Governance and management of veterinary laboratories.

This paper describes those components of governance and management of a public veterinary laboratory that are deemed essential for the effective delivery of a diagnostic service. Whilst broadly applicable to all veterinary diagnostic services, there is a focus on publicly supported veterinary diagnostic laboratories in developing countries, highlighting the critical components that should be established as a minimum. The need for establishing overarching ownership, governance and resourcing is emphasised but followed by a detailed account of the components of diagnostic service management and delivery, linked to a description of the key support services that are essential in assisting delivery. Elements of quality assurance and compliance are described, with an emphasis on the need to both understand and meet the regulatory environment in which a diagnostic laboratory now operates. The outputs from a veterinary laboratory must be rooted in sound science, and mechanisms must be in place to prevent corrupt practices and inappropriate political influences.

Rinderpest surveillance performance monitoring using quantifiable indicators.

This paper describes an objective system of monitoring the performance of disease surveillance. The system was developed through dialogue with a number of countries in Africa and adopted as part of the Global Rinderpest Eradication Programme of the Food and Agriculture Organization of the United Nations. The performance monitoring system uses a clinical stomatitis-enteritis case definition, an outbreak investigation classification scheme, and a series of eight performance indicators to measure the sensitivity, specificity and timeliness of the surveillance system. Field-testing indicates that the approach is successful when good record-keeping is practiced and highlights the importance of dialogue in helping to ensure that the system is simple and acceptable. The system provides a quantitative measure of the efficacy of national disease surveillance programmes and of the quality of data derived from such programmes for use in international disease control, animal health information exchange and trade risk analysis.

An international approach to laboratory diagnosis of animal diseases.

During the past thirteen years, and as part of an overall support program for developing countries, the Joint FAO/IAEA Division has developed a series of standardized and internationally validated ELISA-based systems for the diagnosis and surveillance of the major epizootics affecting livestock in the developing world. Linked to comprehensive internal and external quality assurance protocols and to the use of standardized equipment and data management software, veterinary laboratories in many developing countries are now able to provide quality assured results on the prevalence of the major diseases affecting their livestock. This internationally standardized approach can rapidly enable developing countries to both monitor the effectiveness of their disease control and eradication programs and to meet requirements for international disease status recognition and for trade in livestock and livestock products.

Veterinary diagnostic laboratories in developing countries: the challenge of credibility.

The problems facing veterinary diagnostic laboratories in developing countries range from simple problems, such as limited funds to keep good personnel or obtain equipment, supplies, reagents or training, to the more complex problems of designing and executing appropriate sample collection schemes for disease surveillance of evaluating the performance characteristics of a new diagnostic assay. While many developing countries are addressing these problems independently and a number of international and national organisations provide various forms of external support, the Office International des Epizooties (OIE) must continue in its critical role to provide guidelines for the most appropriate diagnostic assays for trade purposes and the development of the primary reference reagents for these assays. In addition, the OIE should take the lead in development of quality management guidelines for veterinary diagnostic testing laboratories. Without these guidelines and standards, diagnostic laboratories in developing countries will have difficulty in gaining the credibility necessary to help improve the positions of their countries in the international trade of livestock and livestock commodities.

International reference standards: antibody standards for the indirect enzyme-linked immunosorbent assay.

Reference standards are used to calibrate similar assay systems against an international reference protocol and to provide a template for the preparation of secondary and/or working standards. Three reference standards are recommended for the indirect enzyme-linked immunosorbent assay: a strong positive standard, a weak positive standard and a negative serum standard. The negative standard should be derived from a single serum or from a serum pool which exhibits typical background activity in the reference protocol. The strong and weak positive standards should be derived from a single serum or from a serum pool which typifies the humoral response (antibody) to natural infection. Suitable candidates for the positive reference standards should exhibit dose/response curves in the mid-range of antibody activity. The strong and weak positive standards should each be prepared from a one-time dilution in the negative standard, to yield antibody activities which are defined by specific points on the linear portion of the dose/response curve. The strong positive standard should represent an antibody activity (absorbance value) midway between the upper and central points and the weak positive standard should represent an antibody activity midway between the central and lower points of the linear portion of the curve. Owing to inherent differences among assay systems, antibody activities should be expressed in relative rather than in absolute terms. It is recommended that the antibody activity of the strong positive standard should denote 100% positivity. The activities of the weak positive and negative standards should then be expressed as relative percentages. Every set of international reference standards should be accompanied by an information sheet which includes, among other things, a plot of the dose/response curve and an indication of the dilutions used to prepare the standards.

Standardisation and validation of enzyme-linked immunosorbent assay techniques for the detection of antibody in infectious disease diagnosis.

