P. Morrondo

Seroprevalence of Toxoplasma gondii and Neospora caninum in wild and domestic ruminants sharing pastures in Galicia (Northwest Spain).

The prevalence of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and cELISA, respectively, in 160 roe deer (Capreolus capreolus), 177 sheep and 178 cattle sharing pastures in Galicia (Northwest Spain). The seroprevalence for T. gondii was 13.7% in roe deer, 57% in sheep and 7.3% in cattle. The seroprevalence for N. canimum was 6.8%, 10.1% and 24.1% in roe deer, sheep and cattle, respectively. Statistically significant differences were observed between sheep and the other species for T. gondii and between cattle and the other ruminants for N. caninum. Only 19/515 animals were positive for both, T. gondii and N. caninum. Statistically significant differences were observed among different geographical areas for T. gondii but not for Neospora, seroprevalence being higher in the coastal area lower than in other areas. This study reveals a widespread exposure to T. gondii in Galician ruminants, and therefore, those species, particularly sheep, should be regarded as a potential source of infection for humans.

Use of a sandwich-enzyme-linked immunosorbent assay (SEA) for the diagnosis of natural Fasciola hepatica infection in cattle from Galicia (NW Spain) ☆

An indirect-enzyme immune-linked immunosorbent assay (IEA) was compared to a sandwich-enzyme-linked immunosorbent assay (SEA) to diagnose fasciolosis in naturally infected cattle. By means of a rabbit polyclonal IgG antibody to F. hepatica antigens, captured circulating antigens were detected by an SEA technique. A total of 85.1% of the samples examined was considered positive by using the IEA-test, whereas this percentage was of 37.3% by the SEA test. Using the two enzymatic probes, only the 38.3% resulted positive to both immunoassay probes. Our data indicate that circulating antigen detection assay in combination with results of antibody detection may offer a more reliable diagnostic technique than classic coprologic ones, with results very appropriate for epidemiologic studies carried out on a wide area.

Diagnosis of Parasitic Zoonoses by Immunoenzymatic Assays—Analysis of Cross‐Reactivity Among the Excretory/Secretory Antigens of Fasciola hepatica, Toxocara canis, and Ascaris suum

Several parasitic infections such fasciolosis, toxocariosis or ascariosis are important zoonoses. During the infection with Fasciola hepatica, Toxocara canis and Ascaris suum, an important intraorganic phase in their hosts takes place, releasing antigens responsible for a humoral immune response, which enables the diagnosis of that parasitosis. A study to identify the existence of cross‐reactivity among the excretory/secretory antigens of F. hepatica, T. canis and A. suum was developed. One group of Sprague–Dawley rats was infected with 20 metacercariae of F. hepatica and another group remained uninfected as control. By means of an Indirect‐ELISA, the rat humoral immune response (IgG and IgM) against the excretory/secretory antigens of F. hepatica was analysed and measured for cross reactivity with T. canis and A. suum. IgM cross‐reaction was mainly observed in the first 10 weeks post‐infection. IgG cross‐reaction was observed throughout the study, and was maximal at the 2–3 weeks and 3–6 weeks post‐infection, which corresponds to the intraorganic migratory phase of these parasites. The western‐blot showed that the rat IgG recognised three proteins of 190, 160 and 33 kDa in the antigens from F. hepatica, T. canis and A. suum. The existence of cross‐reactivity among these antigens seems to demonstrate also the presence of structural similarities, such as tegumental proteins. These results should be consider when immunoassay probes are used in the diagnosis of parasitic infections.

Genotype and subtype analysis of Cryptosporidium isolates from calves and lambs in Galicia (NW Spain).

