Echinococcus multilocularis in Belgium: prevalence in red foxes (Vulpes vulpes) and in different species of potential intermediate hosts

Echinococcus multilocularis causes a rare but potentially lethal zoonotic infection in humans. This tapeworm is known to be endemic in foxes in several countries of Western and Central Europe. In Western Europe, the common vole (Microtus arvalis) and the water vole (Arvicola terrestris) are considered to be the most important intermediate host species of this cestode whereas the red fox is by far the most important final host. The purpose of this study was to provide data on the prevalences in Wallonia (Southern part of Belgium) both in the red fox and in different potential intermediate hosts. A total of 990 red foxes were examined between January 2003 and December 2004 for the presence of E. multilocularis. The average prevalence was 24.55% (22.38-27.87). Out of 1249 rodents or insectivores belonging to the species Apodemus sylvaticus, Arvicola terrestris, Clethrionomys glareolus, Microtus arvalis, Microtus agrestris and Sorex araneus, only one M. arvalis (out of 914-0.11% (0.003-0.61) and one C. glareolus (out of 23-4.3% (0.1-21.9) were found to be infected. However, the muskrat (Ondatra zibethicus) seems to be a good intermediate host as 11.18% (9.72-12.76) of the animals (n=1718) were found to be infected. A positive correlation was found between the prevalences in foxes and in muskrats in each of the different geological regions. This study indicates that the muskrat is highly sensitive to this zoonotic tapeworm and could perhaps represent a good bioindicator when studying the epidemiology of this parasitic infection in Belgium and in other countries where the muskrat is present.     

Echinococcus multilocularis (Cestoda,Taeniidae) in Red foxes (Vulpes vulpes) in northern Belgium

The first record of the tapeworm Echinococcus multilocularis (Cestoda, Taeniidae) in Red foxes (Vulpes vulpes) in northern Belgium is described. Between 1996 and 1999, 237 dead foxes were examined for the presence of this tapeworm using the intestinal scraping technique. Four foxes (1.7%) were found to be infected with E. multilocularis and showed medium to very high parasitic burdens. Three infected foxes originated from the south of the study area and the fourth animal came from the north of the study area near the border with The Netherlands. These findings are discussed in relation to the high endemicity of E. multilocularis in southern Belgium and to the increased distribution of the Red fox (V. vulpes) in northern Belgium during the last two decades.     

Controlling Echinococcus multilocularis-ecological implications of field trials

Two field trials to reduce the prevalence of Echinococcus multilocularis in foxes have been conducted in recent years. Although both trials reduced prevalence considerably, they failed to eradicate the parasite in the study region. Following the control trial in northern Germany, prevalence recovered unexpectedly and rapidly, reaching pre-control levels five quarters (15 months) after the end of control. To understand the internal dynamics of the parasite-host system's reaction to control, we developed a spatially explicit simulation model, Echi. The simulation model incorporates the information available concerning fox tapeworm population dynamics.Using epidemiological parameters to adjust pre-control prevalence, the model predicts the temporal evolution of the prevalence of E. multilocularis in controlled foxes without departing from the range of uncertainty of the field data. However, the model does not predict the rapid pre-control recovery observed in the field trial.The deviation of the model's prediction from field data indicates the involvement of processes not yet taken into account. We modified the model step by step to mimic processes with the potential to cause the rapid post-control recovery of the prevalence of E. multilocularis in foxes.Neither the longevity of tapeworm eggs nor the migratory behaviour of foxes showed any influence on the post-control reaction of the parasite-host system. However, landscape structures leading to a heterogeneous distribution of infected foxes have the potential to alter the system's reaction to control. If infected foxes are concentrated in multiple clusters in the landscape, the model prediction tallied with the range of uncertainty of the field data. Such spatial distribution of infected foxes may be caused by differential abiotic conditions influencing the survival of tapeworm eggs.The model was found to comply best with field data if the foxes acquire partial immunity by being exposed to the fox tapeworm.Both hypotheses explaining the rapid post-control recovery of the prevalence of E. multilocularis observed in the fox population were supported by field data.Both hypotheses have far-reaching consequences for future control trials. The spatial aggregation of infected foxes would enable control efforts to be concentrated on these highly infected areas. However, the acquisition of immunity acts as a buffer to control, necessitating intensified control measures.     