Enzyme-linked immunosorbent assay (ELISA) techniques for the detection of antibodies are now widely used throughout the world for the diagnosis of infectious diseases in veterinary medicine. Although many laboratories have independently developed ELISA techniques for their own purposes, little progress has been made with respect to the international standardisation and validation of these techniques. This lack of international conformity is of major concern to organisations such as the Office International des Epizooties (OIE), the United Nations Food and Agriculture Organisation (FAO), the World Health Organisation (WHO) and the International Atomic Energy Agency (IAEA) which are involved in the establishment of international guidelines and programmes for the control, surveillance and/or eradication of infectious diseases. In this regard, a Joint FAO/IAEA Meeting of Consultants was convened in Vienna in January 1992 to review aspects of ELISA data expression, primary reference standards, quality assurance and diagnostic validation. Based on the consensus derived from this meeting, the authors describe procedures which are recommended as a platform on which to build definitive guidelines for international standardisation of ELISA protocols and reagents, in cooperation with the OIE and the OIE Reference Laboratories.

Failure to establish congenital bluetongue virus infection by infecting cows in early pregnancy.

Two groups of 10 pregnant cows were inoculated with bluetongue virus type 11 at either 40 or 60 days of gestation. All the cows became infected as judged by the detection of viraemia and seroconversion but they showed no clinical signs. Seventeen of the cows produced live calves none of which showed any evidence of prenatal infection. After challenge with the same virus all the calves became viraemic and seroconverted. The response to challenge of the two groups did not differ from that of a control group challenged at the same time. It was concluded that the infection of pregnant cows in early gestation with this virus did not result in the transplacental infection of the fetuses and did not produce immunotolerant, latently infected calves.

Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype.

Analyses of reassortant and parental strains of BTV serotypes 3 and 10, in serum neutralization tests, confirmed the major role of outer capsid protein VP2 in determination of virus serotype and its involvement in serum neutralization. However, a reassortant BTV strain (R70), containing protein VP5 derived from BTV 3 and VP2 derived from BTV 10, cross-neutralized with both parental virus strains (BTV 3 and BTV 10). It is concluded that VP5 also plays some part in serotype determination of these virus isolates, as analyzed by serum-neutralization, but its role may be less significant than that of VP2.

Cultivation of bluetongue virus-specific ovine T cells and their cross-reactivity with different serotype viruses.

Bluetongue (BT) virus-specific ovine T-cell lines were prepared from BT virus-infected sheep by three cycles of alternate stimulation and resting culture in vitro. In antigen-specific proliferation assays and/or cytotoxicity assays, most of these T-cell lines responded not only to homologous serotype virus but also heterologous serotype viruses. This cross-reactivity did not correlate with the relatedness of serotypes as defined by cross-neutralizing antibodies. One cell line, 58-014, has grown continuously for more than 7 months without loss of antigen specificity. However, most of the cell lines lost their antigen specificity 2-4 months after cultivation. Certain BT virus-specific T-cell lines were able to reduce BT virus replication in autologous skin fibroblast cell culture.

A reassessment of the dual vaccine against rinderpest and contagious bovine pleuropneumonia.

In the light of the recent outbreaks of rinderpest in Africa a further assessment of the efficacy of the simultaneous inoculation of rinderpest virus vaccine and contagious bovine pleuropneumonia vaccine was undertaken. Groups of cattle were inoculated with a dual preparation of rinderpest vaccine virus and Mycoplasma mycoides subspecies mycoides or M mycoides alone. These groups were then challenged with M mycoides, first unsuccessfully by an in-contact challenge method and then by subcutaneous challenge. All animals were examined clinically after challenge for evidence of contagious bovine pleuropneumonia and serologically for rinderpest virus and M mycoides mycoides antibodies. There was no evidence that the serological response to the dual vaccine was in any way less than that to either agent given alone and no clinical disease was detected in these animals after in-contact challenge. However, after subcutaneous challenge, the dual vaccinated groups reacted similarly to an unvaccinated control group and unlike the group vaccinated only with M mycoides. This would indicate that the rinderpest virus component of the dual vaccine interfered with the ability of the M mycoides component to induce a fully effective immune response. In the pan African rinderpest campaign the use of the dual vaccine in areas where contagious bovine pleuropneumonia occurs should be carefully considered; in areas where the disease does not occur it is contraindicated.

Serial inoculation of sheep with two bluetongue virus types.

Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.

Bluetongue vaccine: cells and/or antibodies.

