemper virus and the novel bocavirus, previously identified using our metagenomics approach. Our novel approach using metagenomics for viral screening in noninvasive samples of wolves and dogs has profound implications on the knowledge of both virology and wildlife diseases, establishing a complementary tool to traditional screening methods for the conservation of threatened species.     

Arctic Lineage-Canine Distemper Virus as a Cause of Death in Apennine Wolves (Canis lupus) in Italy

Canine distemper virus (CDV) infection is a primary threat affecting a wide number of carnivore species, including wild animals. In January 2013, two carcasses of Apennine wolves (Canis lupus) were collected in Ortona dei Marsi (L''Aquila province, Italy) by the local Veterinary Services. CDV was immediately identified either by RT-PCR or immunohistochemistry in lung and central nervous tissue samples. At the same time, severe clinical signs consistent with CDV infection were identified and taped (Videos S1–S3) from three wolves rescued in the areas surrounding the National Parks of the Abruzzi region by the Veterinary Services. The samples collected from these symptomatic animals also turned out CDV positive by RT-PCR. So far, 30 carcasses of wolves were screened and CDV was detected in 20 of them. The sequencing of the haemagglutinin gene and subsequent phylogenetic analysis demonstrated that the identified virus belonged to the CDV Arctic lineage. Strains belonging to this lineage are known to circulate in Italy and in Eastern Europe amongst domestic dogs. To the best of our knowledge this is the first report of CDV Arctic lineage epidemics in the wild population in Europe.     

Musca domestica is not a vector of Thelazia callipaeda in experimental or natural conditions

Thelazia callipaeda Railliet and Henry (Spirurida: Thelaziidae), commonly called oriental eyeworm for its widespread presence in the Far East, has been recently found to affect dogs, cats and foxes in northern and southern Italy. Although the biology of T. callipaeda in the definitive hosts has been recently investigated, many doubts still remain about its biology in insect vectors. It has been suggested that more than one species of Diptera, namely Musca domestica Linnaeus (Diptera: Muscidae) and Amiota okadai Maca (Diptera: Drosophilidae), may be involved in the transmission of T. callipaeda in China. The aim of the work described here was to verify the role of M. domestica as a vector of T. callipaeda both in experimental and natural conditions. A total of 310 m. domestica (Group 1) were put in a cage and allowed to feed for 14 days around the eyes of a dog naturally infected by T. callipaeda. Ten flies were collected daily for 14 days. A total of 149 houseflies (Group 2) were fed with T. callipaeda first stage larvae (L1) and dissected at 1, 2 and 7 days post-infection. From June to August 2003, flies were netted (Group 3) in two different sites every 10 days both from the environment and directly from the periocular region of dogs affected by thelaziosis. Musca flies were examined for eyeworms by dissection and visual inspection of house flies (Groups 1 and 2) and using a molecular approach (Groups 1-3) via a specific amplification of the ribosomal internal transcribed spacer 1 (ITS1) sequence of T. callipaeda. On the whole, 180 pools of M. domestica flies were processed molecularly and all the experimentally infected flies (Groups 1 and 2) were found to be negative both at the visual dissection and at the molecular assay. Similarly, the 234 M. domestica collected from Group 3 were negative for T. callipaeda. The results clearly suggest that M. domestica is unlikely to act as a vector of T. callipaeda in southern Europe, in contrast with a single previous report.     

Marked host specificity and lack of phylogeographic population structure of Campylobacter jejuni in wild birds

Zoonotic pathogens often infect several animal species, and gene flow among populations infecting different host species may affect the biological traits of the pathogen including host specificity, transmissibility and virulence. The bacterium Campylobacter jejuni is a widespread zoonotic multihost pathogen, which frequently causes gastroenteritis in humans. Poultry products are important transmission vehicles to humans, but the bacterium is common in other domestic and wild animals, particularly birds, which are a potential infection source. Population genetic studies of C. jejuni have mainly investigated isolates from humans and domestic animals, so to assess C. jejuni population structure more broadly and investigate host adaptation, 928 wild bird isolates from Europe and Australia were genotyped by multilocus sequencing and compared to the genotypes recovered from 1366 domestic animal and human isolates. Campylobacter jejuni populations from different wild bird species were distinct from each other and from those from domestic animals and humans, and the host species of wild bird was the major determinant of C. jejuni genotype, while geographic origin was of little importance. By comparison, C. jejuni differentiation was restricted between more phylogenetically diverse farm animals, indicating that domesticated animals may represent a novel niche for C. jejuni and thereby driving the evolution of those bacteria as they exploit this niche. Human disease is dominated by isolates from this novel domesticated animal niche.     

