Detection and characterization of a Cryptosporidium isolate from a southern elephant seal (Mirounga leonina) from the Antarctic peninsula.

The presence of Cryptosporidium and Giardia in 221 fecal samples from different species of Antarctic pinnipeds was investigated by immunofluorescence microscopy and PCR. Cryptosporidium, a skunk-like genotype, was detected only in a southern elephant seal. Giardia was not detected. This is the first report of a Cryptosporidium sp. in Antarctic marine mammals.

Neospora caninum infection in stray and farm dogs: seroepidemiological study and oocyst shedding.

In a previous study, farm and stray dogs were considered potential high risk populations of Neospora caninum infection in Spain. Consequently, we decided to investigate the significance of N. caninum infection in these populations. Specific antibodies were detected in 120 out of 275 dog sera (43.6%), with titres ranging from 1:50 to 1:800. Differences in seroprevalence between farm (47.5%, 67/141) and stray (39.5%, 53/134) dogs were not significant (P>0.05; χ(2) test), but farm dogs showed significantly higher titres (P<0.01; Student's t-test). N. caninum seroprevalence in farm dogs was associated with increasing age (P<0.01; χ(2) test) and dogs with free access to the farm were more likely to be seropositive than controlled-dogs (P<0.05; χ(2) test). The presence of anti-N. caninum antibodies was more often detected in dogs from farms with 5-20% N. caninum within-herd seroprevalence (56.9%, 37/65) than those from farms with 0-5% seroprevalence (38%, 23/60) (P<0.05; χ(2) test). We microscopically observed N. caninum-like oocysts in the faeces from one farm dog, but the number of oocysts was very low, and the aetiology could not be confirmed. Also, parasite isolation was attempted from fresh neural tissue from stray dogs but was unsuccessful.

Pathogenic characterization in mice of Neospora caninum isolates obtained from asymptomatic calves.

In this study, we characterized 8 new isolates obtained from healthy but congenitally infected calves using a BALB/c mouse model. Neospora caninum-infected mice survived without exhibiting any clinical signs of disease. Nevertheless, differences among isolates in parasite organ distribution, parasite burden and the severity of histopathological lesions were determined. Mice infected with the Nc-Spain 5H, Nc-Spain 7 and Nc-Spain 9 isolates showed higher parasite burdens and more severe brain lesions during the late phase of infection compared to mice infected with the Nc-Spain 2H, Nc-Spain 3H or Nc-Spain 6 isolates. Furthermore, differences in the immunoglobulin IgG1 and IgG2a isotype kinetics induced by these isolates were observed, with a more rapid IgG2a response seen in mice infected with the Nc-Spain 2H and Nc-Spain 3H isolates. These results confirm the intra-species variability of N. caninum pathogenicity.

First isolation of Besnoitia besnoiti from a chronically infected cow in Spain.

Besnoitia besnoiti was isolated from a skin biopsy of a chronically infected cow from central Spain. Zoites released from macroscopic cysts were adapted to its culture in vitro on a MARC-145 cell monolayer. Tachyzoites produced in vitro were either cryopreserved or used for genomic DNA isolation. A 2206 nt sequence containing 18S ribosomal RNA gene, internal transcribed spacer 1 (ITS 1), and a partial sequence of 5.8S ribosomal RNA gene was amplified by PCR and sequenced. This sequence showed a 99-100% identity to 18S, ITS1, and 5.8S sequences of B. besnoiti published in databases. After analysis by transmission and scanning electron microscopy of isolated bradyzoites and tachyzoites, it was observed that their ultrastructural morphology coincided with B. besnoiti. The isolate characterized in this study was identified as B. besnoiti on the basis of the disease produced, molecular characteristics, and morphology. The B. besnoiti isolate was denoted as BbSpain-1; it is the first isolate obtained and characterized in Spain and one of the first European isolates adapted to grow in vitro. The isolation and in vitro production of this B. besnoiti isolate offers a good opportunity to study general aspects of bovine besnoitiosis, including epidemiology, pathogenesis, and diagnosis of this re-emergent disease.

Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain.

Neospora caninum is a cyst-forming parasite that causes abortion in cattle. Despite this parasite's ubiquitous distribution and wide host range, the number of N. caninum isolates obtained to date is limited. In vitro isolation of the parasite is arduous and often unsuccessful. In addition, most isolates have been obtained from clinically affected hosts and therefore could be biased towards more virulent isolates. In this report, an improved isolation approach from transplacentally infected newborn calves was undertaken and 9 new isolates were obtained. Moreover, a microsatellite technique was applied to investigate the genetic diversity of these isolates. Most isolates showed specific genetic profiles. However, the Nc-Spain10 isolate was identical to the previously described Nc-Spain1H isolate and Nc-Spain3H was identical to Nc-Spain4H. These isolates were likely to have identical genotypes because they were isolated from distinct calves of the same herd. Future pathogenic characterization of these isolates will contribute to the investigation of the relationship between isolate virulence and the outcome of infection, as well as other epidemiological features, such as transmission.

Seroprevalence and risk factors associated with Neospora caninum infection in different dog populations in Spain.

In this study, Neospora caninum seroprevalence and some associated risk factors were investigated in four different dog populations in Spain. N. caninum seropositivity was significantly higher in farm dogs (51%, 51/100) (P<0.001) and lower in household dogs (2.9%, 3/102) (P<0.0001). The seroprevalence in hunting (23%, 23/100) and stray (24.5%, 23/94) dogs was moderate, and no significant differences were observed between these two populations (P>0.05). A significantly higher number of dogs showed titres of 1:50-1:100 (68%, 68/100) than >or=1:200 (33%, 33/100) titres (P<0.0001). N. caninum antibodies were more often detected in mixed breed than pure breed dogs (P<0.01), but when data were stratified by dog type a significant association was not found (P>0.05). A significantly higher prevalence of N. caninum was observed in dogs over 1 year old (P<0.01), indicating that horizontal transmission may be the most important route of infection. The presence of N. caninum antibodies was significantly more frequent in Leishmania infantum-seropositive hunting (P<0.05) and stray dogs (P<0.00001). This study confirms that farm, stray and hunting dogs can be considered at-risk dog populations for N. caninum infection in Spain.

The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum.

A Neospora caninum 17-19 kDa antigenic protein fraction (p17) in one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) is the immunodominant antigen recognized by sera from bovines naturally infected by N. caninum. To identify the proteins making up the p17 fraction, we screened a new N. caninum tachyzoite cDNA library with an affinity-purified antibody against p17 (APA17). We isolated several cDNA clones with 100% sequence identity to the NcGRA7 gene. This previously described gene encodes a dense granule protein with an apparent molecular mass of 33 kDa. A second line of evidence emerged through a combined proteomic approach associating two-dimensional PAGE (2D-PAGE) to Western blotting and to mass spectrometry to characterize the p17 fraction. Two acidic immunodominant but minority protein spots were recognized by APA17 and by bovine sera. These antigens of 17 and 33 kDa are respectively composed of 4 and 2 isoforms. Furthermore, p17 isolation by 2D-PAGE and peptide sequencing by tandem mass spectrometry yielded a partial sequence of 17 amino acids, which allowed the putative amino terminal region of the NcGRA7 protein to be identified unambiguously. The NcGRA7 protein, without the putative signal peptide at the NH2-terminus, was cloned and expressed in Escherichia coli and when the purified recombinant protein (rNcGRA7) was analysed by SDS-PAGE and mass spectrometry, 2 bands of 24 and 33 kDa were resolved and identified as NcGRA7. These results demonstrate that the immunodominant 17 kDa antigen of N. caninum is encoded by the NcGRA7 gene.

Toxoplasma gondii infection in adult llamas (Lama glama) and vicunas (Vicugnavicugna) in the Peruvian Andean region.

