Protozoal Reproduction Losses in Farm Ruminants

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A. BACKGROUND

Infectious organisms can cause significant losses in farm ruminant production as a result of abortion, embryonic damage or maternal infertility. The two principal agents causing protozoal abortion in ruminants are Neospora caninum in cattle and Toxoplasma gondii in sheep and goats. Both pathogens are closely related. Tritrichomonas foetus is a serious cause of cattle infertility. Furthermore, parasites of the genus Sarcocystis are widely distributed and may inflict infections affecting the reproductive tract of ruminants, which also may result in abortion.

Abortion in ruminants may pose a very considerable public health risk as many of the pathogens that cause disease in ruminants may pose a significant danger to humans. Thus rapid, accurate diagnosis is vital in order to be able to assess the degree of risk caused by potential ruminant abortifacients with zoonotic potential such as T. gondii, Chlamydophila abortus (Chlamydia psittaci), Coxiella burnetii, Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Brucella spp.. While N. caninum and Sarcocystis spp. are not currently considered to be zoonotic, rapid accurate diagnostic methods for protozoal causes are essential to rule in or out more dangerous pathogens, to allow meaningful risk assessments.

Neospora caninum is an important cause of infectious abortion and stillbirth in cattle world-wide. Infection is common and may frequently be passed from mother to calf (vertical transmission) with no signs of disease. Disease occurs when the parasite multiplies in the developing calf and its placenta and causes sufficient damage to trigger abortion or stillbirth. Preliminary research suggests that infection of the foetus early in gestation is more likely to be fatal to the conceptus than infection later in gestation. However, it also appears that infection is more likely to be transmitted in

late rather than early pregnancy. Thus, the majority of infections are not fatal and in this way inapparent infections are maintained in a herd. Vertical transmission is the major route of transmission, but it is likely that oocysts of N. caninum, produced by dogs and excreted in their faeces, may also infect cattle if they ingest contaminated food or water (horizontal transmission). Control of bovine neosporosis is difficult. Pharmaceutical preparations are known that will kill Neospora, but their use to control infection/disease in cattle have not been sufficiently evaluated yet, and no effective vaccine is currently available. Control measures therefore rely on applying certain management strategies, which are only partially satisfactory. Current knowledge suggests, that Neospora does not cause disease in human beings.

Toxoplasma gondii is an important zoonotic infection as well as being a major cause of abortion in sheep and goats. The majority of cases of human toxoplasmosis follow the consumption of uncooked or lightly cooked meat (sheep, goat, pig, cattle, deer). There is also an added risk from drinking unpasteurised goats milk as well as from the ingestion of fruit and vegetables contaminated with soil containing Toxoplasma oocysts. Control is very difficult as it depends on preventing a primary infection in a pregnant sheep or goat. While certain management procedures may reduce the risk, elimination is not possible. A live commercial vaccine - for use in sheep - is sold in some EU member states. Use of a vaccine sufficiently effective to protect against abortion in sheep and goats would be considered very likely to reduce, if not prevent, the development of tissue cysts in muscle. This would make the meat (and milk) very much safer for human consumption. However, this is a neglected area of research.

Tritrichomonas foetus, a venereally transmitted bovine infection, is an important cause of pregnancy loss and abortion in naturally bred cattle throughout the world. Trichomonosis has been a list B disease by OIE classification for many years and is therefore the subject of animals disease control in several countries. Since the introduction of artificial insemination the economic importance of this disease has decreased, but there is evidence of a re-emergence in extensive husbandry in some European states. As T. foetus may have spread inapparently also among other livestock animals such as pigs, a basic reconsideration of the epidemiological situation in Europe and the classification of virulence and pathogenicity among Tritrichomonas isolates is strongly required.

To limit the impact of protozoal reproduction failure in farm ruminants, research into the biochemistry, biology and genetics of these protozoa is urgently required to answer questions relevant for diagnosis and control. This, combined with a practical understanding of the immune responses of sheep, goats and cattle to these organisms will provide some understanding of important differences in the pathogenesis of the infections. For instance, such studies may be able to answer the question why do sheep not abort more than once with T. gondii, while cattle may pass N. caninum infection to their offspring in subsequent pregnancies, sometimes with no clinical effect and sometimes with fatal effect. Diagnosis of protozoal abortifacients is not always easy and the diagnostic criteria may vary from lab to lab. Standard procedures and parameters need to be set between labs and different countries so that epidemiological data can be improved and consolidated. The knowledge of risk factors for infection needs to be increased (e.g. presence of dogs or cats, which may shed infectious Neospora or Toxoplasma oocysts in their faeces, lactogenic infection, seasonality, factors causing recrudescence in bovine neosporosis, a-symptomatic carriers of T. foetus within or outside the ruminant host range etc). On the basis of a quantitative assessment of the consequences of protozoal reproduction failure it will be possible to establish models for the estimation of the economic losses and to perform cost-benefit analyses for potential control programmes. In this way management procedures to reduce the impact of disease can be developed. The ultimate goal is to enable the development of control measures including vaccine strategies for these diseases.