Serologic evidence of Leishmania infection in free-ranging wild and domestic canids around a Brazilian National Park

Transmission of disease between wildlife, domestic animals, and humans is of great concern to conservation issues and public health. Here we report on the prevalence of anti-Leishmania sp. antibodies in 21 wild canids (7 Chrysocyon brachyurus, 12 Cerdocyon thous, and 2 Lycalopex vetulus) and 74 free domestic dogs (Canis familiaris) sampled around the Serra do Cipó National Park. In dogs, the apparent prevalence was 8.1% and in wild canids it was 19% (2 crab-eating foxes, C. thous, and 2 maned wolves, C. brachyurus). Management of the domestic dog population with evaluation of incidence changes in humans and wildlife, and enlightenment on the role of wild reservoirs are essential issues for future action and research.     

Molecular Characterization of Enterocytozoon bieneusi in Wild Carnivores in Spain

Microsporidia comprises a diverse group of obligate intracellular parasites that infect a broad range of invertebrates and vertebrates. Among Microsporidia, Enterocytozoon bieneusi is the most frequently detected species in humans and animals worldwide bringing into question the possible role of animal reservoirs in the epidemiology of this pathogen. Although E. bieneusi is an emerging zoonotic pathogen able to infect many domestic and wild mammals that could act as reservoir of infection for humans and other animals, only few studies have documented its occurrence in wild carnivores. To determine the occurrence of E. bieneusi in wild carnivores, we examined 190 wild carnivores collected from different locations in Spain. Twenty‐five fecal samples (13.2%) from three host species (European badger, beech marten, and red fox) were E. bieneusi‐positive by PCR. Nucleotide sequence analysis of the ITS region revealed a high degree of genetic diversity with a total of eight distinct genotypes including four known (PtEbIX, S5, S9, and WildBoar3) and four novel (EbCar1‐EbCar4) genotypes identified. Phylogenetic analysis showed that the four novel genotypes (EbCar1‐EbCar4), S5, S9, and WildBoar3 clustered within the previously designated zoonotic Group 1. Our results demonstrate that human‐pathogenic genotypes are present in wild carnivores, corroborating their potential role as a source of human infection and environmental contamination.     

Presence of Thelazia callipaeda Railliet & Henry, 1910, in Piedmont, Italy

A sample of 314 dogs from a zone of 80,000 ha in Piedmont, Italy, were examined for the presence of thelaziae under the nictitating membrane. Sixteen animals from eight districts were infected, the foothills being the main location of the parasitosis. All 44 nematodes collected were identified as Thelazia callipaeda.     

Human thelaziosis--a neglected parasitic disease of the eye

The oriental eyeworm, Thelazia callipaeda (Spirurida, Thelaziidae), infects a range of definitive hosts, such as dogs, cats, foxes, rabbits, and humans. This parasite usually lives under the nictitating membrane of the eye, where the adult females release first-stage larvae into the lachrymal secretions; these larvae are subsequently ingested by the intermediate arthropod host within which they develop to the infective, third-stage larvae. The latter larvae are then deposited into the eyes of the definitive host. Recently, T. callipaeda has been reported to infect dogs, foxes, and/or cats in Europe (Italy, France, and Germany). Human thelaziosis (HT) is considered to be an underestimated parasitic disease, whose prevalence appears to have increased in poor socioeconomic settings in many Asian countries, including China. In humans, the disease can be subclinical or symptomatic, exhibiting epiphora, conjunctivitis, keratitis, excessive lachrymation, corneal opacity, and/or ulcers. Knowledge about HT is presently fragmentary and mainly limited to clinical case reports. This article provides a background on the parasite and its life cycle, reviews cases of human thelaziosis, summarizes key aspects regarding the diagnosis of thelaziosis, and proposes future research and methods of control of the disease in humans, particularly in Asia.     

