Increased specificity of Fasciola hepatica excretory-secretory antigens combining negative selection on hydroxyapatite and salt precipitation.

A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.

Bayesian hierarchical modelling of the geospatial distribution of fasciolosis in dairy cattle and the impact on production: Application to the main milk-producing region (Galicia) in Spain.

Fasciolosis caused by Fasciola hepatica is a common parasitic infection among cattle in many countries. Although infected adult cows rarely show overt clinical signs, milk production may be impaired. Thus, significant production losses may occur in dairy herds with a high prevalence of fasciolosis. In this study, Bayesian hierarchical modelling was used to estimate the geospatial distribution of dairy cattle fasciolosis and its impact on milk production. The study was conducted in Galicia, the main milk producing region in Spain and a geographically heterogeneous area. The aims were: 1) to model the geospatial distribution of fasciolosis in dairy herds in the study area, 2) to identify clusters of herds with a high prevalence of fasciolosis, and 3) to assess the effect of fasciolosis on milk yield and quality. A large number of dairy cattle farms (n = 4907), of which 1660 provided production records, were surveyed. Fasciola infection status was determined by applying the MM3-SERO ELISA test to bulk tank milk samples. A high probability of infection was predicted in several zones, particularly in the centre, northeast and southeast of Galicia. Conversely, the predicted probability was very low in some parts of the northwest of the region. Infections with high within-herd prevalence (> 25% lactating cows infected) predominated. High within-herd prevalence was associated with loss of milk production (-1.387 kg/cow/ day, on average). No association between Fasciola infection and either milk fat or protein content was observed. This study has generated the first maps of the spatial distribution of the probability of Fasciola infection in dairy cattle herds in Galicia. The maps presented here can be used for reference purposes, enabling the design of better targeted fasciolosis control programmes in the region. Use of Bayesian hierarchical statistical analysis enabled us to ascertain the uncertainty of the predictions and to account for the spatial autocorrelation in the data. It also enabled us to generate maps showing the residual spatial variation in milk production, a topic that may deserve more detailed study.

Field evaluation of the enhanced MM3-COPRO ELISA test for the diagnosis of Fasciola hepatica infection in sheep.

Fasciolosis is a severe zoonosis responsible for major economic losses in livestock. The enhanced MM3-COPRO test (eMM3-COPRO) and the commercial version BIO K 201 (Bio-X Diagnostics, Rochefort, Belgium) are widely used as immunodiagnostic tools for the specific detection of coproantigens released by Fasciola during the late prepatent and patent stages of infection. However, performance of the eMM3-COPRO has never been evaluated under field conditions. To address this gap, a large number of ovine faecal samples, collected in a region where fasciolosis is endemic (Galicia, NW Spain), were analyzed. Two groups of sheep flocks were selected according to the Fasciola infection status: 'Fasciola-free' and 'Fasciola-infected' flocks. 'Fasciola-free' flocks were seronegative flocks with no history of fasciolosis detected by either coproscopy or necropsy in the last 5 years. Faecal samples from these sheep were used to calculate a cut-off value for infection (OD = 0.021). The cut-off was calculated using a bootstrap resampling method that enables estimation of the sampling distribution of the statistical parameters without making assumptions about the underlying data distribution. 'Fasciola-infected' flocks were characterized by high seroprevalence, a history of fasciolosis and periodical treatment with flukicides. Samples from these flocks were used to estimate the diagnostic accuracy of the eMM3-COPRO relative to coproscopy, which although limited by poor sensitivity is the only reference test available for diagnosing fasciolosis in vivo. To overcome this limitation, all animals classified positive by eMM3-COPRO were treated with triclabendazole and then retested. The eMM3-COPRO displayed higher sensitivity than coproscopy, as it detected coproantigens in all samples with positive coproscopy and in 12% of samples with negative coproscopy. The test also proved highly specific as coproantigens disappeared after the treatment. The eMM3-COPRO was less time consuming than coproscopy, particularly when the procedure involved numerous samples, and showed promise as a tool for monitoring flukicide efficacy.

Current Challenges for Fasciolicide Treatment in Ruminant Livestock.

Pharmacological treatment remains essential to control fasciolosis in areas where infection is endemic. However, there are major constraints to treating food-producing animals. Of particular concern is the lack of flukicides for treating early Fasciola infections in ruminant livestock in some countries. In addition, the information provided in package leaflets, particularly regarding withdrawal periods, is often incomplete, confusing, and/or contradictory. International regulatory bodies should harmonize the use of flukicides in livestock in favor of fairer, safer international trade. In addition, monitoring the efficacy of fasciolicides on farms is also essential to minimize the spread of drug-resistant populations of Fasciola. The current situation regarding flukicide formulations in the European Union and other, non-European countries is analyzed in this review paper.