With a move to more extensive systems of agriculture the risk of protozoal abortion in ruminants would remain and perhaps attain greater significance. In the case of toxoplasmosis in sheep and goats, infection is picked up from contaminated grass, hay and water as well as from concentrated loose feed. It occurs just as commonly in extensive as in intensive farming systems. With bovine neosporosis evidence is accumulating to indicate, that the incidence of abortion is exacerbated by stress. In some situations this may occur with more intensive farming methods (such as the feedlot systems encountered in California), while in other cases stress may occur in extensive systems of agriculture due to severe environmental conditions, such as caused by extremes of weather. Cases of fatal sarcocystiosis occur more frequently when extensively reared animals are moved to grassland nearer the farm or to locations visited by people, due to contamination of the ground by dog and cat faeces.

Despite considerable scientific efforts currently being undertaken in several European countries with these infections, no formal framework exists at the European level to permit an ongoing, structured discussion process in this rapidly evolving field. COST provides an excellent basis for such a co-operation, particularly because it allows the inclusion of groups from countries outside the European Union. This has been demonstrated for the particular aspect of vaccines against animal coccidioses in COST 820, where a Working Group on sarcocystiosis, toxoplasmosis and neosporosis existed.

Relationship to the activities of COST 820 and relevant conclusions

While COST 820 ("Vaccines against Animal Coccidioses") originally focused on parasites of the genus Eimeria and Cryptosporidium (mainly parasites of poultry, ruminants, pigs and rabbits), the so-called cyst-forming coccidia (Toxoplasma gondii, Sarcocystis spp., Neospora caninum) gained considerable attention during the course of the Action, which was mainly due to the fact, that N. caninum had emerged as one of the most frequently diagnosed causes of abortion in cattle.

B. OBJECTIVES AND BENEFITS

The main objective of the Action is to develop strategies to control reproductive diseases caused by protozoa in farm ruminants.

The following main topics will be covered:

1.Biology biochemistry and genetic background of virulence
- Study biological and biochemical key factors determining infection and disease
-Characterise parasite surface molecules that interact with the host cell
- Characterise the interplay of metabolic products of the parasite with the host cell
- Understand parasite differentiation from tachyzoite to bradyzoite, and vice versa (in case of N. caninum, T. gondii and Sarcocystis spp.).
Determine the genetic background of virulence

2.Pathogenesis and host immune responses
- Define and compare the development of pathology in the different animal hosts
- Determine the relative role of the immune responses of the dam and the foetus and their effect on infection, transmission and disease (in the case of N. caninum, T. gondii and Sarcocystis spp.)
- Determine the relative role of the immune responses of the dam in pathology or to control or prevent mucosal T. foetus infection
- Identify protective and diagnostic antigens

3.Diagnosis
- Identify and improve tools to diagnose infection and/or disease
- Standardise diagnostic techniques

4.Epidemiology economics and control
- Define the prevalence and/or incidence of infection or disease
- Identify risk factors
- Calculate the economic losses on farm ruminant production
- Develop cost-effective preventive and therapeutic control measures

These topics (1-4) will also become the titles of the working groups.

C. SCIENTIFIC PROGRAMME

1.Biology biochemistry and genetic background of virulence

1.1.Biology and biochemistry of Neospora

Core studies will focus on host cell infection by N. caninum and subsequent pathogenesis. Three lines of investigation will be pursued:

(a)Tachyzoite surface and secretory molecules are important in host cell adhesion and invasion, in which parasite ligand(s) interact with host cell receptor(s). Study of both protein antigens and the less immunogenic carbohydrate-based molecules and the molecular characterisation of these receptor-ligand systems will inform the development of vaccines against Neospora and related parasites.

(b) Certain important parasite metabolites modulate host physiology and/or immunological responses. These survival strategies evolved by N. caninum need to be identified as they are thought to influence the host cytokine and chemokine pattern with regard to Th-cell polarisation.