Toxoplasma gondii in humans and animals in Japan: An epidemiological overview

Toxoplasmosis is a cosmopolitan protozoan zoonosis caused by Toxoplasma gondii infamous for inducing severe clinical manifestations in humans. Although the disease affects at least one billion people worldwide, it is neglected in many countries including developed ones. In literature, the epidemiological data documenting the actual incidence of the disease in humans and domestic animals from Japan are limited and importantly many earlier papers on T. gondii infections were published in Japanese and a considerable part is not available online. Herein, we review the current summary about the epidemiological situation of T. gondii infection in Japan and the potential associated risk factors in humans and animals as well as the different T. gondii genotypes isolated in Japan. Several T. gondii isolates have been identified among cats (TgCatJpTy1/k-3, TgCatJpGi1/TaJ, TgCatJpObi1 and TgCatJpOk1–4) and goats (TgGoatJpOk1–13). This literature review underscores the need for a nationwide investigation of T. gondii infection in Japanese people and assessment of the socioeconomic impact of the disease burden. Furthermore, epidemiological studies in domestic and wild animals and estimation of degree of contamination of soil or water with T. gondii oocysts are needed, for a better understanding of the scope of this public health concern.     

The helminth fauna of wild and domestic ruminants in Mongolia—a review

The helminth fauna of eight species of wild ruminants and five species of domestic ruminants sharing common grassland in Mongolia was reviewed. A total of 108 species of helminths, belonging to 40 genera, 23 families, 11 superfamilies, 6 orders and 3 classes were reported in wild and domestic ruminants in Mongolia. Several new host–helminth associations have been recorded. Twenty seven helminth species have been found in certain host species for the first time in Mongolia. These included 2 species of trematodes, 2 species of cestodes and 23 species of nematodes. Helminths that are specific for some of the examined ruminant species were determined. However, wild animals may serve as a reservoir of helminth infections for domestic animals when sharing grassland in Mongolia. The helminth fauna of the ruminants with respect to occurrence, distribution, prevalence and intensity of infection was considered for six geographic regions of Mongolia.     

Innate immunity in free-ranging African buffalo (Syncerus caffer): associations with parasite infection and white blood cell counts

Mammalian immunology has been studied in great detail in laboratory animals, but few of the tools and less of the insight derived from these studies have been placed in the context of natural, outbred wildlife populations subject to variable environments. We investigated patterns of innate immunity in free-ranging African buffalo in relation to host traits (age, reproductive status, body condition, white blood cell counts) and disease status (bovine tuberculosis [BTB], gastrointestinal nematodes, coccidia, ticks). We evaluated and used an in vitro assay measuring bactericidal competence of blood to assess a component of innate immunity in 200 female buffalo captured at Kruger National Park, South Africa, in June/July and October 2008. Animals with BTB had higher bactericidal competence of blood. Animals with higher neutrophil counts had higher bactericidal competence, whereas animals with lower lymphocyte counts had higher bactericidal competence. This pattern was driven by animals captured at the end of the dry season (October) and may be evidence of immune polarization, whereby individuals are unable to upregulate multiple components of immunity simultaneously. Bactericidal competence did not vary with host pregnancy status, body condition, age, lactation, tick infestation, nematode egg count, or coccidia oocyst count. Overall, we demonstrate that the bactericidal competence assay is practical and informative for field-based studies in wild bovids. Our results also show a correlation between bactericidal competence and bovine tuberculosis infection and reveal possible functional polarizations between different types of immune response in a free-ranging mammal.     

Using Molecular Epidemiology to Track Toxoplasma gondii from Terrestrial Carnivores to Marine Hosts: Implications for Public Health and Conservation

Background

Environmental transmission of the zoonotic parasite Toxoplasma gondii, which is shed only by felids, poses risks to human and animal health in temperate and tropical ecosystems. Atypical T. gondii genotypes have been linked to severe disease in people and the threatened population of California sea otters. To investigate land-to-sea parasite transmission, we screened 373 carnivores (feral domestic cats, mountain lions, bobcats, foxes, and coyotes) for T. gondii infection and examined the distribution of genotypes in 85 infected animals sampled near the sea otter range.