Maternal immune response in the placenta of sheep during recrudescence of natural congenital infection of Neospora caninum.

In order to gain further insight into the pathogenesis and transmission of ovine neosporosis, the serological response of 13 naturally infected pregnant sheep was monitored. All sheep were euthanized upon the detection of a sharp increase in the level of specific antibodies against N. caninum in order to study the maternal immune response after the recrudescence of a chronic infection. Ten sheep were euthanized between 84 and 118 days of gestation, whereas the three remaining and three control not infected, pregnant sheep were euthanized at 135 days of gestation after no sharp increase in antibodies was detected. Vertical transmission was confirmed in 11 sheep by detection of N. caninum-DNA in at least one fetus, confirming recrudescence. Not all of fetuses showed pathologic microscopic lesions, however, multifocal non-purulent encephalitis was the main finding. Furthermore, nine out of the 11 vertical transmission positive sheep had lesions in placentomes (mainly multifocal necrotic foci), and the parasite was detected in eight out of 11 placentas by PCR and/or immunohistochemestry. The placentomes from sheep that suffered recrudescence showed an increased number of T lymphocytes CD3+ (CD4/CD8 < 1) and macrophages (MHC-II+), assessed by immunohistochemestry, together with an up-regulation of IFN-γ, IL-10, IL-4, TNFα, IL-2 and IL-18. IL-17 was only upregulated in the three infected sheep that did not have a sharp increase in antibody levels. In the sheep that showed fetal death at the time of euthanasia (n = 3) the placental microscopic lesions were more severe, the inflammatory infiltrate was higher, and the upregulation of cytokines was greater than in those sheep carrying viable fetuses. This study suggests that, similarly to bovine neosporosis, the time of gestation when recrudescence occurs determines the viability of the fetuses and, thus, seems to be related to the severity of lesions and immune response in the placenta. These results suggest that there might be a correlation, either as cause or as a consequence, between protection against vertical transmission of the parasite and a milder maternal serological response together with a high level of transcription of IL-17 in the placenta.

Endogenous transplacental transmission of Neospora caninum during successive pregnancies across three generations of naturally infected sheep.

Endogenous transplacental transmission, which occurs during pregnancy as the result of reactivation of a latent infection in the dam, is the main mechanism of propagation of Neospora caninum within cattle herds. However, the importance of this propagation mechanism has not yet been evaluated in relation to ovine neosporosis. In this study, involving three generations of ewes naturally infected by N. caninum, we demonstrated that endogenous transplacental transmission may also be highly efficient in the ovine host since transmission of infection occurred in 96.6% of gestations and the congenital infection rate ranged between 66.7 and 93%. Nevertheless, parasite burdens decreased gradually in consecutive generations. Reactivation of latent infections had a strong impact on the pregnancy outcome, with high mortality rates recorded in the offspring of the two first generations of ewes (21.4-46.1%). Histological examination of the brain revealed that all aborted foetuses had characteristic lesions of neosporosis (necrotic glial foci) and a few parasite cysts, whereas most stillborn and newborn lambs that died shortly after birth had non-specific lesions (mild glial foci without necrosis) and parasite cysts were more frequent. Microsatellite analysis revealed scarce genetic variability in the N. caninum population, in accordance with a scenario in which infections were of a single origin and were exclusively maintained by clonal propagation through endogenous transplacental transmission.

Vaccination of sheep with Quil-A® adjuvant expands the antibody repertoire to the Fasciola MF6p/FhHDM-1 antigen and administered together impair the growth and antigen release of flukes.