Detection of Echinococcus multilocularis in foxes in The Netherlands

Echinococcus multilocularis was demonstrated in 5 out of 272 foxes in The Netherlands close to the border with Germany and Belgium. Besides microscopic examination of mucosal scrapings, two different PCR assays were used based on the detection of E. multilocularis DNA in colon content. Two distinct areas in The Netherlands were positive for E. multilocularis. Two positive foxes were found in the northern province of Groningen and three positive foxes were found in the southern province of Limburg. Both PCR assays detected more positive foxes compared to microscopic examination of the intestinal content. This is the first report of E. multilocularis in foxes occurring in The Netherlands.     

Increase in the prevalence of Echinococcus multilocularis infection in red foxes in Lower Saxony.]

Echinococcus multilocularis is a tapeworm having carnivores as final hosts, the red fox in particular, dog and cat less frequent. Its two host life cycle consists of a larval cycle which predominantly takes place in the liver of rodents such as mice but it can also develop in musk rats as intermediate hosts. Man can also be infected and serves as a wrong intermediate host. He develops an alveolar echinococcosis which usually ends lethal without medical treatment. The prevalence of E. multilocularis among 5.365 red foxes in Lower Saxony was monitored from 1991 to 1997. The data were analysed using spatial epidemiological methods. This evaluation is based on a significance test which was applied to the parameters of spatial autoregressive regression models (CAR) fitting to the data of two successive sampling periods from 1991-1994 and 1994-1997. The mean prevalence (spatial median) increased from 6% to 11%. The results of this epidemiological study which was restricted to Lower Saxony support for the first time the earlier reported assumption that the prevalence of foxes infected with E. multilocularis has risen in Europe in the last decade. The reasons for this development are still unknown.     

Aleutian mink disease virus in furbearing mammals in Nova Scotia,Canada

Background

Aleutian mink disease virus (AMDV) is widespread among ranched and free-ranging American mink in Canada, but there is no information on its prevalence in other wild animal species. This paper describes the prevalence of AMDV of 12 furbearing species in Nova Scotia (NS), Canada.

Methods

Samples were collected from carcasses of 462 wild animals of 12 furbearing species, trapped in 10 NS counties between November 2009 and February 2011. Viral DNA was tested by PCR using two primer pairs, and anti-viral antibodies were tested by counterimmunoelectrophoresis (CIEP) on spleen homogenates.

Results

Positive PCR or CIEP samples were detected in 56 of 60 (93.3%) American mink, 43 of 61 (70.5%) short-tailed weasels, 2 of 8 (25.0%) striped skunks, 2 of 11 (18.2%) North American river otters, 9 of 85 (10.6%) raccoons, and 2 of 20 (10.0%) bobcats. Samples from six fishers, 24 coyotes, 25 red foxes, 58 beavers, 45 red-squirrels and 59 muskrats were negative. Antibodies to AMDV were detected by CIEP in 16 of 56 (28.6%) mink and one of the 8 skunks (12.5%). Thirteen of the mink were positive for PCR and CIEP, but three mink and one skunk were CIEP positive and PCR negative. Positive CIEP or PCR animals were present in all nine counties from which mink or weasel samples were collected.

Conclusions

The presence of AMDV in so many species across the province has important epidemiological ramifications and could pose a serious health problem for the captive mink, as well as for susceptible wildlife. The mechanism of virus transmission between wildlife and captive mink and the effects of AMDV exposure on the viability of the susceptible species deserve further investigation.     