Methodology/Principal Findings

Nested PCR-RFLP analyses and direct DNA sequencing at six independent polymorphic genetic loci (B1, SAG1, SAG3, GRA6, L358, and Apico) were used to characterize T. gondii strains in infected animals. Strains consistent with Type X, a novel genotype previously identified in over 70% of infected sea otters and four terrestrial wild carnivores along the California coast, were detected in all sampled species, including domestic cats. However, odds of Type X infection were 14 times higher (95% CI: 1.3–148.6) for wild felids than feral domestic cats. Type X infection was also linked to undeveloped lands (OR = 22, 95% CI: 2.3–250.7). A spatial cluster of terrestrial Type II infection (P = 0.04) was identified in developed lands bordering an area of increased risk for sea otter Type II infection. Two spatial clusters of animals infected with strains consistent with Type X (P≤0.01) were detected in less developed landscapes.

Conclusions

Differences in T. gondii genotype prevalence among domestic and wild felids, as well as the spatial distribution of genotypes, suggest co-existing domestic and wild T. gondii transmission cycles that likely overlap at the interface of developed and undeveloped lands. Anthropogenic development driving contact between these cycles may increase atypical T. gondii genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife.     

Filarids (Spirurida: Onchocercidae) in wild carnivores and domestic dogs from the Brazilian Atlantic forest

Onchocercidae nematodes are heteroxenous parasites with worldwide distribution, and some of the species associated to animals may present zoonotic potential. Climatic changes and anthropic influences on the environment may result in vectors’ proliferation, facilitating the spillover to humans and/or non-typical animal hosts. The Iguaçu National Park (PARNA Iguaçu), one of the most important Brazilian natural remanescents of Atlantic rainforest, is strongly affected by human activities such as tourism and agriculture. The complexity of this area is especially characterized by the close nexus between the rich wildlife, humans, and domestic animals, especially domestic dogs. Based on this, this research aimed to diagnose the Onchocercidae nematodes in wild carnivores and domestic dogs in the PARNA Iguaçu and the surrounding areas. For this, we collected 162 samples of seven species of wild carnivores and 225 samples of domestic dogs. The presence of microfilariae in the blood samples was diagnosed by the modified Knott’s test and molecular screening, and the specific identification was based on sequencing of the myoHC and hsp70 genes. Microfilariae were detected only in ring-tailed coatis, in which we found five species: Mansonella sp. 1, Mansonela sp. 2, Onchocercidade gen. sp. 1, Onchocercidade gen. sp. 2, and Dirofilaria immitis. The morphological analysis supported the molecular findings. The domestic dogs were parasitized by Acanthocheilonema reconditum, representing a new locality record for this species. Phylogenetic analysis showed high genetic similarity among the four undetermined species and Mansonella spp., Brugia spp., and Wuchereria bancrofti. The presence of D. immitis in ring-tailed coatis may be result of spillover from dogs, even though the parasite was not diagnosed in the sampled dogs. The presence of several undetermined Onchocercidae species indicates the necessity of continuous investigations on wild and domestic animals from Neotropical area, especially considering the growing anthropic influence on forest remnants.     

Comparison of Campylobacter populations in wild geese with those in starlings and free-range poultry on the same farm

Wild geese are a potential source of Campylobacter infection for humans and farm animals and have been implicated in at least two large waterborne disease outbreaks. There have been few investigations into the population biology of Campylobacter in geese, carriage rates are reported to vary (0 to 100%), and no genetic characterization of isolates has been performed. Fecal samples collected from wild geese in Oxfordshire, United Kingdom, were culture positive for C. jejuni (50.2%) and C. coli (0.3%). The C. jejuni (n = 166) isolates were characterized by using multilocus sequence typing and were compared with isolates collected from free-range broiler chickens and wild starlings sampled at the same location. A total of 38 STs, six clonal complexes, and 23 flaA SVR nucleotide STs were identified. The ST-21 and ST-45 complexes (5.4% of isolates) were the only complexes to be identified among isolates from the geese and the other bird species sampled in the same location. These clonal complexes were also identified among human disease isolates collected in the same health care region. The results indicate that large numbers of wild geese carry Campylobacter; however, there was limited mixing of Campylobacter populations among the different sources examined, and the host source could be predicted with high probability from the allelic profile of a C. jejuni isolate. In conclusion, genotypes of C. jejuni isolated from geese are highly host specific, and a comparison with isolates from Oxfordshire cases of human disease revealed that while geese cannot be excluded as a source of infection for humans and farm animals, their contribution is likely to be minor.