Faecal specimens from diarrhoeic pre-weaned calves (n=61) and lambs (n=127) collected over a 1-year period (2008-2009) at 27 cattle and 28 sheep farms in Galicia (NW Spain) were examined for the presence of Cryptosporidium oocysts and positive specimens were selected for molecular examination. Overall, 30 calves (49.2%) and 39 lambs (30.7%) tested positive for Cryptosporidium by microscopy. PCR products of the SSU rRNA locus were obtained for 27 Cryptosporidium positive calf isolates and 23 lamb isolates. Restriction analyses generated profiles of C. parvum in all isolates except for 9 lamb specimens from 5 farms that yielded banding patterns and sequences indicative of the Cryptosporidium cervine genotype. Sequence analyses of the glycoprotein (GP60) gene revealed that all but 1 C. parvum isolate from calves belonged to the subtype IIaA15G2R1 and 1 isolate was identified as the novel subtype IIaA13G1R1. Two different subtypes were identified in sheep flocks including IIaA16G3R1, which was seen in 7 lamb isolates from a single farm and subtype IIaA15G2R1, identified in 3 isolates from 2 farms. These findings suggest a limited genetic diversity within C. parvum in ruminant livestock from this geographical area, although both calves and lambs should be considered as a reservoir for zoonotic subtypes.

Influence of Age and Breed on Natural Bovine Fasciolosis in an Endemic Area (Galicia, NW Spain)

An analysis was undertaken of the effect of breed and age on bovine fasciolosis using antibody and antigen detection ELISAs. A total of 84.3% of the 1284 serum samples examined had positive antibody values and 20.4% exhibited antigenaemia. The seroprevalence of antibodies in crossbred cattle was higher than that in autochthonous Rubia Gallega, Friesian or Brown Swiss cows. The highest percentage antigenaemia occurred in the Brown Swiss cattle, but the breed differences were not statistically significant. Cattle aged 3–5 years had the highest antibody and circulating antigen prevalence and the age differences were significant. It was concluded that the apparent influence of breed was probably closely associated with the husbandry system. The autochthonous Rubia Gallega may be better adapted to fasciolosis as its percentage of antigenaemia was the lowest.

Identification of Cryptosporidium xiaoi in diarrhoeic goat kids (Capra hircus) in Spain.

Faecal specimens from five diarrhoeic goat kids (Capra hircus) younger than 21 days were collected in a goat farm in Galicia (NW Spain) and examined for the presence of Cryptosporidium oocysts. Two Cryptosporidium-positive isolates were detected and selected for molecular examination. A banding pattern indicative of Cryptosporidium bovis was obtained after restriction analyses of PCR products from small-subunit rRNA genes. However, both positive isolates were identified as Cryptosporidium xiaoi by sequence analyses of SSU rRNA and actin genes. Our results demonstrate that domestic goats are a host for C. xiaoi and suggest that this parasite species may be involved in the aetiology of neonatal diarrhoeic outbreaks in goat farms. This is the first published description of C. xiaoi in goat kids in Spain.

Field Evaluation for Anthelmintic-Resistant Ovine Gastrointestinal Nematodes by In Vitro and In Vivo Assays

A coprological survey to analyze the presence of flock resistance to benzimidazoles (BZ) and macrocyclic lactones (ML) was performed in sheep under field conditions. Fecal samples were collected from 2,625 sheep in 72 commercial farms from Galicia (NW Spain). The in vitro (FECRT, fecal egg count reduction test) and in vivo (EHA, egg hatch assay, and LFIA, larval feeding inhibition assay) tests were used to assess the efficacy of these anthelmintics. Coprocultures were also developed to obtain knowledge on the main genera of trichostrongylid nematoda prior to, and after, the administration of the anthelmintics. By using the FECRT, BZ resistance was observed in 13 (18%) flocks, whereas ML resistance was only detected in 2 (3%) farms. The number of resistant flocks to BZ was 21 (29%) by using the EHA and 7 (10%) by means of the LFIA. None of the flocks used in this study showed simultaneous resistance to both employed anthelmintics. The results from the in vitro and in vivo tests revealed that 92% of the flocks FECRT resistant to BZ were also resistant with the EHA. The LFIA confirmed all the farms resistant to ML by using the in vivo test. After the administration of BZ, nematode larvae belonging to Teladorsagia circumcincta (32.2%), Trichostrongylus spp. (29%), Nematodirus spp. (6.5%), and Chabertia ovis (3.2%) were identified. In the flocks receiving ML, only T. circumcincta was identified (57%). We recommend the use of in vitro tests because they are more efficient. As the use of macrocyclic lactones is increasing in this region, further investigation is needed for detecting resistance to the anthelmintic family compounds by the LFIA.

