V. Risco-Castillo

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 parasites 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.

Development and use of an indirect ELISA in an outbreak of bovine besnoitiosis in Spain

An indirect ELISA based on a soluble extract of Besnoitia besnoiti tachyzoites was developed and standardised. A set of positive and negative reference bovine sera were characterised using an immunofluorescence antibody test and Western blot. A cut-off with a relative index per cent of 8.1 was determined for equal sensitivity and specificity (100 per cent) by twograph receiver operating characteristic analysis. Cross-reactions with other closely related Apicomplexan parasites were discarded. The standardised ELISA was then used during an outbreak of bovine besnoitiosis in a mountainous area of central Spain. The outbreak occurred in nine herds, and 358 animals that shared grazing lands during the summer season were affected. Clinical examination and blood sampling were carried out for all animals, and skin biopsies were obtained from animals with skin lesions. The confirmatory diagnosis was carried out by means of the indirect ELISA, together with the identification of tissue cysts by microscopy. Most of the animals were seropositive (90·5 per cent), but only 43 per cent of seropositive cattle developed clinical signs compatible with besnoitiosis. Additionally, a significant increase in seroprevalence and clinical signs was found to be associated with the increasing age of the animals, suggesting rapid horizontal transmission of the disease.

First Isolation of Besnoitia besnoiti from a Chronically Infected Cow in Spain

Abstract 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 (ITS1), 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.

Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy

The Nc-Spain 1H isolate of Neospora caninum, which was newly obtained from the brain of a congenitally asymptomatic infected calf, demonstrated a reduced in vitro tachyzoite yield and viability rate, as well as low virulence in mouse models. The objective of the present study was to determine the ability of this isolate to induce foetal death in a pregnant bovine model. For this purpose, 13 naïve pregnant heifers were divided into three groups and were experimentally challenged with either 107 tachyzoites of Nc-1 (group 1, n = 5), Nc-Spain 1H (group 2, n = 5) isolates or phosphate-buffered saline (group 3, n = 3) intravenously at 70 days of gestation. After inoculation, pregnancy was monitored and dams were sacrificed when foetal death was detected. The remaining animals were slaughtered at 45 days post-infection. Maternal and foetal samples were collected for examination by histology and parasite DNA detection. Parasitaemia, specific anti-N. caninum IgG and interferon γ responses were also studied. At 3–4 weeks after infection, foetal death was detected in 3 out of 5 Nc-1-infected dams. However, no evidence of foetal death was observed in either Nc-Spain 1H-infected or control groups during the period studied. The most severe histopathological lesions were observed in the placenta and foetal organs from Nc-1-infected cattle that exhibited foetal death. It was in these animals that N. caninum DNA was more frequently detected. Parasitaemia was observed in all Nc-1-infected dams and in only 3 out of 5 Nc-Spain 1H-infected animals. The magnitude of the immune response was significantly higher in the Nc-1-inoculated group than in the group inoculated with the Nc-Spain 1H isolate. These data reveal the reduced virulence of the Nc-Spain 1H isolate in cattle.

Usefulness of rNcGRA7-and rNcSAG4-based ELISA tests for distinguishing primo-infection, recrudescence, and chronic bovine neosporosis

Bovine reproductive failure caused by the parasite Neospora caninum is a major problem and is responsible for severe economic losses worldwide. Currently, appropriate control measures depend on the predominant transmission route in a particular herd. Therefore, the development of diagnostic tools capable of discriminating between primo-infection, recrudescence, re-infection, and chronic infection is a major challenge in the serodiagnosis of bovine neosporosis. Here, two recombinant protein-based ELISAs utilizing the immunodominant NcGRA7 dense granule protein and the NcSAG4 bradyzoite stage-specific protein were developed and showed good diagnostic performances. Their usefulness for discerning between primo-infection, recrudescence, re-infection, and chronic infection was also studied by analyzing an appropriate panel of serum samples belonging to different groups of experimentally and naturally infected bovines. Our results suggest that anti-rNcGRA7 antibody levels may be indicative of acute infection (primo-infection, re-infection, and recrudescence), whereas the presence of anti-rNcSAG4 antibodies may be associated with chronic infection and could be a good indicator of infection establishment (tachyzoite-bradyzoite conversion). Moreover, primo-infection associated with a Neospora-associated epidemic abortion pattern is characterized by the detection of anti-rNcGRA7 antibodies together with the absence or detection of anti-rNcSAG4 antibody levels around the cut-off point. In contrast, the detection of antibody levels directed against both recombinant proteins may be quite indicative of recrudescence or re-infection associated with abortion and/or vertical transmission in herds with a Neospora-associated endemic abortion pattern. In conclusion, both serological tests developed in the present study offer additional information to conventional avidity tests and, consequently, improve the diagnosis of bovine neosporosis with perspectives for control measures.

Comparative analysis of stress agents in a simplified in vitro system of Neospora caninum bradyzoite production.

