Development of improved diagnostics for the detection of Neospora caninum infected carrier cattle (PhD)

Two N. caninum antigens expressed as recombinant proteins (tNcSRS12A-B and tNcSRS44-A) may represent a supplementary diagnostic tool for bovine neosporosis. When associated with assays targeting tachyzoite-specific antibody responses, these tests could provide additional information particularly on those animals which tested antibody negative with tests based exclusively on tachyzoite antigens. Seropositivity with the tNcSRS12A-B and tNcSRS44-A Western Blots would suggest persistent infection whilst antibody negative results would enable to classify N. caninum seronegative cattle more confidently. This method could be applied in those field conditions in which in-depth serological investigations are required and economically justifiable. For example, it would be beneficial within the selection of breeding stock in high genetic merit herds. Further investigations are required for the development of functional ELISAs based on the antigens tNcSRS12A-B and tNcSRS44-A described in this study.

Tests based on tachyzoite antigens alone showed generally better performances than tests based on bradyzoite antigens. However, the use of tests based on bradyzoite antigens should be considered because of the evidence that a proportion of animals may display antibodies against antigens expressed by the quiescent stage of the parasite but not against the rapidly multiplying tachyzoite stage. These findings confirmed the concerns related to the potential lack of sensitivity of serological assays based exclusively on tachyzoite antigens. Such shortcoming may be overcome by carrying out multiple tests based on both N. caninum tachyzoite and bradyzoite antigens in parallel. This strategy would be particularly beneficial in those clinical scenarios in which high sensitivity is required, for example, to identify N. caninum infected cattle to cull or exclude from breeding replacement stock, or to screen prospective replacement cattle.

The cross-sectional seroprevalence study showed that N. caninum is widespread amongst British dairy herds. In addition, the data collected did not support that testing animals in late pregnancy would increase the likelihood of detecting cattle infected with N. caninum. However, further longitudinal studies are required to corroborate this finding.

The novel microsatellite markers described and evaluated in the present study were highly polymorphic and showed their potential applicability to the study of the genetic diversity of N. caninum. Importantly, the microsatellite amplification and fragment analysis protocols developed were successfully applied to DNA samples obtained from bovine aborted foetuses and placentas in which the parasite was previously detected.

The MLG offers a very useful discrimination typing tool although the analysis is highly dependent on the resolution of the markers used and sampling strategies applied. Further investigations, including the analysis of a larger number of laboratory-maintained, clinical and non-clinical (e.g. domestic and wild asymptomatic intermediate hosts) samples, are required to enable the selection of optimal markers that may contribute, in association with previously described microsatellites (Regidor-Cerrillo et al., 2006, Al-Qassab et al., 2010a), to the generation of a standardised multilodus genotypes (MLG) framework that can be used at the inter-laboratory level as previously described for other apicomplexans of human and veterinary importance such as C. parvum (Chalmers et al., 2017, Hotchkiss et al., 2015).

Refined MLG methods based on microsatellite markers would be beneficial to investigate the source of infection and infection dynamics at the herd or regional levels. In addition, they would aid the study of the association between the genetic heterogeneity of N. caninum and the diverse biological features in vitro and in vivo (i.e. virulence and pathogenicity) that are seen amongst different isolates.

An international workshop to define and universally approve the number and choice of microsatellite markers to be used for genotyping N. caninum as well as determine the genotypic characteristic of the N. caninum laboratory-maintained isolates commonly used in research would be highly advantageous.

Besides highlighting the need for more reliable serological diagnostics, the questionnaire-based survey evidenced the demand for enhanced guidance in the interpretation of test results, especially those obtained with serological assays.

Continuing professional development (CPD) programmes for large animal veterinarians may be required to update on the characteristics and limitations of the diagnostic options, help the interpretation of the outcome of different tests and standardise the management advice given to farmers. A nationwide industry stakeholder group, similar to the COWS (Control of Worms Sustainability) and the SCOPS (Sustainable Control of Parasites in Sheep) initiatives which promote best practice in the control of ruminants’ parasites, may be created for providing veterinarians and farmers with up to date evidence-based information and learning opportunities related to the diagnosis and control bovine neosporosis.

The Challenge

The protozoan parasite Neospora caninum is the most frequently diagnosed infectious cause of bovine abortion in the United Kingdom and worldwide. The cost of neosporosis, which involves not just the loss of the calf but also reduced milk production and additional breeding costs, is significant to the UK cattle industry. Since there are no effective vaccines or treatments available that can prevent abortion, transmission or cure infection, the only option is to minimise the risk of N. caninum infection at a farm level by implementing control strategies based on biosecurity and reliable diagnostics.

Diagnosis of N. caninum infection is usually made by detection of the parasite in abortion material or by revealing parasite specific antibodies in infected animals. According to the stage of infection and the effectiveness of host’s immune responses, N. caninum can be found in two different forms: the tachyzoite, characterised by rapid replication and the bradyzoite, a quiescent stage observed in persistently infected cattle. Most, if not all, currently available serologic tests utilise tachyzoite antigens to detect parasite specific antibodies. As a consequence the tests can give false negative results for persistently infected animals, when the parasite hides from the host’s immune system by differentiating into bradyzoites, which express different antigens.

The Project

The project aims to identify immunogenic stage-specific antigens that are expressed in the bradyzoite stage, in order to set up and validate an improved test able to recognise both recently and persistently infected animals. A reliable test will allow farmers to identify correctly their uninfected cattle. This is essential for breeding out N. caninum from infected herds. In addition, it will be beneficial for testing of replacement cattle, ensuring that no carrier animals are introduced into uninfected herds.

The Student

Stefano Guido