The present study was designed to investigate Toxoplasma gondii infection in adult llamas (Lama glama) and vicunas (Vicugna vicugna) in the Peruvian Andean region, for which to date no information has been available. Serum samples from 43 llamas (L. glama) and 200 vicunas were tested by IFAT detecting titres of 1:50 or higher in 55.8% (33.9-70.9%) and 5.5% (2.8-9.6%), respectively. IFAT titres ranged from 1:50 to 1:6400. In order to avoid cross reactions with closely related coccidian parasites and to confirm the existence of T. gondii specific antibodies, IFAT positive sera from both ruminant species were also analysed by western blot. T. gondii specific antigens were recognised by IFAT positive sera, although different IFAT cut-off points could be selected for llamas (1:200) and vicunas (1:50) meaning seroprevalence of 44.2% (29.1-60.1%) and 5.5% (2.8-9.6%), respectively. Based on the frequency and intensity of tachyzoite antigen recognition, at least three immunodominant antigens with apparent molecular weights of 22-24, 30, and 38-40 kDa were detected, together with other minor protein fractions located in the 18-73 kDa range. This study documents for the first time the presence of T. gondii infection and reports the target T. gondii antigens in adult llamas and vicunas in Peru.

Pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses.

Different aspects of Neospora tachyzoite antigen recognition by Neospora-infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora-antigen recognition was studied in Neospora-infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy. Based on the frequency and intensity of recognition, four IDAs-17-18, 34-35, 37 and 60-62kDa antigens-have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17-18kDa antigen. We emphasize the important role that the 17-18kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.

Rotavirus and concurrent infections with other enteropathogens in neonatal diarrheic dairy calves in Spain.

Faeces samples from 218, one to 30 days old, diarrheic dairy calves in 65 dairy herds were screened for the presence of rotavirus and concurrent infections with coronavirus, Cryptosporidium, F5+ Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1-7, 8-14, 15-21 and 22-30 days. Rotavirus infection was detected in 46.9%, 45.6%, 33.8% and 48.3% of the calves in the respective age-groups. No significant differences in the detection rate of rotavirus were found among calves on the different age-groups. Rotavirus was the only enteropathogen detected in 39 of the 93 (41.9%) diarrheic calves positive to this agent. Concurrent infections with other enteropathogen(s) were detected in 31.3%, 33.3%, 20.6% and 3.4% of the rotavirus infected calves in the age-groups 1-7, 8-14, 15-21 and 22-30 d, respectively. A significant age-associated decrease in the detection rate of mixed infections (p < 0.01) was found. The detection rates of the other enteropathogens considered in calves with rotavirus infection were 20.4% for coronavirus, 85.2% for Cryptosporidium, 16.7% for F5+ E. coli and 1.8% for Salmonella.

Detection of infectious Cryptosporidium parvum oocysts in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule).

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10(3) oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.

Pathological alterations caused by Anoplocephala perfoliata infection in the ileocaecal junction of equids.

The pathological alterations caused by Anoplocephala perfoliata in the ileocaecal junction of 28 equids slaughtered in an abattoir in Madrid (Central Spain) are described. The lesions were scored in grades based on the intensity of the damage and were related to the tapeworm number observed. The first grade (grade I) of alterations consisted of a slight enteritis associated with focal erosions observed in 43% of parasitized animals with low parasitic burden (1-26 tapeworms). The second grade (grade II) was a focal pseudomembranous enteritis, present in the ileocaecal junctions of 36% infected animals with moderate to high burden (23-188 tapeworms), and the third grade (grade III) was a regional necrotizing enteritis, present in the animals (21%) with the highest burden (72-248 tapeworms). The possible role of the lesions caused by this parasite in the aetiology of colic is discussed.

Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain.

