Antibody responses to chimeric peptides derived from parasite antigens in mice and other animal species.

Peptide vaccines constitute an interesting alternative to classical vaccines due to the possibility of selecting specific epitopes, easy of production and safety. However, an inadequate design may render these peptides poorly immunogenic or lead to undesirable outcomes (e.g., formation of B neoepitopes). As an approach to vaccine development, we evaluated the antibody response to chimeras composed of two or three known B epitopes from Trichinella and Fasciola, and several linkers (GSGSG, GPGPG and KK) in species as different as mice, sheep and turbot. All these species could mount an effective immune response to the short chimeric peptides. Nevertheless, this response depended on several factors including a favorable orientation of B-cell epitopes, adequateness of linkers and/or probability of formation of T neoepitopes. We also observed that, at least in mice, the inclusion of a decoy epitope may have favorable consequences on the antibody response to other epitopes in the chimera.

Helminth infections on organic dairy farms in Spain.

The aim of this study was to assess the prevalence of the major helminth infections affecting organic dairy cattle in northern Spain. Milk and faecal samples were obtained from 443 milking cows. Ostertagia ostertargi and Fasciola hepatica exposure was assessed by detection of specific antibodies in milk samples and F. hepatica infection was diagnosed by the detection of coproantigens in faecal samples. Dictyocaulus viviparus and Calicophoron daubneyi infections were diagnosed by conventional coprological techniques. The prevalence of infections caused by F. hepatica was considerable low, but similar to data reported from conventional farming in the same area. The prevalence rate of C. daubneyi infection was higher than previous data mirroring an increase of the prevalence that was also reported in other European countries in recent years. Specific antibodies against O. ostertargi were detected in all herds and the median levels of antibodies, determined by ELISA, exceeded the thresholds indicating milk production losses. The prevalence of D. viviparus was almost negligible. For each parasite, an ordinal logistic-regression analysis was used to assess the risk of infection by taking into account the administration of effective anthelmintics and the number of lactations. Treatment of cows with fasciolicides decreased the risk of F. hepatica infection in multiparous cows, whereas treatment with oxiclozanide or albendazol did not decrease the risk of C. daubneyi infection or O. ostertargi exposure, respectively. The study findings demonstrate that helminth infection in organic dairy farming is similar or even lower than previous data reported from conventional farming. Special attention should be paid to the impact of these infections on milk production.

Development and validation of a mtDNA multiplex PCR for identification and discrimination of Calicophoron daubneyi and Fasciola hepatica in the Galba truncatula snail.

Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C. daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C. daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1 ng of DNA from C. daubneyi and 0.001 ng of DNA from F. hepatica. This allowed infection by either F. hepatica or C. daubneyi to be detected even when pools made up with only 1 µl (60 ng of DNA) from infected snail plus 99 µl from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98 µl), C. daubneyi infected (1 µl) and F. hepatica infected (1 µl) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological models of both infections to be established and consequently a better strategic control.

Molecular and immunological characterization of Fasciola antigens recognized by the MM3 monoclonal antibody.

Fascioliasis is a re-emerging parasitosis produced by liver flukes of the genus Fasciola. In this study we used protein fingerprinting (PMF) and MS/MS analysis to investigate the Fasciola secretory antigens that are recognized by mAb MM3. The results showed that mAb MM3 binds to several Fasciola cathepsins L1 and L2, but also co-purifies a Kunitz-type protein previously described in F. hepatica, which appears to bind to Fasciola cathepsins L. After identifying the target antigens for mAb MM3, we cloned and expressed a cathepsin L1 isoform in E. coli (gb|FR848428), which after refolding exhibited the MM3-recognized epitope and displayed cysteine protease activity. Using native, folded-recombinant and denatured-recombinant Fasciola cathepsins L as targets, we demonstrated that during F. hepatica infections in sheep, antibody responses to linear and conformational epitopes present on cathepsins L are promoted. However, the antibody response to linear epitopes was only detected in significant amounts in animals suffering from repeated infections. A different antibody response to linear and conformational epitopes also appears to occur in rabbits immunized with native or recombinant unfolded cathepsins, as sera from animals immunized with the latter did not react with native cathepsins and vice versa. In addition, the ELISA inhibitions showed that the MM3 epitope is not recognized by rabbits, which explains the usefulness of these species for producing capture antibodies for use in MM3-ELISA assays.

Different humoral mechanisms against Neospora caninum infection in purebreed and crossbreed beef/dairy cattle pregnancies.

The antigen-specific IgG subclass response may be a convenient indicator of the underlying nature of T helper cell regulation. The aim of the present study was to identify possible differences in Neospora caninum-specific total plasma IgG, IgG1 and IgG2 antibody levels in purebreed and crossbreed pregnancies throughout gestation in beef and dairy cattle chronically infected with N. caninum. Comparisons were also made between aborting and non-aborting dams. The population examined comprised 96 pregnant parous cows seropositive for N. caninum. Plasma antibodies were determined on Days 90, 120, 150, 180 and 210 of gestation or until abortion. Of the 96 pregnancies examined, 12 ended in abortion. None of the 14 Holstein-Friesian (HF) cows inseminated with HF semen (HF-HF group) aborted, whereas 6 (11.0%) of the 54 HF cows inseminated with Limousin semen (HF-L group) and 6 (21.4%) of the 28 Rubia Gallega (RG) beef cows inseminated with RG semen (RG-RG group) aborted. In the 84 non-aborting cows, a significant positive effect of gestation day was observed on total IgG, IgG1 and IgG2 antibodies levels (P<0.0001 for the three variables). In RG-RG cows, significantly higher levels of IgG (P=0.003; d.f.=2; F-value=6.41), IgG1 (P<0.001; d.f.=2; F-value=10.55) and IgG2 (P=0.004; d.f.=2; F-value=5.82) antibodies against N. caninum were recorded throughout gestation compared to the other groups, whereas the levels of these antibodies were significantly lower in HF-HF on Days 180 and 210 of gestation. In aborting cows, significantly lower IgG (P=0.001; d.f.=1; F-value=25.21) and IgG2 (P=0.001; d.f.=1; F-value=20.39) antibody levels were observed in the RG-RG cows compared to the HF-L cows, whereas no significant effect on IgG1 antibody levels was detected in the two groups with aborting animals (RG-RG and HF-L). Our findings indicate that humoral mechanisms against N. caninum infection and abortion differ in purebreed pregnancies and crossbreed pregnancies in beef/dairy cattle.

Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks.

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%-99.9%), 100% (95% CI: 98.6%-100%) and 0.997 (95% CI: 0.987-1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R(2)=0.84; p<0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa=0.92; p<0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection.

Dynamics of anti-Neospora caninum antibodies during gestation in chronically infected dairy cows.

The dynamics of antibody production against Neospora caninum during the gestation period was examined in chronically infected dairy cows. Data were obtained from 86 pregnant parous dairy cows, 21 of which had suffered abortion. The cows belonged to two herds in which a diagnosis of N. caninum infection had been previously confirmed in aborted foetuses. Pregnancy diagnosis and blood collection were performed on post-insemination Days 40, 90, 120, 150, 180, 210, and at parturition or until the time of abortion detection. Blood plasma was tested for antibodies against N. caninum using ELISA. The non-aborting cows were divided into two groups according to whether their antibody values in the second half of gestation had increased or not, while aborting cows were classified as those showing an antibody peak before abortion or those not showing a pre-abortion peak. Differences in antibody values throughout pregnancy in each group of non-aborting and aborting cows were analysed by GLM repeated measures of analysis of variance. While 32 non-aborting cows (49%) showed a significant and consistent increase in anti-Neospora antibody values during the second half of gestation, antibody values in the remaining 33 non-aborting cows were practically constant throughout gestation. An antibody peak around abortion was observed in 11 aborting cows (52%), while antibody values in the remaining 10 aborting cows were similar before and at abortion. Seroprevalence fluctuations, defined as seronegative blood samples at some point during the gestation period, were, furthermore, observed in 2 aborting and 11 non-aborting cows. Our results indicate two clearly distinguishable types of humoral immune dynamics throughout gestation: an increased or flat production of antibodies during the second half of gestation in non-aborting animals and before abortion in aborting cows. The observation that some Neospora-infected dams can exhibit negative antibody values at any time during gestation, particularly at parturition or abortion, prompts future studies designed to explore the use of new ELISA strategies at the farm level.

Prevalence and intensity of infection of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Galicia (NW Spain).

Faecal samples were collected from 734 cattle selected at random from 60 dairy farms in Galicia (NW Spain). The animals studied were classified into 12 age groups: <1 month (53); 1-5 months (30); 6-11 months (31); 12-16 months (72); 17-20 months (64); 21-24 months (96); 3 years (94); 4 years (74); 5 years (67); 6 years (67); 7-8 years (63) and 9-13 years (23). Oocysts of Cryptosporidium spp. were identified in 104 animals (14.2%) distributed throughout all of the age groups and from 40 different farms (66.7%). The percentage of cattle infected ranged between 58.5% in calves <1 month and 7.9% in 7 to 8-year-old cows, i.e. the percentage of infection decreased significantly (P < 0.05) with increasing age. The intensity of infection in animals older than 1 month ranged between 10 and 5924 oocysts/g of faeces and there were no significant differences between the different groups. Cysts of Giardia duodenalis were identified in 221 animals (30.1%) from 56 farms (93.3%). The parasite was detected in all age groups, at rates of infection ranging between 21.8% (9-13 years) and 56.7% (1-5 months), although these differences were not statistically significant. The intensity of infection ranged between 7 and 15 412 cysts/g of faeces, with the number of cysts shed being significantly higher (P < 0.05) in calves <1 month than in calves aged 1-5 months. Significant associations between parasitisation by Cryptosporidium spp. or G. duodenalis and the consistency of the faeces were only found in calves aged <1 month and 1-5 months. Concurrent infections were more prevalent in the groups of calves of 1-5 months (23.3%) and 6-11 months (25.8%).

First isolation of Neospora caninum from an aborted bovine fetus in Spain.

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.

Efficacy of beta-cyclodextrin against experimental cryptosporidiosis in neonatal lambs.

The efficacy of beta-cyclodextrin against experimental Cryptosporidium parvum infection was evaluated in neonatal lambs. The animals were treated by oral administration of the drug at 1 g/kg of body weight during 3 consecutive days. Preventive treatment was started within 1 day of birth, and therapeutic treatment was initiated at the onset of diarrhea following confirmation of infection. Disease development and drug efficacy were evaluated by monitoring the presence or absence of diarrhea and oocyst shedding from birth until 30 days of age. Weight gains at 15 and 30 days of age were also recorded. Beta-cyclodextrin was highly effective as a prophylactic treatment; 1 animal did not acquire the infection, diarrhea was prevented in infected animals, and there was a considerable decrease in oocyst shedding. The therapeutic treatment was effective in decreasing the severity of diarrhea and the duration of oocyst shedding. The animals tolerated the drug well, and there was a significant increase in their body weights.