Reduced egg production of Echinococcus multilocularis in experimentally infected and re-infected red foxes (Vulpes vulpes)

Ingestion of eggs of the small fox tapeworm, Echinococcus multilocularis, causes the severe human disease alveolar echinococcosis. Previously, the dynamics of the egg excretion from infected carnivores have been studied only where the host animals have been exposed to a single experimental infection. In nature, foxes are most likely repeatedly infected. To study the effect of repeated exposure, twenty-one foxes were inoculated with a high dose of E. multilocularis protoscoleces three times over a 1-month period. For comparative purposes, three groups of twenty-one foxes were respectively inoculated with low, medium, or high single dose of protoscoleces. For each group, worm number and morphology were analyzed after necropsy of seven foxes at 1, 2, and 4 months after last inoculation. The establishment of intestinal worms was very low in all foxes, and surprisingly, most of the worms did not produce eggs. Although most reproductive structures were detectable, the genital pore and the cirrus pouch often had abnormal enlargements that spread internally, most likely preventing the reproductive function. The reason for this abnormality could not be determined, but the preparation and storage conditions of the inoculated protoscoleces may have contributed to the stunted development. Physical stress of E. multilocularis at the larval stage in rodents may later adversely affect the reproductive success of the adult tapeworm in the carnivore definitive host; as in the present study where a worm establishment in the definitive host was only followed by a neglectable egg production.     

Survey for Trichinella spp. in red foxes (Vulpes vulpes) in Belgium.

Concurrently with a survey for Echinococcus multilocularis in the red fox (Vulpes vulpes) in Flanders, northern Belgium, serological and parasitological analyses for Trichinella spp. were carried out from 1996 to 1999. Muscle samples from foxes in Wallonia, southern Belgium, were obtained during a survey for rabies and alveolar echinococcosis from 1998 to 2000.In muscle samples from tongue, diaphragm, hindlegs and tail of 179 Flemish foxes no larvae were found by trichinoscopy. Serum and muscle juice of, respectively 176 and 26 animals were examined using an ELISA for the detection of antibodies against excretory-secretory (ES) antigen. There were eight (4.5%) positive sera, but no positive muscle juice samples.All muscle samples from 639 foxes in Wallonia proved to be negative for larvae in artificial digestion. Serum and muscle juice of 130 and 478 foxes, respectively were examined in ES-ELISA. There were 61 (46.9%) positive sera and 90 (18.8%) positive muscle juice samples. A comparison between 88 serum and muscle juice samples of the same foxes showed that only half of the serum-positive animals were detected using muscle juice. However, for establishing the true meaning of these results, a more profound epidemiological study on the vulpine population in Belgium is necessary.     

Liver fluke infection and sarcoptic mange in red foxes in Berlin.]

396 red foxes originating from the city of Berlin were examined for opisthorchiid liver flukes and clinical sarcoptic mange between January 1997 and March 1998. Out of 232 (= 58.6%) foxes positive for opisthorchiid flukes 221 animals harboured Metorchis bilis and 70 were infected with Opisthorchis felineus. Pseudamphistomum truncatum was found only in 8 foxes. M. bilis occurred as mono-infection in 154 animals. M. bilis in combination with O. felineus was found in 61 cases. Pure Opisthorchis infection as well as other fluke combinations were found in a small number of animals only. 85 (= 21.5%) foxes showed clinical sarcoptic mange. Liver fluke positive foxes showed a higher mange prevalence than uninfected animals. However, significant associations between flukes and manage were only found when comparing uninfected foxes with those having the highest worm burden. The association of liver flukes and mange could be established for adult female foxes by a significant Odds Ratio of 4.3.     

