Clearance and persistence of Escherichia coli in the freshwater mussel Unio mancus.

The excessive use of antibiotics has led to the emergence of resistant bacteria, mainly from the Enterobacterales group, with high pathogenic/zoonotic potentials that can lead to problems in public health. The increasing presence in freshwater ecosystems highlights the need to evaluate potential sentinel species as risk indicators for both ecosystem and human health. The freshwater mussels provide several ecosystem services, may represent potential sentinel species due to their ability to filter water and retain both organic and inorganic particles. We tested the capability of U. mancus to retain Escherichia coli as a model bacterial organism. Under experimental conditions, the mussels could clear suspended E. coli, facilitating its rapid elimination from water within the first 24 h after exposure. The species also presented a maximum retention time of 4 days. We also provide allometric equations correlating the filtering capacity with the length and the weight of mussel body parts often used in biometric studies. We provide a first assessment of the potential of the bivalve Unio mancus to act as a sentinel species for the detection of Enterobacterales and demonstrate the ability to act as a water cleaner.

Ruminant pestiviruses in North Africa.

Ruminant pestiviruses are widely distributed worldwide, causing congenital disease and massive economic losses. Although ruminant production is an important economic sector in North Africa, the knowledge about pestiviruses is scarce. The present study aimed at assessing the presence of Pestivirus in cattle in Algeria, and to review the data available on ruminant pestiviruses in North Africa. A cross-sectional study was conducted on dairy farms from North-Western Algeria. Blood samples from 234 dairy cattle from 31 herds were collected. All sera were analysed for the presence of antibodies using a commercial iELISA. The presence of Pestivirus RNA was also assessed by using a Reverse Transcription-PCR, and PCR-positive samples were sequenced. Risk factors related to Pestivirus infection were also analysed. The review of the presence of ruminant pestiviruses in North Africa was performed using a systematic search and compilation methodology of the peer-reviewed literature available in order to identify gaps of knowledge for future research. The seroprevalence at population and farm levels obtained in the present study (59.9% and 93.5%, respectively) concur with data reported in neighbouring countries. Risk factors associated with Pestivirus infection in cattle were the presence of sheep in the herd and the animal category (cow vs heifer). Furthermore, we confirmed the presence of BVDV-1a in Algeria. The scarce data suggest an endemic epidemiological scenario of pestivirus in livestock. The lack of studies about the epidemiology and molecular variability of ruminant pestiviruses in livestock and wildlife in North Africa is of concern for animal health and wildlife conservation, and needs to be addressed.

Toxoplasma gondii in sympatric domestic and wild ungulates in the Mediterranean ecosystem.

Toxoplasma gondii is a zoonotic protozoan of worldwide distribution. The present study provides information on risk factors affecting T. gondii infection in domestic and free-ranging wild ungulates sharing habitats in Mediterranean ecosystems in Spain. Serum samples from 482 extensively reared domestic ruminants and 2351 wild ungulates were tested for T. gondii antibodies using the modified agglutination test (MAT, cut-off 1:25). Toxoplasma gondii seroprevalence was 41.2% of 194 sheep, 18.6% of 199 cattle and 5.6% of 89 goats. The main risk factors associated with infection in livestock were the presence of cats, feeding on the ground and at stubble fields. In wild ungulates, T. gondii antibodies were detected in 10.5% of 1063 red deer, 15.6% of 294 fallow deer, 5.6% of 216 European mouflon, 5.6% of 90 Spanish ibex, 13.6% of 22 roe deer and 18.6% of 666 wild boars. The risk factors affecting T. gondii infection in wildlife were species, age and hunting season. Significantly higher seroprevalence was found in domestic ruminants, particularly in sheep, compared to the wild species tested. The present study indicates widespread exposure to T. gondii among domestic and wild ungulates in Southern Spain, with significant differences among species sharing the same ecosystem. The high seroprevalence observed in domestic ruminants, particularly in sheep, reinforces the need for farm management practices to control the risk factors associated with T. gondii infection in extensively reared livestock. Consumption of raw and undercooked food products from domestic and wildlife species may have important implications for public health.

First report of the novel atypical porcine pestivirus in Spain and a retrospective study.