Fasciolosis continues to be a major cause of economic losses in the livestock industry and a growing threat to humans. The limited spectrum of effective anthelmintics and the appearance of resistances urge the need for developing an effective vaccine. Most studies have been focused on the use of TH1-polarizing adjuvants and the use of recombinant Fasciola critical molecules and, despite the efforts, no reproducible protections have been achieved. The F. hepatica MF6p/FhHDM-1 protein is a heme-binding protein also reported to have immunomodulatory properties, constituting a promising target for vaccination and/or as target for the development of new flukicides. Thus, in this study, we investigated the effects of the TH1-polarizing adjuvant Quil A® on sheep immune response to MF6p/FhHDM-1, and the vaccine potential of both native and synthetic forms of this protein against ovine fasciolosis. Subcutaneous injection of Quil A® alone, i.e., without co-injecting any antigen, expands the antibody repertoire to MF6p/FhHDM-1 triggered by a subsequent primoinfection with metacercariae. This effect was not observed with aluminum hydroxide, the most frequently adjuvant used in commercial vaccines. On the other hand, vaccination with synthetic MF6p/FhHDM-1 in Quil A® prompted a 2-4-week delay in the antibody response induced in sheep by a challenge experimental infection. Moreover, fluke populations stablished showed stunted growth and low antigen release probably due to reduced metabolic activity. These observations suggest that primary circulating antibodies induced by the immunization had harmful effects on fluke development. Such effects could not be demonstrated to be associated to TH1 immune response linked events (production of IgG2 isotype antibodies and IFN-γ).

Transmission of Calicophoron daubneyi and Fasciola hepatica in Galicia (Spain): Temporal follow-up in the intermediate and definitive hosts.

BACKGROUND: Paramphistomosis caused by Calicophoron daubneyi and fasciolosis caused by Fasciola hepatica are common parasitic diseases of livestock animals. Transmission of the diseases depends on the presence of intermediate hosts, i.e. freshwater gastropods such as lymnaeids. We carried out a 2-year-long study of the dynamics of the snail population acting as the intermediate host for these parasites, considering the population structure in terms of size/age and infection status. In addition, we determined the kinetics of trematode egg excretion in grazing cows. Generalized Additive Models (GAMs) were used to analyze the associations between different response variables and snail size, sampling month and weather-related variables. RESULTS: Of the molluscan species examined, Galba truncatula, Radix peregra, Anisus (Anisus) leucostoma and Pisidium casertanum (n = 2802), only G. truncatula was infected with C. daubneyi or F. hepatica, at prevalence rates of 8.2% and 4.4% respectively. The probability of infection with C. daubneyi or F. hepatica was linearly related to snail size, although in different ways (negative for C. daubneyi and positive for F. hepatica). The total snail population increased in winter, when specimens of all size classes were found. Infected snails were more abundant during spring-autumn. Mature cercariae of both parasites were found in most seasons. In the statistical models, the sampling month accounted for a high percentage (71.9-78.2%) of the observed variability in snail abundance. The inclusion of climatic variables in the models moderately increased the percentage of deviance explained (77.7-91.9%). Excretion of C. daubneyi eggs in cow faeces was always higher than that of F. hepatica eggs. CONCLUSIONS: Particular care should be taken to prevent pastures and the surrounding environment being contaminated with parasite eggs during winter-spring, when the number of snails susceptible to miracidial infections is maximal. This is therefore the optimal time for treating grazing animals. Nevertheless, control of trematodosis based only on chemotherapy is difficult in an area such as the study area, where environmental factors favour the regular appearance of snail populations harbouring mature cercariae.

Cryptosporidium spp. and Giardia duodenalis as pathogenic contaminants of water in Galicia, Spain: the need for safe drinking water.

The objectives of this cross-sectional study were to detect the presence of Cryptosporidium spp. and Giardia duodenalis in drinking water treatments plants (DWTPs) in Galicia (NW Spain) and to identify which species and genotype of these pathogenic protozoans are present in the water. Samples of untreated water (surface or ground water sources) and of treated drinking water (in total, 254 samples) were collected from 127 DWTPs and analysed by an immunofluorescence antibody test (IFAT) and by PCR. Considering the untreated water samples, Cryptosporidium spp. were detected in 69 samples (54.3%) by IFAT, and DNA of this parasite was detected in 57 samples (44.8%) by PCR, whereas G. duodenalis was detected in 76 samples (59.8%) by IFAT and in 56 samples (44.0%) by PCR. Considering the treated drinking water samples, Cryptosporidium spp. was detected in 52 samples (40.9%) by IFAT, and the parasite DNA was detected in 51 samples (40.1%) by PCR, whereas G. duodenalis was detected in 58 samples (45.6%) by IFAT and in 43 samples (33.8%) by PCR. The percentage viability of the (oo)cysts ranged between 90.0% and 95.0% in all samples analysed. Cryptosporidium andersoni, C. hominis, C. parvum and assemblages A-I, A-II, E of G. duodenalis were identified. The results indicate that Cryptosporidium spp. and G. duodenalis are widespread in the environment and that DWTPs are largely ineffective in reducing/inactivating these pathogens in drinking water destined for human and animal consumption in Galicia. In conclusion, the findings suggest the need for better monitoring of water quality and identification of sources of contamination.