(c)Stage conversion is vital for parasite development and transmission, thus mechanisms leading to gamogony and oocyst/sporozoite formation play a crucial role in horizontal transmission. Knowledge of tachyzoite-bradyzoite stage conversion will also help to explain persistent infection in bovine neosporosis, ovine toxoplasmosis and sarcocystiosis, and illuminate why recrudescence does or does not occur during or outside pregnancy.

1.2.Genetic background and factors of virulence in all parasites

1.2.1. N. caninum and T. gondii

In neosporosis a key question is how abortion and recrudescence of infection are triggered. Differences in parasite strains, subspecies or taxonomic variants, which may exhibit different degrees of virulence, could be important. Parasite isolates obtained from field studies and related protozoa (e.g. Hammondia heydorni) will thus be compared at the genetic level for potential virulence factors such as genes coding for proteases, surface membrane proteins, and secretory products. Comparisons will be made between bovine neosporosis and ovine and caprine toxoplasmosis. Recrudescence does not feature significantly in the pathogenesis of the latter, although it is a crucial factor in AIDS patients, when it is often fatal.

1.2.2. T.foetus

Besides T. foetus in cattle, T. suis is found in swine in Europe. Both have similar cultural and ultrastructural properties as well as resembling physiological and antigenic characteristics. Cross-infection experiments suggest little difference in host-specificity of T. foetus and T. suis. Thus the validity of defining tritrichomonads from cattle and swine as separate species is in doubt and recent molecular findings support this conclusion. The relationship between T. foetus, T. suis and other tritrichomonads raises not only a taxonomic question, but one of virulence, as demonstrated by different cross-infecting tritrichomonads isolated from cattle, swine or other farm animals. The research plan focuses on parasite molecules central to the pathogenesis of T. foetus and other tritrichomonads, so as to identify vaccine candidates.

2.Pathogenesis and host immune responses

In ruminants foetal immune competence develops from very low levels in early gestation to a good ability to control both T. gondii and N. caninum at the time of birth. Thus the stage of gestation at which the placenta and the foetus are challenged by these parasites is a crucial factor in the survival of the foetus.

Toxoplasmosis. Models of infection and disease have been developed for ovine toxoplasmosis in pregnant and non-pregnant sheep and mice, but recent exciting advances in the understanding of the parasite and ovine immunology now permit further precise questions to be addressed. The results from studies in sheep will be compared with those from goats. While sheep and goats may transmit infection in the milk, goats may also abort due to toxoplasmosis on more than one occasion. The reasons for these subtle but important differences will be used to inform improved management as well as vaccine development.

Neosporosis. Over the next few years both non-pregnant and pregnant bovine models of Neospora infection and disease will be developed to answer how and when, in pregnancy, Neospora is transmitted to the foetus and whether it is possible to induce protective immunity in a cow to prevent this transmission. The local immune response to both live and killed N. caninum as well as selected antigens will be examined by means of lymphatic cannulations in non-pregnant cattle.

Sarcocystiosis. Sarcocystis species are ubiquitous but their role in causing abortion is still unclear. It has been suggested that infection is maintained by vertical transmission and that the parasite may cause abortion, but conclusive evidence is lacking. Understanding the differences between Sarcocystis spp., T. gondii and N. caninum will point to important disease mechanisms, which in turn will aid the formulation of novel control strategies.

Tritrichomonosis. In adult cattle, T. foetus dwells mainly on the surface of mucosal tissues, where in female cattle it causes inflammation. The mechanisms of invasion used by T. foetus and subsequent pathogenesis have not yet been elucidated in detail, but they appear to rely on : (i) adherence, (ii) the production of hydrolases, and (iii) iron acquisition. Assessing these factors will contribute to the understanding of pathogenesis and will provide the basis for novel diagnostic tools able to discriminate between virulent and non-virulent strains.

Maternal and foetal immune responses during neosporosis and toxoplasmosis. In vitro, specific antibodies able to neutralise infection or opsonise parasites for uptake by macrophages will be measured and the effect of T-cell derived cytokines on parasite growth determined. The cytotoxic effects of CD4+ and CD8+ T-cells for parasite-infected target cells will be assessed. Experiments in mice will be used to indicate, which critical immune responses are involved in protection against the parasites and in ruminant models these will be closely monitored in "protected "vs" non-protected" animals.