This study aimed to provide information regarding viral pathogenesis and molecular epidemiology linked with recently reported atypical porcine pestivirus (APPV) strains and to determine the circulation of APPV in Spain from 1997 to 2016. Two-day-old piglets with moderate-severe congenital tremor (CT) from a Spanish farm were received for diagnostic purposes. Sera, nasal and rectal swabs and tissue samples were collected. qRT-PCR was performed in these samples, and a retrospective study to detect APPV RNA was carried out using a serum collection from 1997 to 2016. APPV genome was identified with high and moderate RNA loads in different tissues of the CT affected pigs. High APPV RNA load was detected in lymphoid organs, suggesting that these constitute a target for APPV replication. In 89 of the 642 retrospectively analysed samples (13.9%), APPV genome was detected. CT cases were related to the presence of APPV in viraemic piglets below 1 week of age, in which the viral RNA load was the highest. A considerable number of animals between 4 and 14 weeks of age and some 1-week-old piglets were viraemic in the absence of CT, which can act as carriers of the virus. The relative risk of APPV and CT was 8.5 (CI 95% 5.8-12.5). Thus, our data show that APPV infection is epidemiologically related to CT. Phylogenetic analysis from 1615 NS2-3 nucleotides showed only one defined APPV clade, grouping the most phylogenetically related strains from Europe and China. Of this clade, there are other strains from Europe, USA and China. This data confirm the high APPV genetic diversity, not being able to cluster this virus according to the geographic area. Our result showed that APPV has been circulating in Spain at least since 1997, being the earliest date of detection of this virus worldwide and suggesting that APPV may be widespread.

Post-Natal Persistent Infection With Classical Swine Fever Virus in Wild Boar: A Strategy for Viral Maintenance?

In this study, fifteen wild boar piglets were intranasally inoculated <10 h after birth with the moderately virulent classical swine fever virus (CSFV) strain Catalonia 01. At 5 days post-inoculation, seven other animals within 48 h of birth were put in contact with them. Viral replication and innate and specific immune responses were evaluated. Of the inoculated animals, 46.67% remained post-natally persistently infected and were apparently healthy with neither humoral nor cellular immunological responses specific to CSFV and with high viral loads in their blood, organs and body secretions. Moreover, the present data extend the time period to 48 h after birth when a moderately virulent CSFV strain could lead to post-natal persistent infection given the generation of persistently infected wild boars in the contact group (33.33%). The innate immune response to the virus, as measured by type I IFN-α in serum, was mostly not impaired in the persistently infected wild boars. Interestingly, a decrease and lack of IFN-γ-producing cells against CSFV and PHA was observed. In endemic countries where wild swine species are increasing and low and moderate virulence CSFV strains are prevalent, the possible generation of this form of disease cannot be ruled out.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain.

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Influenza A Virus Surveillance in the Invasive American Mink (Neovison vison) from Freshwater Ecosystems, Northern Spain.

Influenza A viruses (IAVs) are negative-sense, single-stranded and segmented RNA viruses of the Orthomyxoviridae family that may cause acute respiratory disease in a wide range of birds and mammals. Susceptibility of several species within the family Mustelidae to IAVs has been reported as a result of natural or experimental infections. The objectives of this study were to assess whether free-ranging American mink populations from Northern Spain were infected with IAV and try to define the role of this species in the epidemiology of IAV. Sera from 689 American mink from Northern Spain captured between 2011 and 2014 were tested for the presence of antibodies against IAVs using a commercial competition cELISA. Positive sera were further analysed with haemagglutination inhibition (HI) assay. Fifteen of the 689 (2.2%, 1.3-3.6 CI95% ) of the American minks analysed were ELISA positive. No significant differences were observed between years of capture, provinces, river basins, sexes or ages of the animals. All seropositive sera resulted negative to the panel strains used in the HI assay, showing that the most relevant strains circulating in swine, the most relevant avian subtypes (H5 and H7) and the H10N4 subtype isolated in minks have not been circulating in this free-ranging exotic carnivore from Spain. In the light of these results, the free-range American mink from Northern Spain do not seem to have an important role in the epidemiology of IAVs.

Cytokine gene expression in aborting and non-aborting dams and in their foetuses after experimental infection with Neospora caninum at 110 days of gestation.