Neospora caninum infection as a cause of reproductive failure in a sheep flock.

Neospora caninum has been detected only sporadically in cases of ovine abortion, and it has therefore traditionally been considered as an unimportant parasite in small ruminants. This study was carried out with the aim of identifying the pathogen causing serious reproductive problems on a commercial sheep farm. Sera from all rams and ewes tested negative for antibodies against Border disease virus, Schmallenberg virus and Coxiella burnetii, and infections by these agents were therefore ruled out. Nevertheless, seropositivity to N. caninum and/or Toxoplasma gondii was detected, although the seroprevalence was higher in the case of N. caninum. The percentage of lambings and the number of lambs per dam were significantly lower in ewes that were seropositive to N. caninum while no effect on these parameters was detected in ewes that were seropositive to T. gondii. There was also no evidence of infection by T. gondii in the foetal/lamb tissues analyzed by PCR and/or immunohistopathological techniques. On the contrary, the DNA of N. caninum was detected in 13 out of 14 foetuses/lambs descendant from dams seropositive to this parasite. Characteristic lesions caused by N. caninum and/or its antigen were also detected. Genotyping of the N. caninum DNA revealed only two closely related microsatellite multilocus genotypes. The results clearly demonstrate that infection by N. caninum was the cause of the low reproductive performance of this sheep flock.

Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics, clinical outcome and peripheral and local immune responses.

This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.

The wild boar (Sus scrofa Linnaeus, 1758) as secondary reservoir of Fasciola hepatica in Galicia (NW Spain).

Fasciolosis is an emerging or reemerging human and animal disease in numerous parts of the world. In Galicia (NW, Spain), the wild boar (Sus scrofa) is the main wild ungulate in terms of abundance and distribution. Its population has continuously increased over the past decades and this population growth has been accompanied by a reduction of habitats, so that the wild boar populations encroach more and more frequently onto agricultural lands. The increase of the interface area between livestock and the wild boars frequently involves the sharing of pastures and water sources, so that the circulation of common pathogens is propitiated. This is the first report concerning the importance of the wild boar as a possible reservoir of Fasciola hepatica infection in Spain. Livers from 358 hunted wild boars were analyzed showing that 11.2% were parasitized by F. hepatica, with burdens ranging from 1 to 14 flukes (mean=2.3). Fecal analysis demonstrated that 40.0% of parasitized animals shed F. hepatica eggs with a mean excretion of 6.1 eggs per gram of feces (epg). The presence of coproantigens analyzed by MM3-COPRO ELISA was positive in 62.9% of infected wild boars. After incubation, the percentage of hatched eggs ranged between 41.0% and 90.0% suggesting that the wild boar is very likely to contribute to the environmental contamination with viable parasite eggs. Comparative morphometric data were obtained using a computer image analysis system (CIAS) on the basis of standardized measurements. F. hepatica from cattle, sheep and wild boars from the same geographical area presents a similar body development and gravidity. Our study shows for the first time that the F. hepatica uterus from the wild boar presents an intermediate size between that found in primary reservoir hosts such as cattle and sheep, i.e., the individual potential egg output capacity of the wild boar does not greatly differ from that detected in Galician livestock. These results show that F. hepatica in Galicia has a normal development in wild boars, presenting its own characteristics in shape and size in comparison with other host species. The high prevalence of infection detected in the wild boar, the normal fluke development in the liver, and the possibility of shedding F. hepatica eggs capable of embryonating and giving rise to viable miracidia with the potential to infect intermediate hosts suggest a possible role of this species as a secondary reservoir in this Spanish region.

The first report of Cryptosporidium bovis, C. ryanae and Giardia duodenalis sub-assemblage A-II in roe deer (Capreolus capreolus) in Spain.