Immunological studies with bluetongue virus have indicated that protection from re-infection involves components of both the humoral and cellular immune response. However, it was found that the humoral response was type-specific, whilst the cellular immune response, particularly through the action of cross-reactive cytotoxic T lymphocytes, gave rise to heterotypic protection. Work involving simultaneous and sequential inoculation of live virus and the inoculation of various inactivated preparations has further characterized the type of vaccine formulation needed for efficient protection in multitype endemic areas. The authors cite these studies on bluetongue virus as an example of an immunological approach to vaccine design that is too often ignored by vaccine manufacturers and yet clearly yields results.

Importance of ovine cytotoxic T cells in protection against bluetongue virus infection.

In sheep, bluetongue virus (BTV) was shown to induce anti-BTV cytotoxic T lymphocytes (CTL) and their effect to be maximal around 14 days post inoculation (p.i.) of virus. Using cellular adoptive transfer techniques in monozygotic sheep, such cells were shown to partially protect animals from BTV challenge. A short-lived cross-protective mechanism was identified involving thoracic duct lymphocytes (TDL) and nonneutralising antibody. These observations suggest that T lymphocytes play an important role in protection against BTV and that current vaccine design based on in vitro serological typing of BTV can be improved.

Clinical and serological outcome following the simultaneous inoculation of three bluetongue virus types into sheep.

The simultaneous inoculation of sheep with three different bluetongue virus types resulted in the replication of only two of the virus types and the formation of neutralising antibodies to only those two types and a failure in the production of heterotypic antibodies. This suggests that the present system of control, using multivalent vaccines in areas in which a number of bluetongue serotypes exist, should be reappraised.

A study of the role of cell-mediated immunity in bluetongue virus infection in sheep, using cellular adoptive transfer techniques.

The transfer of thoracic duct lymphocytes from sheep inoculated 14 days, but not 7 days previously with bluetongue virus into their monozygotic twin resulted in some protection from challenge with bluetongue virus. T cell enrichment of the 14 day thoracic duct lymphocyte population resulted in a similar effect, indicating the T cell basis of the observed protection. Animals recovered from infection with bluetongue virus type 3 and which received thoracic duct lymphocytes from an identical twin recently infected with the same bluetongue virus type were protected from challenge with bluetongue type 4. These observations suggest that T lymphocytes play an important role in protection against bluetongue virus.

Role of neutralising antibody in passive immunity to bluetongue infection.

A group of sheep inoculated with serum obtained from sheep which had recovered from bluetongue virus type 3 infection were protected from challenge with the homologous virus type but not from heterologous challenge. Twin lambs which had received colostrum containing virus antibodies were shown to be only partially protected against homologous challenge. A monoclonal antibody directed against the type-determining protein of the virus was also shown to give partial protection against challenge. From this series of experiments it was concluded that antibody has a significant role in protection from bluetongue but that the outcome of challenge will depend on several interacting factors.

Clinical and serological response of sheep to serial challenge with different bluetongue virus types.

A group of British sheep was infected with bluetongue virus 5 (BTV5) and subsequently challenged with the same virus type. Protection from this challenge and a homotypic BTV neutralising antibody response were observed. A second group of sheep was infected serially with three different BTV types. Animals previously exposed to BTV4 and BTV3 were found to be resistant to challenge by BTV6. Animals infected with BTV4 and challenged with BTV3 were shown to produce a transient heterotypic neutralising antibody response to a number of types. Although the level of this heterotypic response diminished with time, after challenge with BTV6 these animals developed a similar broad heterotypic response. The nature of this response and its implications in terms of observed protection merit consideration in future vaccine design and evaluation of field survey work.

Production of murine cytotoxic T lymphocytes by bluetongue virus following various immunisation procedures.

The induction of bluetongue virus specific cytotoxic T lymphocytes (CTLs) in C3H mice by various live and inactivated bluetongue virus preparations was studied. Live virus preparations were shown to induce good levels of CTLs; however, inactivation of virus preparations either by beta propriolactone or glutaraldehyde induced only a low level response. The use of Freund's adjuvants and double immunisation procedures failed to improve the response of the inactivated preparations. These findings are discussed in relationship to protection from bluetongue disease with various bluetongue virus vaccines.

Generation of cross-reactive cytotoxic T lymphocytes following immunization of mice with various bluetongue virus types.

Mice immunized with a single bluetongue (BT) virus type were shown to produce cytotoxic T lymphocytes (CTL's) which cross-reactive with a number of BT virus types. These cross-reactive CTL's could be induced by both primary in vivo and secondary in vitro stimulation. A varying degree of cross-reactivity occurred with the six BT types examined. Aspects of the character of this cross-reactivity were examined and its role in protection from disease and vaccination strategy is discussed.