Neospora caninum is a major cause of abortion in cattle. However, it is not known why not all infected animals abort. In this study, Th1 (IFN-γ), Th2 (IL4) and T reg (IL-10) cytokine gene expression was examined by real time PCR using the TaqMan approach in all of these dams and their foetuses after experimental infection with the isolate Nc-Spain7 at 110 days of pregnancy and euthanasia 6 weeks after infection. In prior published work, foetal death was observed in three of six infected dams and transplacental infection in all the 6 infected foetuses. In the spleen of the dams, IL-4 expression was down-regulated in dams with aborted/non viable foetuses compared to both uninfected dams (controls, n=3) and infected dams with live fetuses at euthanasia. In the lymph nodes draining the placenta, up-regulated expression of IL-4 was observed in infected dams with live foetuses compared to control dams. In the placenta, infected dams with live foetuses had significantly up-regulated IFN-γ in both caruncle and cotyledon and up-regulated IL-10 in cotyledon compared to control dams. Infected live foetuses showed up-regulated expression of IFN-γ and IL-10 in foetal spleen, and showed downregulated expression of IL-4 in the thymus compared to control uninfected foetuses. Expression of any cytokine in the thymus was significantly lower compared to the levels observed in foetal spleen. The results indicate an up-regulated expression of Th1, Th2 and Treg in infected dams with live foetuses and in their foetuses. On the other hand, down-regulation of Th2 immune responses and Treg cytokines were observed in infected dams which had aborted or had non-viable foetuses at euthanasia, suggesting an immunological recovery of cytokine gene expression levels in dams a few weeks after an abortion occurred.

The European hare (Lepus europaeus) as a potential wild reservoir for ruminant pestiviruses.

Ruminant pestiviruses cause important economic losses in livestock and the epidemiological role of free-ranging sympatric wildlife is of special interest for the implementation of pestivirus eradication plans. Moreover, the emergence of high mortality outbreaks of pestivirus in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) since 2001 in the border between Spain and France has increased the value of knowing the hosts that role pestivirus infection. In the present study, pestivirus infection was assessed in 94 sera from wild hunted European hares (Lepus europaeus) collected in two different areas: Pyrenees (alpine and subalpine ecosystems) versus Non Pyrenees (non alpine and subalpine ecosystems). The presence of antibodies against Border Disease Virus (BDV) and Bovine Viral Diarrhea Virus (BVDV) was evaluated by means of the Virus Neutralization Test and the presence of viral RNA in sera samples was assessed by Reverse transcription-polymerase chain reaction (RT-PCR). A total of 34 out of 94 (36.2%; CI95 0.26-0.46) sera presented neutralizing antibodies against ruminant pestiviruses, and significant differences between BDV4 and BVDV1 titres were found in 7 hares. In the Pyrenean area not statistically significant seroprevalence was observed when comparing with the Non Pyrenean area. RT-PCR analysis of sera samples resulted all negative. The results of the present study indicate that the European hare is susceptible to pestivirus infection and that could be involved in the epidemiology of ruminant pestiviruses. To the authors' knowledge, this is the third wild non-artiodactyl with reported antibodies against ruminant pestivirus after the rabbit and Bennet's wallaby.

Maternal and foetal cytokine production in dams naturally and experimentally infected with Neospora caninum on gestation day 110.

In the present study, IFN-γ (Th1), IL-17A (Th17) and IL-4 (Th2) concentrations in response to concanavalin (ConA) and Neospora caninum antigen (Nc-1) stimulation were determined in cultures of cells from control uninfected (n=4), naturally N. caninum-infected (n=3) and experimentally N. caninum-infected (n=6) pregnant dams and their foetuses. Experimental animals were infected at 110days of gestation and euthanized 6weeks post-infection. In culture supernatants from the dams, significantly higher IFN-γ and IL-4 levels were found in the experimentally-infected animals compared to the control or naturally-infected dams. However, among the experimentally-infected dams no significant differences in IFN-γ production were observed regardless of the incidence of live or aborted/dead foetuses, though spleen cultures of dams carrying live foetuses showed the highest levels of IFN-γ. IL-17A production was very low and occasional in the dams infected with N. caninum and did not seem to be a major regulator of IFN-γ production in this model. Experimentally infected dams with live foetuses showed higher IL-4 levels and accordingly IFN-γ/IL-4 ratios were significantly lower than ratios recorded for cows with aborted/dead foetuses. In the infected foetuses of these dams, only spleen cultures showed high levels of IFN-γ and IL-4 after Nc-1 antigen and ConA stimulation, respectively. No IL-17A was detected in the foetuses. As conclusion, although we could not clearly relate a protective immune response against N. caninum abortion only to IFN-γ levels in cell cultures, our results highlight the important role of an inverse IFN-γ/IL-4 balance in conferring protection against abortion induced by this parasite.

Pestivirus in alpine wild ruminants and sympatric livestock from the Cantabrian Mountains, Spain.