During pregnancy, immuno-modulation occurs, which allows the mother to carry what is essentially a foreign graft. At the interface between placenta and foetus inflammatory responses, such as those induced by IFN(which inhibits T. gondii and N. caninum), are potentially very damaging and may cause rejection or abortion of the foetus directly. In human pregnancy foetal trophoblast cells produce the cytokine IL-10 that floods the maternal immune response locally. IL-10 suppresses production of IFN(and may result in the mother being less able to control infections such as T. gondii and N. caninum whilst pregnant. Thus IFN( and certain other pro-inflammatory cytokines, important in protection against the parasites, may compromise the pregnancy. This hypothesis will be examined in non-pregnant and pregnant animals.

Maternal immune responses during tritrichomonosis: In infected heifers a nonsuppurative endometritis with nodular lymphoid aggregates develops. The latter may represent sites of local IgA production in response to T. foetus, associated with its clearance. Bovine IgG2 may also be involved in extracellular cysteine proteinase cleavage, which in turn reduces the severity of the pathology of infection. Bovine antibody and complement are also involved in the neutrophil-mediated killing of T. foetus and this knowledge will be used to guide identification of protective antigens for trial mucosal vaccines.

3.Diagnosis

Two main problems will be targeted in this particular area: firstly, the diagnostic criteria and techniques in the aborted ruminant foetus and, secondly, the diagnosis of infection and/or disease in the live animal, including the development of new diagnostic tools for detection of diseased animals. In protozoa with a heteroxeneous life cycle, parallel research into the diagnosis of infection in the definitive host will be undertaken. These problems will be approached by the development of two main lines of research:

The standardisation of available diagnostic techniques. Standard procedures and parameters for protozoal abortion in ruminants need to be set between laboratories and countries in order to be able to compare basic epidemiological data and to produce accurate figures of its economic impact. Diagnosis standardisation is of the utmost importance since some of these aetiological agents are a very frequent cause of abortion in ruminants, some are zoonotic (e.g. T. gondii) and some cause diseases regulated by EU health policies (e.g. T. foetus) and affect the ruminant products' trade. At present, diagnosis of abortion can be achieved by examination of foetal tissues for parasite-specific lesions, detection of parasite antigens or DNA and parasite-specific antibodies in foetal fluids. In the live animal, diagnosis is accomplished by detection of specific antibodies using a broad panel of serological techniques, including differences in the type of antigen used, type of immunoglobulin detected and the system to express the result (titre, optical density, percentage of positivity) or by detecting the parasite (or its products) in different tissues (Toxoplasma and Neospora in brain and muscles) and fluids (Tritrichomonas in preputial smegma/vaginal smears). Research tasks include

the comparison and standardisation of existing diagnostic tests across Europe by a multi-centred study that compares the different serological, immunohistochemical and PCR techniques as well as cultivation methods using a standard panel of sera, fluids and tissues. Moreover, results will be summarised in Guidelines for the Diagnosis of Protozoal Abortifacients in Farm Ruminants. These guidelines will contain recommendations on the diagnostic procedures to be followed by official and private institutions across Europe (e.g. diagnostic laboratories at state veterinary investigation centres and university veterinary schools, as well as private diagnostic laboratories), when dealing with this group of diseases, including diagnostic methodology in the adult population (e.g. sampling of bull studs for trichomonosis in artificial insemination centres).

The development of new tools to diagnose infection and/or disease. During the life span of the action, other important research tasks to be achieved in the area of diagnosis are the development of more refined tests, which permit:

- differentiation of acute and chronic infection in the live animal by testing the immune response to stage-specific antigens previously purified from the parasite or expressed as recombinant antigens or by examining the quality of the immune response (affinity, isotype, recognition of particular polypeptides) and its relationship to clinical consequences (abortion, transmission).

- efficient and cheap mass screening of the ruminant population (e.g. detection of parasite specific antibodies in bulk milk samples in neosporosis), quantification of parasite loads in aborted foetal tissues and adult fluids (preputial smegma and vaginal smears for Tritrichomonas) by developing quantitative PCR techniques and the development of molecular typing methods (e.g. RAPD, AFLP, RFLP) to characterise different parasite strains.

4.Epidemiology risk assessments, economics and control

Results of Working Groups 1, 2 and 3 will also be used to advance the knowledge of the epidemiology of the defined infections and will be incorporated in risk assessments.