Effect of early treatment with ivermectin and doramectin on the dynamics of antibody response in cattle naturally infested by Hypoderma lineatum and H. bovis

The field efficacy of two avermectins (ivermectin and doramectin) and the subsequent development of the antibody response were assessed in cows naturally infested with first-instar larvae (L-1) of Hypoderma sp. Twenty-eight Frisian cows were randomly divided into three groups while the first-instar larvae were still in migration: Group 1 (G-C) untreated control; Group 2 (G-Iv), treated with ivermectin injectable (0.2 mg kg-1 body weight) and Group 3 (G-Dor), which received doramectin injectable (0.2 mg kg-1 body weight). Serum antibody response was studied by an indirect ELISA test using, as antigen, the hypodermin C obtained from L-1 of H. lineatum. In treated animals no grubs were present on the back at any time during the trial, whereas a variable number of nodules were found in untreated animals. Both avermectins showed total efficacy against L-1 of Hypoderma sp., and there were no local or general reactions. In ivermectin-treated cattle serum antibody levels declined from one month p.t., while in those treated with doramectin they started to fall two weeks later, but no differences were found among both groups. On the other hand, G-C antibody levels progressively increased and remained high until December, when the first grubs became detectable on the back; after that, they began to decline. Early treatments against Hypoderma sp. have an influence on the results of ELISA, so they must be considered to determine the most suitable time for blood sampling.

Immunomodulatory effect of Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production

This study examines the immunomodulatory effect of a crude larval extract (CLE), obtained from first stage larvae (L1) of H. lineatum, and the purified fractions hypodermin A (HyA), HyB and HyC. Proliferative responses of peripheral blood mononuclear cells (PBMC) from uninfested and previously infested cattle and the production of the cytokines IL‐10, IL‐4 and IFN‐γ, in response to concanavalin A (Con A), were determined. The stimulation index of peripheral blood mononuclear cells from uninfested cattle was significantly lower than that from infested animals with the different antigens assayed. The HyA was the antigen that most inhibited the proliferative response, followed by the HyB, the HyC and the CLE. This hypodermin provoked an increase of IFN‐γ and a suppression of IL‐10 production that would support a Th1‐like cytokine response. The HyB reduced the production of IL‐10 stimulated by the Con A in cultures from infested animals. The HyC did not modulate the production of cytokines. Finally, the CLE induced a marked suppression in the production of the different cytokines in cultures from naïve and previously sensitized animals. Our results indicate that Hypoderma larval secretions are comprised of different components (hypodermins) that individually induce distinct but partially overlapping modulatory responses.

Risk periods of infection by Calicophoron daubneyi (Digenea:Paramphistomidae) in cattle from oceanic climate areas.

From March 2001 to February 2004, 724 faecal samples of cattle under field conditions were collected in 121 farms from Lugo (NW Spain), an area with oceanic climate. The risk periods of infection by Paramphistomum trematoda in cattle was assessed by analyzing the prevalence of paramphistomosis, egg-output and climatic data. A significantly higher percentage of cattle passing trematode eggs by faeces was recorded firstly in September and secondly in March. The greatest numbers of eggs were achieved in May. Finally, the individual average prevalence was 19% and 36% of the farms had Calicophoron daubneyi-positive cattle. We conclude that the periods of high risk for C. daubneyi infection in areas with oceanic climate are those following periods with elevated rainfall, which occurs in May–June and November–December in these areas. Appropriate measures for reducing the risk for paramphistomosis are needed and the administration of chemotherapy with efficient anthelmintic in June and in November is strongly recommended.