Bovine viral diarrhoea virus (BVDV) and Border disease virus (BDV) were investigated at the wildlife-livestock interface in the distribution area of chamois in the Cantabrian Mountains, North-Western Spain. From 2010 to 2014, sera from sympatric wild (n=167) and domestic (n=272) ruminants were analysed for pestivirus antibodies by cELISA, virus neutralisation test (VNT) and for the presence of pestiviral RNA using a reverse transcription (RT)-PCR. Results showed a higher seroprevalence in cattle (59.4 per cent, 13/13 of herds) than in domestic small ruminants (5.9 per cent sheep, 2/8 of flocks; 0 per cent goats of 4 flocks) and wildlife (10.8 per cent in red deer, 0 per cent in roe deer and 0 per cent in Cantabrian chamois). High VNT titres were detected in two cattle herds, suggesting the circulation of BVDV-1 and BVDV-2 strains. BVDV-1 RNA was detected in one cattle calf by RT-PCR and sequencing. Conversely to other similar grazing systems, sheep flocks did not play a relevant role in the pestivirus epidemiology in this region. Pestivirus infections in wild ruminants were sporadic and most probably dependent on a domestic source.

Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy-Associated Glycoproteins 1 and 2 in Foetal Fluids.

Plasma concentrations of PAG-1 are used for pregnancy diagnosis and as a marker of placental/foetal well-being, while those of PAG-2 may be an indicator of abortion risk in Neospora caninum-infected cows. Studies have shown that N. caninum infection modifies PAG-1 and PAG-2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG-1, PAG-2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG-2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non-infected foetuses. In infected foetuses, significantly higher concentrations of PAG-1 (p < 0.001) and PAG-2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non-infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG-1 and PAG-2 levels and pH values than fluids from non-infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH.

Absence of circulation of Pestivirus between wild and domestic ruminants in southern Spain.

Ruminant pestiviruses (family Flaviviridae) affect both wild and domestic ruminants worldwide, causing reproductive disorders and severe economic losses. Wild (n=1442) and domestic (n=373) ruminants from southern Spain were tested for the presence of antibodies to pestiviruses. Seropositivity was detected by both ELISA and virus neutralisation test in 1/892 (0.1 per cent) red deer, 29/125 (23.2 per cent) cattle and 17/157 (10.8 per cent) sheep. Pestivirus-specific antibodies to bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) genotypes 1, 4 and 5 were detected. Significantly higher antibody titres to BDV-4 compared with BVDV-NADL were found in one cattle farm. Results indicate that wild ruminants may act as spillover hosts from domestic ruminants, and therefore they do not represent a risk for domestic ruminants in southern Spain. The wide distribution of pestiviruses among sheep and cattle suggests that transmission between these species may occur.

Antigenic and molecular characterisation of Border disease virus associated with high mortality in lambs in Spain.

INTRODUCTION: Border disease virus (BDV) causes congenital disorders in sheep and results in severe, but underestimated, economic losses worldwide. However, information about BDV strains affecting several ruminants worldwide is scarce. Therefore, antigenic and genetic classification of isolates from different geographical regions is important to enhance the knowledge of the epidemiology of BDV. MATERIALS AND METHODS: Five pestiviruses isolated from lambs in an epidemic outbreak with an unusually high mortality in Spain in 1997 were characterised antigenically with a panel of monoclonal antibodies and genetically by sequencing within the 50 untranslated (50UTR) region of the genome. RESULTS: All the isolates were classified as BDV and showed a high homology with the Aveyron strain (Av), which was associated with an epidemic reported in sheep from the Aveyron region of France in 1984. CONCLUSIONS: Classification of the isolates from this study provides valuable information on the molecular epidemiology of BDV.

Epidemiology and prevalence of Toxoplasma gondii infection in the Iberian hare (Lepus granatensis).

Hares are important game species in Europe and their meat is consumed by humans. Toxoplasmosis is an important cause of mortality in hares in Nordic countries but little is known of Toxoplasma gondii infection in the Iberian hare (Lepus granatensis). Sera from 298 Iberian hares from Navarra (North Spain) were assayed for antibodies against T. gondii by the modified agglutination test. The samples were collected at necropsy during three consecutive years (2009-2011). Titers of 1:25 and higher were found in 34 hares (11.4%; CI95%: 7.8-14.9). Significant differences in seroprevalence were observed among geographical areas, years of sampling and age groups. The highest seroprevalence and the highest titers were observed in 2009, indicating fluctuating rates of transmission. Significantly higher seroprevalence was observed in the juvenile age group compared to the adult hare group. More than 40% of juvenile hares were seropositive the first year of study, seroprevalence drastically decreased in the juvenile animals sampled in the second year and was negligible in those sampled in the third year in this group. No clinical cases were detected in the study area and T. gondii was not detected by PCR in the liver of the 34 seropositive hares tested. These data may indicate that most juvenile seropositive hares did not survive as adults in the consecutive year or that there is a short-term humoral immune response against T. gondii in Iberian hares.