The aim of the present study was to identify the species and/or genotypes of Cryptosporidium and Giardia duodenalis infecting roe deer (Capreolus capreolus) in Galicia (NW Spain). The presence of both enteropathogens was investigated in 212 faecal samples from roe deer shot in diverse game preserves in three different areas of Galicia. The samples were analyzed by immunofluorescence microscopy and PCR amplification, and fragments of the 18S SSU rRNA gene of Cryptosporidium and the ß-giardin gene of G. duodenalis were sequenced. In total, 9 samples (4.2%) were positive for Cryptosporidium and 19 samples (8.9%) for G. duodenalis. These samples tested positive with both techniques. However, gene sequencing was only possible for Cryptosporidium in 6 of the samples and for G. duodenalis in 7 of the samples. Cryptosporidium bovis was identified in 3 samples and C. ryanae oocysts were detected in another 3 samples. Sequencing of the amplicons identified G. duodenalis sub-assemblage A-II in 7 samples. Both Cryptosporidium and G. duodenalis infections were more prevalent in juvenile than in adult animals, although the differences were not significant. G. duodenalis was more prevalent than Cryptosporidium in both age groups, although again the differences were not statistically significant. The mean intensity of infection by Cryptosporidium and G. duodenalis was similar in both age groups and ranged between 5 and 225 oocysts/g and 5 and 320 cysts/g of faeces, respectively. This study represents the first molecular characterization of these parasites in Spanish roe deer. Identification of C. bovis and G. duodenalis sub-assemblage A-II indicates that zoonotic transmission of these enteropathogens between roe deer and humans is possible and that cross transmission of some Cryptosporidium species and G. duodenalis (sub-assemblage A-II) may occur between related animal species sharing the same habitats.

Presence of Cryptosporidium scrofarum, C. suis and C. parvum subtypes IIaA16G2R1 and IIaA13G1R1 in Eurasian wild boars (Sus scrofa).

The aim of the present study was to identify the species of Cryptosporidium infecting Eurasian wild boars (Sus scrofa) in Galicia (NW, Spain). A sampling of 209 wild boars shot in different game preserves was carried out during the hunting season in 2009-2010. All samples were examined for Cryptosporidium infection, using both immunological and molecular tools. Cryptosporidium oocysts in faecal samples were identified using a direct immunofluorescence technique with monoclonal antibodies (DFA). The presence of Cryptosporidium DNA was determined using nested PCR involving amplification of a fragment of the small-subunit (SSU) ribosomal RNA gene (SSU rRNA). A total of 35 (16.7%) samples tested positive with both techniques. However, sequencing was only possible in 27 samples. Cryptosporidium scrofarum, Cryptosporidium suis and Cryptosporidium parvum oocysts were identified in 19, 5 and 3 of the samples, respectively. Moreover, C. scrofarum was detected as a dominant species infecting all age groups (juveniles, sub adults and adults). Sequence analyses of the glycoprotein (GP60) gene revealed the presence of C. parvum subtypes IIaA16G2R1 in 2 juveniles and IIaA13G1R1 in 1 sub adult wild boar. These species and subtypes have previously been described in human patients, indicating that isolates from asymptomatic wild boars might have zoonotic potential. This is the first report of the presence of C. scrofarum, C. suis and C. parvum subtypes IIaA16G2R1 and IIaA13G1R1 in wild boars (S. scrofa) in Spain.

Bovine paramphistomosis in Galicia (Spain): prevalence, intensity, aetiology and geospatial distribution of the infection.

The present study explored various basic aspects of the epidemiology of paramphistomosis in Galicia, the main cattle producing region in Spain. In total, 589 cows from different farms located across the region were selected at random in the slaughterhouse for examination of the rumens and reticula for the presence of Paramphistomidae flukes. Paramphistomes were found in 111 of 589 necropsied cows (18.8%; 95% CI: 15.7-21.9%), with higher prevalences of infection in beef cows than in dairy cows (29.2% vs 13.9%). Although the number of flukes per animal was generally low (median=266 flukes), some cows harboured large parasite burdens (up to 11,895 flukes), which may have harmful effects on their health or productivity. Cows with higher parasite burdens also excreted greater numbers of fluke eggs in their faeces, which suggests that heavily parasitized mature cows play an important role in the transmission of paramphistomosis. This role may be particularly important in Galicia, where the roe deer, which is the only wild ruminant in the study area, was found not to be a reservoir for the infection. The use of morpho-anatomical and molecular techniques applied to a large number of fluke specimens provided reliable confirmation that Calicophoron daubneyi is the only species of the family Paramphistomidae that parasitizes cattle in Galicia. The environmental data from the farms of origin of the necropsied cows were used in Bayesian geostatistical models to predict the probability of infection by C. daubneyi throughout the region. The results revealed the role of environmental risk factors in explaining the geographical heterogeneity in the probability of infection in beef and dairy cattle. These explanatory factors were used to construct predictive maps showing the areas with the highest predicted risk of infection as well as the uncertainty associated with the predictions.