Faeces samples from 218, 1 to 30-day-old, diarrheic dairy calves in 65 dairy herds were screened for the presence of Cryptosporidium and concurrent infections with rotavirus, coronavirus, F5 Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1-7, 8-14, 15-21 and 22-30 days. Cryptosporidium infection was detected in 43.8%, 71.9%, 63.2% and 6.9% of the calves in the respective age groups. Significant differences in the detection rate of Cryptosporidium were found between the age group 22-30 days and all other age groups, and between the age group 1-7 days and the age groups 8-14 days and 15-21 days. Cryptosporidium was the only enteropathogen detected in 60 of the 114 (52.6%) diarrheic calves. Concurrent infections with other enteropathogen(s) were detected in 64.3%, 46.3%, 39.5% and 0% of the Cryptosporidium-infected calves in the age groups 1-7, 8-14, 15-21 and 22-30 days, respectively. A significant age-associated decrease in the detection rate of mixed infections (p < 0.05) was found. The detection rates of the other enteropathogens considered in calves with Cryptosporidium infection were 87% for rotavirus, 11.1% for coronavirus, 27.8% for F5+ E. coli and 1.8% for Salmonella.

Proportional morbidity rates of enteropathogens among diarrheic dairy calves in central Spain.

Faecal samples from 218 diarrheic dairy calves in 65 dairy herds, selected by convenience, were screened for the presence of rotavirus, coronavirus, Cryptosporidium spp., F5+ Escherichia coli and Salmonella spp. Animals surveyed were from 1 to 30 days old. Cryptosporidium and rotavirus were the most commonly detected agents (52.3% and 42.7% of the samples positive, respectively). F5+ E. coli was detected in the faeces of 11.9% of the calves and bovine coronavirus was detected in the faeces of 7.3% of the calves. Salmonella spp. was only found in the faeces of two calves (0.9%). Mixed infections with two or more agents occurred in 28% of the calves. Concurrent infection of rotavirus and Cryptosporidium was found in 21.6% of the calves. Two tests were used for the detection of rotavirus (a commercial ELISA and PAGE), F5+ E. coli (ELISA and bacterial culture) and Cryptosporidium (ELISA and microscopy). The validity of the commercial ELISA for the detection of rotavirus, F5+ E. coli and Cryptosporidium in faeces from diarrheic calves was evaluated using PAGE, bacterial culture and microscopy as gold standard, respectively. The ELISA showed a very low sensitivity (28.6%) for the detection of F5+ E. coli compared to bacterial culture.

Reliability of coprological diagnosis of Anoplocephala perfoliata infection.

Three coprological methods were tested to establish the reliability of in vivo diagnosis of Anoplocephala perfoliata. A total of 107 faecal samples were analyzed, and the presence of tapeworms were confirmed postmortem in 24 animals with burdens that ranged from 1 to 248 worms; most of them (71%) with less than 100 parasites. Best results were obtained with a combination of two sedimentation/flotation methods, detecting only half the parasitized animals (54% sensitivity). No relationship could be established between tapeworm burden and egg detection, but results indicate that coprological methods have a lower likelihood of diagnosing cestode infection when horses have less than 100 tapeworms.

Coccidial infection in mouflon, Ovis musimon, in central Spain.

From February to September 1993, ten adult female mouflons (Ovis musimon) and their five offspring from central Spain were examined weekly for coccidial infection. All adult mouflons had Eimeria spp. infections with mean (+/- SD) intensity of 1,869 (+/- 1,264) oocysts per gram of feces the day of capture, increasing progressively during the first two months in captivity and later returning to the initial values (1,869 +/- 1,547). The mean (+/- SD) oocyst shedding in young animals was 16,800 (+/- 966) oocysts per gram at 1 mo and 18,796 (+/- 1,220) at 1.5 mo of age and more than 40,000 (40,250 to 52,000) at 3 mo of age; this high intensity was associated with a transient diarrhea. The species involved, in order of frequency, were E. bakuensis (syn. Eimeria ovina), E. ovinoidalis, E. crandallis, E. caprovina, E. parva, E. faurei, E. granulosa and E. intricata, and one more not previously described and recorded as Eimeria sp.. The predominant species for both age groups was E. bakuensis.