Isolation or detection of Bartonella vinsonii subspecies berkhoffii and Bartonella rochalimae in the endangered island foxes (Urocyon littoralis)

Bartonella rochalimae (B.r.) and Bartonella vinsonii subsp. berkhoffii (B.v.b.) have been isolated from gray foxes (Urocyon cinereoargenteus) in mainland California and high Bartonella seroprevalence was reported in island foxes (U. litorralis), especially from Santa Cruz and Santa Rosa Islands. As a follow-up study, the objectives were to determine the prevalence of Bartonella bacteremia and seropositivity and to identify the Bartonella species infecting a convenience sample of 51 island foxes living on Santa Rosa Island. Using an immuno-fluorescence antibody test directed against B.v.b and Bartonella clarridgeiae (B.c.), used as a substitute for B.r., the overall antibody prevalence was 62.7% with 16 (31.4%) foxes seropositive for B.c. only, 5 (9.8%) for B.v.b. only, and 11 (21.6%) for both antigens. B.v.b. was isolated from 6 (11.8%) foxes using blood culture medium. An additional seropositive fox tested PCR positive for B.v.b. and 3 other seropositive foxes tested PCR positive for B. rochalimae. All of the isolated B.v.b. colonies and the B.v.b. PCR positive sample belonged to type III, the same type found to infect mainland gray foxes. Therefore, Bartonella infection is widespread within this island fox population with evidence for B.v.b. type III reservoir host-specificity. Presence of B. rochalimae in the Channel Islands has been detected for the first time using PCR.     

Occurrence of Giardia and Cryptosporidium in Norwegian red foxes (Vulpes vulpes)

Faecal samples from 269 Norwegian wild red foxes (Vulpes vulpes) shot during the hunting season (October-April) in 2002-2004 were examined for the presence of Giardia and Cryptosporidium. Cryptosporidium oocysts were detected in samples from 6 (2.2%) of the foxes, and Giardia cysts in 13 (4.8%) of the foxes. The prevalence of Giardia infection was significantly higher in juvenile male foxes than in adult male foxes, but no other significant differences between age and sex were found. No significant differences in prevalence related to geographical origin of animals were found. Insufficient nucleated Cryptosporidium oocysts were isolated for successful PCR, but genotyping of Giardia duodenalis isolates from seven foxes demonstrated a high degree of heterogeneity amongst them, with all isolates belonging to the zoonotic Assemblages A and B.     

Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in Hungarian red foxes (Vulpes vulpes)

In the present study we have investigated the seroprevalence to the protozoan parasites Toxoplasma gondii and Neospora caninum in 337 red foxes (Vulpes vulpes) from 16 out of 19 counties in Hungary. The foxes were originally collected within a National vaccination program against rabies. Antibodies to T. gondii were detected in as many as 228 (68%) of the foxes using a commercial direct agglutination test (DAT). In an indirect iscom ELISA, five foxes (1.5%) were positive for antibodies against N. caninum. The high prevalence of foxes positive for T. gondii might be explained by the widespread occurrence of the parasite in the diet of foxes. As a contrast, latent infections of N. caninum among red foxes in Hungary are much less common.     

Toxoplasma gondii in foxes and rodents from the German Federal States of Brandenburg and Saxony-Anhalt: seroprevalence and genotypes

Data on the genotypes of Toxoplasma gondii circulating in wildlife are scarce. In the present study, foxes and rodents from two Federal States in Central or Eastern Germany were examined for T. gondii infections. Body fluids were collected at necropsy or fluids were obtained from frozen tissues of naturally exposed red foxes (Vulpes vulpes), voles (Microtus arvalis), shrews (Neomys anomalus) and a striped field mouse (Apodemus agrarius) and tested for T. gondii by serology. DNA isolated from tissues of seropositive foxes and all the rodents was examined by PCR. In the German Federal States of Brandenburg and Saxony-Anhalt 152/204 (74.5%) and 149/176 (84.7%) of foxes, respectively, but none of the rodents (0/72) had antibodies to T. gondii. Only 28/152 (18.4%) and 20/149 (13.4%) of seropositive foxes from Brandenburg and Saxony-Anhalt, respectively, but none of the rodents tested PCR-positive for T. gondii. The complete T. gondii genotype could be determined for twelve samples using nine PCR-restriction fragment length polymorphism (PCR-RFLP) markers (newSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico). In addition to T. gondii clonal type II (Apico II) and type II (Apico I), type III and T. gondii genotypes showing non-canonical allele patterns were observed in foxes. This suggests that, while T. gondii type II prevails in foxes, other genotypes circulate in wildlife. The population structure of T. gondii in Germany may be more diverse than previously thought.     