Detection of microsporidia in drinking water, wastewater and recreational rivers.

Diarrhea is the main health problem caused by human-related microsporidia, and waterborne transmission is one of the main risk factors for intestinal diseases. Recent studies suggest the involvement of water in the epidemiology of human microsporidiosis. However, studies related to the presence of microsporidia in different types of waters from countries where human microsporidiosis has been described are still scarce. Thirty-eight water samples from 8 drinking water treatment plants (DWTPs), 8 wastewater treatment plants (WWTPs) and 6 recreational river areas (RRAs) from Galicia (NW Spain) have been analyzed. One hundred liters of water from DWTPs and 50 L of water from WWTPs and RRAs were filtered to recover parasites, using the IDEXX Filta-Max® system. Microsporidian spores were identified by Weber's stain and positive samples were analyzed by PCR, using specific primers for Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem. Microsporidia spores were identified by staining protocols in eight samples (21.0%): 2 from DWTPs, 5 from WWTPs, and 1 from an RRA. In the RRA sample, the microsporidia were identified as E. intestinalis. To the best of our knowledge, this is the first report of human-pathogenic microsporidia in water samples from DWTPs, WWTPs and RRAs in Spain. These observations add further evidence to support that new and appropriate control and regulations for drinking, wastewater, and recreational waters should be established to avoid health risks from this pathogen.

Anti-Neospora caninum antibodies in milk in relation to production losses in dairy cattle.

A comprehensive field study was carried out with the following objectives: (a) to assess the usefulness of individual and bulk tank milk analysis for determining Neospora caninum serostatus in individual cows and herds, and (b) to study the associations between N. caninum infection status (based on milk testing), and several productive and reproductive parameters in the animals. Antibodies were detected with a commercially available ELISA test (Bio K 192/5). Analysis of paired serum and milk samples from 1134 lactating cows on 38 farms revealed that 97.6% of the ELISA results were coincident, irrespective of whether serum or milk samples were used. Moreover, multiple linear regression analysis revealed that 86.0% of the variations in ELISA values in milk were due to variations in the serum. The measurement of antibodies in bulk tank milk was a good estimator of the herd level status of N. caninum infection, and enabled detection of infection in 94.7% herds with ≥10.0% seropositive cows and/or in all herds with >4% highly seropositive cows. The odds ratio for abortion in seropositive animals was 9.1 times higher than in seronegative animals. The infection serostatus was also a significant risk factor, as the odds ratio for abortion was even higher (12.0 times) in cows categorized as highly seropositive. ELISA values for the bulk milk from 387 randomly selected herds were negatively associated with average milk production. Moreover, milk production losses mainly occurred on farms categorized as highly positive (i.e. herds with ≥20.0% seropositive cows).

Cryptosporidium spp. and Giardia duodenalis in two areas of Galicia (NW Spain).

The aim of the present study was to investigate the environmental dispersal of Cryptosporidium spp. and Giardia duodenalis in two distinct areas (coastal and inland) in Galicia (NW Spain). Faecal samples were collected from healthy asymptomatic domestic (cows and sheep) and wild animals (deer and wild boars) in the selected areas. In each of the selected areas, samples of untreated water (influent) and of treated water (final effluent) were collected from each of the 12 drinking water treatments plants (DWTPs) and 12 wastewater treatment plants (WTPs) under study. Analysis of a single sample from each of the 635 (coastal) and 851 (inland) domestic and wild animals selected at random revealed that the prevalences of cryptosporidiosis and giardiosis in coastal area were 9.2% and 15.9% respectively, and in inland area, 13.7% and 26.7% respectively. In the coastal area, Cryptosporidium spp. oocysts were detected in influent and effluent samples from 2/12 (16.6%) DWTPs and 8/12 (66.6%) WTPs, while G. duodenalis cysts were detected in influent and effluent samples from 3/12 (25.0%) DWTPs and 12/12 (100%) WTPs. The concentrations were notably higher in WTPs; the mean parasite concentrations in the final treated effluent were 10 oocysts per litre and 137.8 cysts per litre for Cryptosporidium and Giardia, respectively. The mean concentration of G. duodenalis cysts per litre was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. oocysts per litre in both the influent and the effluent samples from all the treatment plants. In the coastal area, C. parvum, C. hominis and G. duodenalis assemblages A (I and II) and E were most repeatedly detected. In the inland area, C. parvum, C. andersoni and G. duodenalis assemblages A (I and II), B and E were most frequently identified.