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.

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.

Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water.

AIM: To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water. METHODS AND RESULTS: Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall. CONCLUSIONS: Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.

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%).

Evaluation of two commercial disinfectants on the viability and infectivity of Cryptosporidium parvum oocysts.

Cryptosporidiosis is mainly a problem in neonatal ruminants. Not only do Cryptosporidium spp. spread ubiquitously in our environment, but the protozoa are highly resistant to harsh environmental conditions and disinfectants, and a control measure is urgently required. This study investigated the potential biocidal activity on Cryptosporidium parvum oocysts of two commercial disinfectants developed originally to be used in farms and food-processing industries. The products, containing formaldehyde and hydrogen peroxide respectively, both had some anticryptosporidial effects. The viability and infectivity of purified C. parvum oocysts exposed to both disinfectants at different concentrations and exposure times were evaluated by inclusion or exclusion of vital dye (propidium iodide), use of an excystation technique and infection of suckling mice. Viability assays showed a decrease in oocyst viability associated with an increase in exposure time for each of the concentrations used. The intensity of infection in neonatal mice was significantly lower (P<0.05) than in the control litters.

Giardia duodenalis and Cryptosporidium parvum infections in adult goats and their implications for neonatal kids.

During the kidding season between January and April 2003, 10 farms were selected and divided into two groups of five. The farms in group A had had serious diarrhoeal illness and losses in neonatal kids the previous year, and there were Cryptosporidium parvum infections in kids associated with diarrhoea during the survey. On the farms in group B, there was no history of diarrhoeal disease the previous year and neither C parvum oocysts nor diarrhoea were detected in neonatal kids during the survey. Faecal samples were collected once from 10 adult goats aged between one and seven years on each farm. To assess more accurately the pattern of output of oocysts of C parvum and cysts of Giardia duodenalis by periparturient adult goats, one farm was selected from each group, faecal samples were collected weekly before and after kidding from 12 goats on the farm in group A and from 10 goats on the farm in group B. There was no significant difference in the prevalence of G duodenalis cysts between the group A farms (14 per cent) and the group B farms (12 per cent), and the numbers of cysts excreted ranged from 143 to 400 cysts per gram of faeces (cpg) on the group A farms and 72 to 334 cpg on the group B farms. There was a significant difference (P=0.03) in the prevalence of C parvum oocysts at the group level between the group A farms (20 per cent) and the group B farms (6 per cent). All the adult goats excreted cysts and oocysts at some date around the kidding period; the number of animals excreting cysts of G duodenalis or oocysts of C parvum increased when they gave birth, and seven to 10 times more cysts and oocysts were shed in the three weeks around kidding than in the period more than three weeks from kidding (P<0.001).

Effect of batch-process solar disinfection on survival of Cryptosporidium parvum oocysts in drinking water.

The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m(-2)) at 40 degrees C. Viability assays (4',6'-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.

Anticryptosporidial activity of furan derivative G1 and its inclusion complex with beta-cyclodextrin.

The capacity of beta-cyclodextrin (betaCD) to form a complex with a new furanic derivative, G1, was investigated. Interactions of the drug and betaCD in solution and in the solid state were studied using phase solubility techniques, thermal methods, X-ray, and IR spectroscopy. Preparation of a kneaded mix of G1/betaCD increased both the aqueous solubility and the dissolution rate of the furan derivative. The anticryptosporidial efficacies of the drug and of the inclusion complex were evaluated using a suckling murine model. Oral administration of G1 considerably decreased the intensity of the infection, but betaCD showed similar anticryptosporidial activity to that of the betaCD-G1 complex and higher activity than G1 alone.

Environmental dispersal of Cryptosporidium parvum oocysts and cross transmission in cultured bivalve molluscs.

Two commercially valuable mollusc species ( Ostrea edulisand Tapes decussatus) were experimentally contaminated with Cryptosporidium parvum oocysts. A direct immunofluorescent antibody technique and inclusion/exclusion of the fluorogenic vital dye propidium iodide were used to test for the presence and viability of the oocysts, showing that transmission of contamination occurred between coexisting species. There was a decrease in the viability of oocysts in the initially uncontaminated molluscs as well as a large decrease in the number of oocysts retained when dead molluscs were used as the source of contamination. The results show the potentially important role that these molluscs play in spreading contamination in depuration plants and areas where aquatic organisms are cultivated.

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.

Survival of Cryptosporidium parvum oocysts recovered from experimentally contaminated oysters (Ostrea edulis) and clams (Tapes decussatus).

Samples of two species of shellfish that form part of the human food chain (the oyster Ostrea edulis and the marine clam Tapes decussatus) were experimentally contaminated with Cryptosporidium parvum oocysts. Changes in the viability of oocysts subsequently recovered from the shellfish were evaluated by means of an immunofluorescent antibody technique (IFAT) and inclusion/exclusion of the fluorogenic vital dye propidium iodide. There was a sharp decrease in oocyst viability during the first 4 days, with 15-25% viable oocysts remaining thereafter. In addition the infectivity of these oocysts at 10 and 31 days post-contamination was demonstrated using a suckling murine model.

