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.

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.

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.

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.

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.

The effect of the host's age on the pathology of Eimeria stiedai infection in rabbits.

The pathology of experimental Eimeria stiedai infection in 2- and 4-month-old rabbits raised coccidia-free was studied. Over 50 days of infection, the serum activities of aspartate (AST) and alanine (ALT) aminotransferases, alkaline phosphatase (AP) and gamma-glutamyltranspeptidase (GGTP) were analyzed as indicators of hepatic lesions and total serum globulins with respect to the immune response of the host; parasite development was followed by oocyst output; weight gain, food intake and relative liver weight were analyzed to control the performance of infected animals. The age of the host strongly affected parasite development and consequently the biochemical and zootechnical parameters differed more markedly in the younger animals; however, the enzyme ALT showed an increase independent of parasite development, and infection did not affect GGTP activity. The increase in age was responsible for resistance of rabbits to hepatic coccidiosis and older animals were less affected than young ones.

The acid phosphatase activity and morphological characteristics of Dipetalonema dracunculoides (Cobbold, 1870) microfilariae.

The acid phosphatase activity and some morphological characteristics of Dipetalonema dracunculoides microfilariae are described. Their morphological features are closely related to those of the pathogenic Dirofilaria immitis when Knott's technique is used for the microfilarial diagnosis. The acid phosphatase activity pattern found in Dip. dracunculoides microfilariae is clearly different from those previously described for D. immitis, D. repens and Dip. reconditum.

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.

Serum antibody response in lambs naturally and experimentally infected with Cryptosporidium parvum.

The immunoglobulin (IgG), IgM and IgA responses in Castellana-Manchega cross-bred colostrum-deprived and colostrum-fed lambs infected neonatally with Cryptosporidium parvum were measured using an enzyme-linked immunosorbent assay. A comparison of oocyst shedding and anti-C. parvum serum IgG levels in lambs suffering either natural or experimental infection was undertaken. Effects on the oocyst shedding and IgG levels of C. parvum rechallenge at 30 and 120 days of age in neonatally infected lambs were also evaluated. Anti-C. parvum immunoglobulin levels in colostrum-deprived animals peaked on Day 30 of life for IgG and on Day 15 for IgM and IgA. Lambs that received maternal colostrum showed elevated anti-C. parvum IgG, IgM and IgA levels at 3 days old indicating a transfer of colostral immunoglobulins. Experimentally infected lambs showed a IgG response similar to naturally infected lambs, suggesting that the serum IgG response is independent of the infective dose. Finally, lambs rechallenged at 30 and 120 days old did not show either appreciable oocyst shedding or any increase in their anti-C. parvum IgG levels when compared with prechallenged animals.

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.

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.

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.

Chemotherapy and chemoprophylaxis of hepatic coccidiosis with sulphadimethoxine and pyrimethamine.

The activity of a mixture of sulphadimethoxine and pyrimethamine (10:3) as prophylactic medication and prophylactic and therapeutic medication was studied in rabbits experimentally infected with Eimeria stiedai. The haematocrit index (packed cell volume) and haemoglobin levels were studied for assessment of drug toxicity. The activity in serum of aspartate aminotransferase (AST) and alanine aminotransferase, alkaline phosphatase and gamma-glutamyltranspeptidase were studied as indicators of hepatic lesions. Parasite development was followed on the basis of the presence of oocysts; other parameters were analysed in order to monitor the performance of infected animals. All the parameters studied showed that the chemoprophylactic medication provided efficient control of the infection and of the hepatic lesions. Serum AST activity was seen to be a good indicator of the effect of the drugs on the liver.

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.

Pharmacokinetics, hepatic biotransformation and biliary and urinary excretion of bromosulfophthalein (BSP) in an experimental liver disease mimicking biliary cirrhosis.

We studied the hepatic handling of bromosulfophthalein in healthy rabbits with hepatic coccidiosis 28 days after an experimental infection with sporulated oocysts of Eimeria stiedai, an experimental model of liver disease histopathologically resembling primary biliary cirrhosis in man. A pharmacokinetic study of the results was performed following a multicompartmental model with 7 transfer constants to describe the physiological disposition of the dye. The study showed that the plasma disappearance, distribution volume (Vi), hepatic biotransformation and the biliary and urinary elimination of conjugated (BSPc) and unconjugated (BSPu) bromosulfophthalein were markedly altered. Whereas Vi and urinary excretion of the dye were significantly increased, the hepatic clearance, biotransformation and biliary excretion of BSPc and BSPu were drastically reduced in infected rabbits. Satisfactory agreement was obtained between the experimental and estimated data, particularly those relating to biotransformation clearance and biliary and urinary excretion of the dye. These results demonstrate that severe liver disease in rabbits with histopathological liver alterations resembling several hepatic dysfunctions in man markedly reduce hepatic uptake, metabolism and biliary excretion of a xenobiotic such as BSP.