Natural infection by gastrointestinal and bronchopulmonary nematodes in mouflons (Ovis musimon) and their response to netobimin treatment.

Gastrointestinal and bronchopulmonary nematode infections and the efficacy of netobimin (Hapasil) were analyzed by way of fecal examination in 10 female mouflons (Ovis musimon), in central Spain, February 1993. Before treatment all 10 mouflons had Trichostrongylus axei, Teladorsagia circumcincta and Marshallagia spp.; sic had Nematodirus spp., two had Trichuris sp., one had Capillaria sp., seven had bronchopulmonary Dictyocaulus filaria and 10 mouflons had protostrongylid lungworms (Muellerius capillaris, Protostrongylus rufescens, Cystocaulus ocreatus or Neostrongylus linearis). Netobimin (7.5 mg/kg) was 100% effective against T. axei, T. circumcincta, Marshallagia spp., and D. filaria infections whereas one animal continued eliminating Nematodirus spp. eggs. The drug also was effective against Capillaria spp. but not against Trichuris spp. or protostrongylid infections.

Seasonal availability of Fasciola hepatica metacercariae in a temperate Mediterranean area (Madrid, Spain).

In this experiment, conducted over a 3-year period (1988-1990), the seasonal availability of Fasciola hepatica metacercariae in a temperate Mediterranean area (Madrid, central Spain) was analysed according to the rhythms of snail infection, the periods of cercarial emission, and the resistance of metacercariae. In this area, snails could be infected from late April to early November. Cercarial emission by spring infection started at the beginning of summer and terminated 3-4 weeks later. Mid-summer temperatures led to the extinction of shedding populations and of the metacercariae emitted by them, but were well tolerated by the snails with undeveloped infections. No aestivation was observed and summer infection led to a progressive shedding wave from August to late December, when it was interrupted. These populations became extinguished in the colder winter of 1988, but overwintered in the mild and belated winter of 1989. Only the snails infected from late summer onwards resumed activity and shed cercariae in spring, from the second half of March to late June. Risk of infection for grazing animals during winter will depend on survival of metacercariae. With a mean mortality of 23% during December and January, and of 38% during the following 2 months, no more than 30% of metacercariae reach the spring alive. Accordingly, risk of infection for grazing animals in spring will depend on overwintering infection of snails. These results indicate that temperature more than humidity could be responsible for different transmission patterns from year to year as winter thermic profiles could be the key for the transmission of fasciolosis during spring.

The overwintering of eggs, intramolluscal stages and metacercariae of Fasciola hepatica under the temperatures of a Mediterranean area (Madrid, Spain).

The survival of embryonated and unembryonated eggs, of snails with mature or immature infection and of metacercariae over the winter of a Mediterranean area was analyzed. Embryonated eggs were more resistant than unembryonated eggs to cold weather, leaving a residual contamination which was responsible for earlier spring infection of the snail. Overwintering was similar in snail populations with both mature and immature infections although the lifespan was shorter in the former. Both snail populations survived in the mildest winter but not in the coldest. Only metacercariae from mid autumn were able to overwinter in an significant proportion (45%) but they were non-viable by mid spring. Our results suggest that in very cold winters no risk for grazing animals should be expected in spring.

Identification of Cryptosporidium parvum oocyst/sporozoite antigens recognized by infected and hyperimmune lambs.

The appearance, persistence and eventual decline of IgG, IgM and IgA antibodies recognizing Cryptosporidium parvum antigens were studied in naturally infected lambs using a Western blot technique, and the results compared with those obtained using sera from immunized lambs. There was an intense recognition of some low molecular weight proteins (15-17 kDa by IgG and IgA; 28-30 kDa by IgA, IgM and IgG) during the infection and early post-infection period. These peptides were not recognized after Days 45-60 of life. Some high molecular weight antigens (94 kDa) were weakly recognized on Day 15 but more intensely recognized from Day 30 onwards, persisting until at least Day 90. Antibody recognition of these C. parvum proteins could be an indicator of recent or past exposure to the parasite.