Multi-centred epidemiological studies will be performed to obtain data on the prevalence and/or incidence of the infections which are covered by this COST Action and the diseases caused by N. caninum, T. gondii, and T. foetus as the basis of risk assessments for these diseases in several countries participating in the Action. A standardised interpretation of diagnostic results obtained with different test systems, is a prerequisite for the comparison of data obtained in different countries. A standardised interpretation of test results will be developed in collaboration with Working Group 3 where standardisation of diagnostic techniques is a main task. Data obtained as a result of the multi-centred studies will be compared and analysed, e.g. for regional and seasonal differences and for factors that may influence them. Moreover, potential risk factors for infection will be identified (case-control studies) and their relative importance assessed (cohort studies). On the basis of these data, provided that sufficient quantitative data becomes available, mathematical models will be developed, which allow quantitative risk assessments.

On the basis of diagnostic results, sensitivity and specificity of the diagnostic tools, (the total economic losses caused by protozoal abortifacients will be estimated, but due to the nature of the data that will become available in the course of this COST Action, it will be necessary to confine the loss calculations here to abortion losses. Calculation of the losses on the basis of reliable epidemiological data will allow rational decisions regarding control measures. The results obtained in Working Groups 1 and 2 relating to new perspectives of immunisation (including the use of novel marker vaccines , and chemotherapy, and the results of the risk assessments will be used to develop recommendations for safe, environmentally friendly and cost-effective control measures. Special attention will also be given to the applicability of herd management measures (e.g. test and removal of seropositive animals) as part of disease control. Recommendations for safe and cost-effective control will be developed and published.

D. ORGANISATION AND TIMETABLE

The organisation of the Action will be according to the document COST 400/01 Rules and Procedures for Implementing COST Actions.

The Management Committee (MC) will be responsible for the organisation and co-ordination of COST Action 854. At the first meeting the MC will establish the general framework and remit of the Working Groups.

There will be four working groups:

- Working Group 1: Biology, biochemistry and genetic background of virulence
- Working Group 2: Pathogenesis and host immune responses
- Working Group 3: Diagnosis
- Working Group 4: Epidemiology, risk assessments, economics and control

Each working group will have one separate meeting per year. In addition, all working groups will meet at an annual conference for exchange between the working groups and with other scientists. Working Group 3 will present "Guidelines for the Diagnosis of Protozoal Abortifacients in Farm Ruminants" at the end of the second year. Working Groups 2, 3 and 4 will present "Recommendations for Safe and Cost-effective Control of Protozoal Abortifacients in Farm Ruminants" at the end of the fourth year (responsible: Working Group 4). To ensure that the necessary interactions within and between the different Working Groups can take place:

The chairperson of the WG will be responsible for ensuring that the goals of the individual Working Groups are achieved including an effective interaction between the Working Groups.

As part of the mechanism to underpin optimal interaction between the Working Groups and to monitor their progress, meetings of the chairpersons of the working groups will be held along with the Management Committee.

Working documents prepared by the working groups (abstracts of papers presented at working groups meetings, conclusions, recommendations etc.) will be made available to all members of the Action through electronic media (e-mail). It is recognised that, due to the crucial role of Working
Group 4 within the Action, the interaction of Working Groups 1-3 with Working Group 4 is of

particular importance, since the quality of the results of the latter will depend, to a considerable extent, on the expertise developed in Working Groups 1-3.

The total duration of the Action will be 4 years.

E. DISSEMINATION OF RESULTS

Papers presented at working group meetings and annual conferences of the COST Action will be published in scientific journals and given at other national and international meetings. The Guidelines will be offered for publication to the European Commission. Annual reports will be prepared as a documentation of the progress of the COST action. Important results and conclusions will be communicated to the veterinary officials of the European Commission.
A web site will be created to communicate between participants and to stimulate dialog and exchange of ideas between formal meetings.

F. ECONOMC DIMENSION

The following COST countries have actively participated in the preparation of the Action or otherwise indicated their interest:

Belgium, Czech Republic, France, Germany, Greece, Ireland, Italy, Poland, Spain, Sweden, Switzerland, The Netherlands, Turkey, United Kingdom

On the basis of national estimates provided by the representatives of these countries the economic dimension of the activities to be carried out under the Action has been estimated, in 2001 prices, at

EURO 25 Million.

This estimate is valid under the assumption that all the countries mentioned above, but no other countries will participate in the Action. Any departure from this will change the total cost accordingly.

Program(-y)

IC-COST - European cooperation in the field of scientific and technical research (COST), 1971-

Temat(-y)

4 - Agriculture and Biotechnology

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N/A

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