Fatal toxoplasmosis associated with an atypical Toxoplasma gondii strain in a Bennett's wallaby (Macropus rufogriseus) in Spain.

Toxoplasmosis is often fatal in captive wallabies, but the causes of this high susceptibility are not well understood. Here, we report fatal toxoplasmosis in a Bennet's wallaby (Macropus rufogriseus) due to an atypical Toxoplasma gondii strain for the first time in Europe. The wallaby was from a colony of 7 Bennet's wallabies that died over a 17-month period at a safari-zoological park in northeastern Spain. Only one of these wallabies was examined at necropsy. T. gondii-like organisms were detected by histological examination in several tissues and the diagnosis was confirmed through detection of T. gondii DNA by PCR. A nested PCR-based assay detected the 200- to 300-fold repetitive 529 bp DNA fragment of T. gondii in a sample of brain tissue. Genotyping analysis with 15 single-copy microsatellite markers was performed on this positive DNA sample and revealed an atypical genotype. Atypical genotypes are frequently associated with severe forms of toxoplasmosis in humans. The present report highlights the possible implications of the introduction of new atypical, more pathogenic T. gondii strains, to non-endemic areas.

Toxoplasma gondii in ruminant species (cattle, sheep, and goats) from southern Spain.

Seroprevalences of Toxoplasma gondii antibodies were assessed with the use of a commercial indirect ELISA in 1,501 domestic ruminants in southern Spain. Antibodies against T. gondii were detected in 420 (83.3%) of 504 cattle, 248 (49.3%) of 503 sheep, and 124 (25.1%) of 494 goats. The herd seroprevalence was 100% (72/72), 84.7% (61/72), and 72.2% (52/72) for cattle, sheep, and goats, respectively. Seropositivity was significantly higher in herds with a low density of animals (P < 0.001). Significant differences (P < 0.05) among municipalities were also found. The seroprevalence observed in the present study indicates a widespread exposure to T. gondii in livestock in southern Spain.

Seroprevalence of Toxoplasma gondii in equids from Southern Spain.

Antibodies to Toxoplasma gondii were determined in serum samples from 616 equids (454 horses, 80 mules and 82 donkeys) in a cross-sectional study of 420 herds in Andalusia (Southern Spain), the region with the highest number of equids in Spain. Antibodies to T. gondii were found in 10.8% horses, 15.0% mules and 25.6% donkeys by using the modified agglutination test (MAT) at a cut-off of 1:25. Herd seroprevalence for horses, mules and donkeys was 14.7% (48/327), 23.9% (11/46) and 34.0% (16/47), respectively, and 75 herds (17.8%) had at least one seropositive animal. Significant differences in T. gondii seroprevalence were observed among species, with donkeys having the highest seroprevalence and horses the lowest (P=0.04). Seroprevalence was significantly higher in herds with presence of domestic ruminants. This study is the first report of the presence of T. gondii antibodies in equine species in Spain and the first reporting T. gondii infection in donkeys in Europe. The presence of antibodies is indication of contact with the parasite and therefore, consumption of equine meat could be a potential source of human infection in Spain.

Presence of Toxoplasma gondii and Neospora caninum DNA in the brain of wild birds.

Toxoplasma gondii infections are prevalent in many avian species and can cause mortality in some bird hosts. Although T. gondii has been isolated from various species of birds, the role of many different species of wild birds in the epidemiology of T. gondii remains unknown. Neospora caninum, a closely related parasite to T. gondii, has been recently confirmed to infect domestic chickens and wild birds such as house sparrows (Passer domesticus). The present study reports the presence of T. gondii and N. caninum DNA by PCR in brain tissues of 14 species of wild birds from Spain. From a total of 200 samples analyzed, 12 samples (6%) were positive for T. gondii [5 Eurasian jays (Garrulus glandarius), 5 magpies (Pica pica), 1 black kite (Milvus migrans) and 1 Griffon vulture (Gyps fulvus)], while 3 samples (1.5%) were positive for N. caninum [2 magpies and 1 common buzzard (Buteo buteo)]. This is the first report of detection of T. gondii in magpies, griffon vulture and black kite and of N. caninum in common buzzard and magpies, extending the list of natural intermediate hosts for T. gondii and N. caninum infections to these species.