Neospora caninum is an apicomplexan parasite identified as a major cause of abortion in cattle and neurological disease in various animal species. It is closely related to Toxoplasma gondii, sharing the ability to persist indefinitely in latent stage within the host as a tissue cyst containing slow-dividing bradyzoites. In this study, we compared different stress methods to induce in vitro bradyzoite conversion, using MARC-145 cells infected with Nc-Liverpool isolate. The tachyzoite-to-bradyzoite conversion rate was monitored at days 3, 5, and 7 after stress in a double-immunofluorescence assay using a monoclonal antibody against the tachyzoite antigen SAG1 (αSAG1) and a rabbit serum directed to the intracytoplasmic bradyzoite antigen BAG1 (αBAG1). Seven days of treatment with 70 μM sodium nitroprusside offered the highest bradyzoite transformation rate and the best yield of total parasitophorous vacuoles observed. In the present work, we introduce an alternative, simplified, and more advantageous method for bradyzoite production of N. caninum, using a reliable cell culture system easy to handle and with promising capacity of parasite purification.

Failure of a vaccine using immunogenic recombinant proteins rNcSAG4 and rNcGRA7 against neosporosis in mice.

The development of an effective vaccine against Neospora caninum infection in cattle is an important issue due to the significant economic impact of this parasitic disease worldwide. In this work, the immune response, safety and efficacy of different vaccine formulations using the N. caninum recombinant proteins rNcSAG4 (the first bradyzoite-specific protein assayed as a vaccine) and rNcGRA7 were evaluated in mouse models. The survival curves of pups from all vaccinated groups showed a slight delay in time to death compared to control groups; this difference was statistically significant for rNcSAG4+adjuvant group. Immune response of mice vaccinated with rNcSAG4 was characterized by reduced specific IgG and cytokine levels with an equilibrated IFN-gamma/IL-10 balance. Regarding mice vaccinated with rNcGRA7, a very strong humoral and cellular immune response was generated characterized by a hyper-production of IFN-gamma. This response was not accompanied by significant protection. Vaccination with a mixture of both recombinant proteins reduced infection in lung and brain during acute and chronic infection, respectively, although it was not statistically significant. In summary, no significant protection was obtained with these vaccine formulations in the present mouse models. However, the study reveals some positive results on immune response and efficacy for both recombinant proteins; these results are being discussed in order to suggest new approaches with new chronic infection mouse models and adjuvants.

Comparative effect of Neospora caninum infection in BALB/c mice at three different gestation periods

Neospora caninum has been recognized as a major cause of infectious bovine abortion worldwide. In the present study, the effect of N. caninum infection in mice at the 3 gestation periods (first, second, and third period) was investigated. In dams, tissue distribution of N. caninum was evaluated by nested polymerase chain reaction. In the progeny, fetal mortality, stillbirth, litter size, neonatal mortality/morbidity, vertical transmission, and parasite burden in neonatal tissues were evaluated. Pregnant BALB/c mice were infected subcutaneously with 2 × 106 NC-1 tachyzoites on days 0, 7, or 14 of gestation. Dams from each group were sequentially killed during gestation and postpartum (PP). Pups were killed on days 1 and 7 PP. Infection on day 0 of gestation produced a high vertical transmission rate, although no changes in fetal mortality, stillbirth, and littermate size were observed. The highest level of vertical transmission, together with an increase in fetal mortality and stillbirth and a decrease in litter size, were observed when infection was done on day 7 of gestation. Finally, infection on day 14 of gestation produced the lowest vertical transmission. Furthermore, infection at any time during gestation compromised the postnatal development of pups, because neonates from infected dams showed less body weight and a delay in the hair development.

Pattern of recognition of Besnoitia besnoiti tachyzoite and bradyzoite antigens by naturally infected cattle.

Bovine besnoitiosis is caused by the protozoan parasite Besnoitia besnoiti. Many recent cases have been described in different European countries, which may be indicative of expansion of the disease in the next few years. Many infected animals remain asymptomatic; therefore, serological tests are essential tools for diagnosis. The objective of the present work was to identify B. besnoiti tachyzoite and bradyzoite immunodominant antigens (IDAs). IDAs were recognised by SDS-PAGE under reducing conditions and Western blot analysis. Positive sera from symptomatic (n=18) and asymptomatic (n=18) cattle came from herds endemically infected by B. besnoiti and were confirmed positive by IFAT, whereas negative sera (n=4) came from besnoitiosis-free herds and were also confirmed negative by IFAT. Up to 28 tachyzoite antigens in the range of 8.5-190.8 kDa were recognised. Based on the frequency of recognition, six IDAs (14.2, 33, 37.1, 39.6, 46.3 and 190.8 kDa) were identified. The 37.1 kDa antigen was recognised by 100% of sera, usually as an intense band. On the other hand, 30 bradyzoite antigens in the range of 8.5-187.9kDa were detected. Seven bradyzoite IDAs (8.5, 15.1, 16.8, 19.0, 34.7, 38.6 and 124.4 kDa) were identified and two of them (15.1 and 16.8 kDa) were considered the most immunogenic ones. Additionally, sera from animals with clinical symptoms recognised a significantly higher number of bradyzoite antigens. Finally, significant cross-reactions with other closely related apicomplexan parasites were not detected. This is the first description of B. besnoiti bradyzoite antigens. In addition, the identification of tachyzoite and bradyzoite IDAs may be useful for the development of vaccines and diagnostic tools for differentiating between acute and chronic infections. Further proteomic studies are needed in order to identify stage-specific proteins.

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.