Evaluation of beta-cyclodextrin against natural infections of cryptosporidiosis in calves.

The effectiveness of beta-cyclodextrin, excipient used in pharmaceutical industry, in the treatment of natural infection by Cryptosporidium parvum in suckling calves, was evaluated. Administration of the drug at a dose of 500 mg/kg body weight for 3 consecutive days from birth (prophylactically) or following confirmation of the infection (therapeutically) decreased the severity of diarrhoea and shortened the duration of oocyst shedding.

Treatment with beta-cyclodextrin of natural Cryptosporidium parvum infections in lambs under field conditions.

Following the unexpected activity of the excipient beta-cyclodextrin against experimental infection by Cryptosporidium parvum in suckling mice, its efficacy in the prevention and treatment of natural infections in lambs was evaluated under field conditions. Fifty-three crossbred neonatal lambs were randomly selected for the study. Treatment consisted of oral administration of an aqueous suspension of beta-cyclodextrin at a dose of 500 mg/kg of body weight. To test prophylactic efficacy, the suspension was administered at 1, 2 and 3 days of age. To evaluate therapeutic efficacy, the suspension was administered on each of the 3 days following onset of diarrhoea. Infection was monitored by daily examination of faecal samples, from birth to 30 days. The criteria studied in evaluating efficacy were: oocyst shedding, the presence of diarrhoea, and weight gain at 15 and 30 days. In the group that received prophylactic treatment with beta-cyclodextrin, there were no mortalities and, compared with control lambs, there was a decrease in the number of animals infected, a longer prepatent period and notable reduction in the patent period and the duration of diarrhoea. Therapeutic treatment also reduced the patent period and the severity of diarrhoea. beta-cyclodextrin was well tolerated by all of the treated animals.

Viability and infectivity of oocysts recovered from clams, Ruditapes philippinarum, experimentally contaminated with Cryptosporidium parvum.

This study confirms the important role of marine bivalve molluscs, destined for human consumption, as transmitters of cryptosporidiosis, zoonotic diarrhoeal disease caused by Cryptosporidium parvum. C. parvum oocysts recovered from seawater clams (Ruditapes philippinarum) were viable and infective in five of eight infected neonatal CD-1 Swiss mice. Oocysts were observed in clam gill and gastrointestinal tract tissue homogenates as well as in gill histological sections, by an immunofluorescent antibody technique. In vitro viability of recovered oocysts was also determined using fluorogenic vital dyes (75% viability).

Inhibition of Cryptosporidium infection in mice treated with a cyclodextrin inclusion complex with diloxanide furoate.

The efficacies of diloxanide furoate, beta-cyclodextrin and a cyclodextrin inclusion complex against Cryptosporidium parvum were evaluated in a suckling murine model. Efficacy was established by numbers of oocysts recovered from the intestinal tract of mice on day 7 postinfection. The level of infection in treated mice was significantly lower than in control mice and, surprisingly, the most efficacious treatment was beta-cyclodextrin, an excipient used in pharmaceutical technology.

Serological response to Cryptosporidium parvum in adult cattle from the Andean region of Colombia.

Single faecal and serum samples were individually collected from 135 asymptomatic adult cows on seven farms in Cundinamarca (Colombian Andean region). Tests for the presence of oocysts of Cryptosporidium parvum (carbol fuchsin stain) and Eimeria spp (flotation in saturated saline solution) revealed that none of the animals had coccidia in their faeces. The IgG antibody levels to C. parvum were measured by an enzyme-linked immunosorbent assay (ELISA) technique and the reactivity to C. parvum antigens by a Western blotting procedure. Cryptosporidial antibodies were detected in cattle from all farms, with 53.3% (72 animals) being seropositive. Sera recognized 5-11 protein fractions with molecular masses ranging from 12 14 kDa to 97-100 kDa. Sera considered as positive by ELISA reacted intensely and more frequently with protein fractions of approximately 20-22, 42-48, 51-57 and 60-69 kDa, whereas only the 42-48 kDa antigen was strongly recognized by sera without IgG antibodies. The presence of IgG antibody against C. parvum in most animals, as well as the reactivity to major proteins of C. parvum, could be indicative of continuous exposure to this parasite.

Unexpected activity of beta-cyclodextrin against experimental infection by Cryptosporidium parvum.

An unexpected activity of beta-cyclodextrin, an excipient used in pharmaceutical technology, was observed against Cryptosporidium parvum. The viability and infectivity of purified oocysts, exposed for 24 hr to beta-cyclodextrin (2.5% suspension), were evaluated by inclusion/exclusion of 2 fluorogenic vital dyes and a suckling murine model, respectively. Results of the viability assay showed a high proportion of nonviable oocysts (81.5%). The intensity of experimental infection, determined 7 days postinoculation by examination of intestinal homogenates, was significantly lower (P < 0.05) than in the control litters. The preventive and curative efficacies of beta-cyclodextrin suspension were also evaluated in experimentally infected neonatal mice. Infection was prevented when the suspension was administered 2 hr before inoculated oocysts and on days 1 and 2 postinoculation.