Leptospira exposure in the human environment in France: A survey in feral rodents and in fresh water

This paper confirms the important role of rodents to be maintenance hosts of leptospires. Their role is related to renal carriage and shedding of leptospires into urine, thus contaminating fresh water. Serological and carriage of feral rodents trapped in France were determined by MAT and hap1PCR specific for pathogenic leptospires. In same areas, fresh water samples were analyzed by hap1PCR. The overall seroprevalence was 44% in 649 rodents and was similar regardless of the species. Seroprevalence for leptospirosis is about 20-53% according to species. hap1PCR (516 kidneys) showed that renal carriage was higher in brown rats (34.7%) and muskrats (15.8%) than in coypus (3.3%). hap1PCR demonstrates a significative difference (P-value > 10(-12)) for the renal carriage between the different species: muskrats and rats are more efficient maintenance hosts than coypu but all infect water. Moreover 5/38 water samples associated with human cases were hap1PCR positive and 1/113 in controlled waters.     

Prevalence of Rabies Virus in Foxes Trapped in the Canadian Arctic

Brains and salivary glands of 521 trapped arctic foxes (Alopex lagopus) submitted from four different settlement areas in the Northwest Territories were examined for rabies by the standard fluorescent antibody and mouse inoculation tests. Rabies antigen was present in 44 of 201 (21.9%) brains from foxes trapped in the Sachs Harbour area, but submissions from Cambridge Bay (127), Spence Bay (93) and Gjoa Haven (100) were negative. Virus was also present in salivary glands from 43 (97.7%) of these 44 positive foxes.

The arctic fox continues to be the main wildlife reservoir of rabies in the Canadian Arctic and foxes in the prodromal stage of the disease pose a particular threat to the trapper. Preexposure vaccination should always be a consideration in this occupational group.

     

Toxoplasma gondii and Neospora caninum in wildlife: common parasites in Belgian foxes and Cervidae?

Sera from Cervidae were tested for the presence of antibodies against Neospora caninum using ELISA; and against Toxoplasma gondii using SAG1-ELISA and a commercially available agglutination test. The T. gondii seroprevalence was 52% (38/73) in roe deer (Capreolus capreolus), 0% in bred fallow deer (0/4) (Dama dama) and red deer (0/7) (Cervus elaphus). We found 2.7% of the roe deer samples and none of the bred deer samples positive for N. caninum. Brain samples from wild roe deer, red deer and red foxes (Vulpes vulpes) were tested for the presence of T. gondii and N. caninum DNA using multiplex real-time PCR. We detected T. gondii in 18.8% (57/304) of the red foxes and in 1 of the 33 deer samples. N. caninum was found in 6.6% of the red foxes and in 2 roe deer samples. Twenty-six of the T. gondii positive DNA extracts from the red fox samples were genotyped. Twenty-five were type II and only one was found to be type III.     

Study on the prevalence of Toxoplasma gondii and Neospora caninum and molecular evidence of Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis infections in red foxes (Vulpes vulpes) in rural Ireland

Thoracic fluid (pleural fluid and clotted blood) from 206 foxes were examined for antibodies to Toxoplasma gondii and 220 thoracic fluid samples were tested for Neospora caninum antibodies using indirect immunofluorescent antibody tests (IFAT). A total of 115 (56%) and six (3%) foxes had antibodies to T. gondii and N. caninum, respectively. The brains from 148 foxes were examined for histological lesions and pathological changes suggestive of parasitic encephalitis were observed in 33 (22%). Two thirds of these foxes had antibodies to T. gondii and one fox had antibodies to both T. gondii and N. caninum. PCR assays carried out on DNA extracted from the 33 brains with histological lesions were negative for N. caninum but one of the brains was positive for T. gondii. Microsporidian DNA was also amplified from the brains of two of these foxes. Sequencing these amplicons revealed 100% homology with Encephalitozoon (Septata) intestinalis in one fox and Encephalitozoon cuniculi in the second fox. This is the first report of Encephalitozoon infections in wildlife in Ireland.     

