A. Fernández-García

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.

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.

Diagnosis of bovine neosporosis: Recent advances and perspectives

Neospora caninum is considered a major cause of abortion in cattle. Appropriate techniques for diagnosis of bovine neosporosis, both in vivo and in aborted foetuses, have been developed in the last ten years and some of them are commercially available. For diagnosis in live animals, detection of antibodies in serum or milk has been shown to be the best option both at the herd and the individual level. These techniques are excellent tools to examine N. caninum-associated abortion problems and to adopt some basic herd-control measures. Concerning foetal diagnosis, detection of compatible lesions by histological examination and parasites by PCR in brain (as well as heart and liver) are the best choices. Diagnostic criteria to distinguish foetal infection and Neospora-associated abortion are based not only on the demonstration of the parasite in the foetus but also on the extent and severity of the lesions in the foetus, foetal age and the assessment of neosporosis at the herd level. In the near future, new tools to diagnose infection should help to detect animals with parasite reactivation by testing the immune response to stage-specific antigens and lead to the development of molecular typing methods to characterise different parasite isolates. Finally, uniform diagnostic procedures need to be established between laboratories and countries in order to standardise result interpretation. The role of National or Regional Reference Laboratories is essential in countries or regions where control programmes for the disease are being developed.

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.

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.

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.

Stage-specific expression of NcSAG4 as a marker of chronic Neospora caninum infection in a mouse model

Neospora caninum infection persists throughout the life of its intermediate host due to the conversion of tachyzoites to slowly dividing bradyzoites that encyst in the brain. This event results in persistent N. caninum infection in bovine herds and partially explains the poor efficacy of many chemotherapeutic agents and vaccine formulations. Thus, there is a need for greater understanding of the tachyzoite-to-bradyzoite conversion mechanisms. Here we studied for the first time the transcription kinetics of the N. caninum bradyzoite-specific gene NcSAG4 in brain samples from chronically infected mice by means of real-time RT-PCR. NcSAG4-messenger RNA (mRNA) levels increased significantly during the chronic phase but followed 2 different expression patterns depending on the isolate used for murine inoculation. NcSAG4-mRNA levels in brains from Nc-1-inoculated mice peaked during late chronic infection (on day 64 post-infection, p.i.), whereas those from Nc-Liv-inoculated mice peaked earlier during the chronic infection (on day 32 p.i.). This difference could be a reflection of the different abilities of these isolates to replicate and form cysts in parasitized brains. These results are consistent with our observations of anti-rNcSAG4 antibody production; low levels were present at seroconversion and slowly increased during the chronic phase. In contrast, NcSAG1 transcription levels, which mark the tachyzoite stage, were maintained without variation in both groups of mice. This suggests the presence of a significant amount of tachyzoites or intermediate zoites expressing NcSAG1 in the brain, even during the late chronic infection.

Seroprevalence of Besnoitia besnoiti infection and associated risk factors in cattle from an endemic region in Europe.

Bovine besnoitiosis, caused by the parasite Besnoitia besnoiti, is a chronic, debilitating disease with both cutaneous and systemic clinical signs that has re-emerged in Europe. This is the first random cross-sectional prevalence study of B. besnoiti infection in cattle carried out in an endemic area in Europe (Navarra, Spain). Dairy (n = 372) and beef (n = 340) cattle >1 year of age were randomly blood sampled. Serum was evaluated using a validated ELISA. True animal prevalence data were restricted to beef cattle (16.0%). The prevalence significantly increased with age and seropositive animals were mostly located in mountainous areas where the disease is endemic. Breed and sex were not found to be risk factors.

Identification of a gene cluster for cell-surface genes of the SRS superfamily in Neospora caninum and characterization of the novel SRS9 gene

Here we present the detection of a gene cluster for Neospora caninum surface genes, similar to the Toxoplasma gondii SRS9 locus, and the cloning and characterization of the NcSRS9 gene. PCR genome walking, using NcBSR4 gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to the SRS5 and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtained in vitro and RT-PCR analysis showed no significant variations of NcSRS9 transcripts during in vitro tachyzoite-bradyzoite switch, probably due to incomplete maturity of in vitro bradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding of N. caninum persistence.