Prevalence of antibodies against Neospora caninum and Toxoplasma gondii in dogs and foxes in Austria

Sera from 1770 dogs and 94 red foxes from Austria were examined for antibodies against Neospora caninum using the indirect immunofluorescent antibody test (IFAT). 3.6% of the dogs were seropositive with titres ranging from 1:50 to 1:6400. Dogs from rural areas were significantly more often seropositive for N. caninum than those from the urban area of Vienna (5.3% versus 2.1%). There were no significant differences in sex or breed, but a slight increase in seropositivity with age was apparent, indicating postnatal infection. None of the foxes had antibodies against N. caninum. Additionally, sera from 242 dogs and 94 foxes were examined for antibodies against Toxoplasma gondii using the IFAT. Thirty-five percent foxes and 26% of the dogs were positive; 1.7% of the dogs were positive for both parasites. This is the first report of the prevalence of N. caninum infections in dogs and foxes in Austria.     

Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum

The present study was undertaken to determine if red foxes are natural intermediate and/or definitive host for Neospora caninum and to study the importance of infection of N. caninum in this species in North-eastern Spain. Faecal samples and brain tissues were obtained from 122 foxes from 21 rural areas of Catalonia. Faeces collected were examined for parasite eggs and coccidian oocysts using sucrose flotation. For PCR-based diagnosis of N. caninum in brain tissues, the specific genomic Nc5 region was selected as the target sequence for DNA amplification. To control for PCR failure and facilitate identification of truly negative samples, the competitor pNc5C molecule was added to all negative samples in a second round of PCR reactions. Of the 122 foxes analysed, 13 (10.7%) were positive by PCR for N. caninum. Signal intensities of all positive samples were relatively weak with the exception of one sample from a 3-month male animal, that also showed the highest repeatability. No differences were observed by sex, age or area of sampling analysis. Detection of stages of N. caninum in brain from naturally infected red foxes demonstrated that red foxes are a natural intermediate host for N. caninum. Faecal samples were analysed for the presence of N. caninum oocysts, however, no oocysts compatible with N. caninum were found. A widespread latent infection of red foxes in North-eastern Spain found in the present study indicates that red foxes could have a very important role in the epidemiology of neosporosis in our area.     

Newly discovered viruses of flying foxes.

Flying foxes have been the focus of research into three newly described viruses from the order Mononegavirales, namely Hendra virus (HeV), Menangle virus and Australian Bat Lyssavirus (ABL). Early investigations indicate that flying foxes are the reservoir host for these viruses. In 1994, two outbreaks of a new zoonotic disease affecting horses and humans occurred in Queensland. The virus which was found to be responsible was called equine morbillivirus (EMV) and has since been renamed HeV. Investigation into the reservoir of HeV has produced evidence that antibodies capable of neutralising HeV have only been detected in flying foxes. Over 20% of flying foxes in eastern Australia have been identified as being seropositive. Additionally six species of flying foxes in Papua New Guinea have tested positive for antibodies to HeV. In 1996 a virus from the family Paramyxoviridae was isolated from the uterine fluid of a female flying fox. Sequencing of 10000 of the 18000 base pairs (bp) has shown that the sequence is identical to the HeV sequence. As part of investigations into HeV, a virus was isolated from a juvenile flying fox which presented with neurological signs in 1996. This virus was characterised as belonging to the family Rhabdoviridae, and was named ABL. Since then four flying fox species and one insectivorous species have tested positive for ABL. The third virus to be detected in flying foxes is Menangle virus, belonging to the family Paramyxoviridae. This virus was responsible for a zoonotic disease affecting pigs and humans in New South Wales in 1997. Antibodies capable of neutralising Menangle virus, were detected in flying foxes.