Global Foot-and-Mouth Disease Research Update and Gap Analysis: 2 - Epidemiology, Wildlife and Economics.

We assessed knowledge gaps in foot-and-mouth disease (FMD) research, and in this study, we consider (i) epidemiology, (ii) wildlife and (iii) economics. The study took the form of a literature review (2011-2015) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD research. During 2011-2015, modelling studies were dominant in the broad field of epidemiology; however, continued efforts are required to develop robust models for use during outbreaks in FMD-free countries, linking epidemiologic and economics models. More guidance is needed for both the evaluation and the setting of targets for vaccine coverage, population immunity and vaccine field efficacy. Similarly, methods for seroprevalence studies need to be improved to obtain more meaningful outputs that allow comparison across studies. To inform control programmes in endemic countries, field trials assessing the effectiveness of vaccination in extensive smallholder systems should be performed to determine whether FMD can be controlled with quality vaccines in settings where implementing effective biosecurity is challenging. Studies need to go beyond measuring only vaccine effects and should extend our knowledge of the impact of FMD and increase our understanding of how to maximize farmer participation in disease control. Where wildlife reservoirs of virus exist, particularly African Buffalo, we need to better understand when and under what circumstances transmission to domestic animals occurs in order to manage this risk appropriately, considering the impact of control measures on livelihoods and wildlife. For settings where FMD eradication is unfeasible, further ground testing of commodity-based trade is recommended. A thorough review of global FMD control programmes, covering successes and failures, would be extremely valuable and could be used to guide other control programmes.

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 1 - Overview of Global Status and Research Needs.

The Global Foot-and-mouth disease (FMD) Research Alliance periodically reviews the state of FMD research to assess progress and to identify new priorities. In this supplement we provide an update of global FMD research, comprising (i) this overview paper, which includes background information with key findings, and papers covering (ii) epidemiology, wildlife and economics, (iii) vaccines, (iv) diagnostics, (v) biotherapeutics and disinfectants, (vi) immunology and (vii) pathogenesis and molecular biology. FMD research publications were reviewed (2011-2015) and activity updates were obtained from 33 FMD research institutes from around the world. Although a continual threat, FMD has been effectively controlled in much of the world using existing tools. However, control remains a challenge in most developing countries, where little has been done to understand the ongoing burden of FMD. More research is needed to support control in endemically infected countries, particularly robust field studies. Traditional FMD vaccines have several limitations including short duration and spectrum of protection, cold chain requirements, and the costs and biosecurity risks associated with vaccine production. Significant progress has been made in the development of novel vaccine candidates, particularly in the use of recombinant vaccines and virus-like particles as an alternative to traditional inactivated whole virus vaccines. Continued investment is needed to turn these developments into improved vaccines produced at scale. Increased knowledge of cellular and mucosal immunity would benefit vaccine development, as would further advances in our ability to enhance vaccine capsid stability. Developments in molecular biology and phylogenetics underlie many of the recent advances in FMD research, including improved vaccines and diagnostics, and improved understanding of FMD epidemiology. Tools for genetic analyses continue to become both more powerful and more affordable enabling them to be used to address an ever-expanding range of questions. This rapidly advancing field potentiates many areas of FMD research and should be prioritized.

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 5 - Biotherapeutics and Disinfectants.

We assessed knowledge gaps in foot-and-mouth disease (FMD) research. Findings are reported in a series of papers, and in this article, we consider biotherapeutics and disinfectants. The study took the form of a literature review (2011-2015) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD research. While vaccines will remain the key immunological intervention used against FMD virus (FMDV) for the foreseeable future, it takes a few days for the immune system to respond to vaccination. In an outbreak situation, protection could potentially be provided during this period by the application of rapid, short-acting biotherapeutics, aiming either to stimulate a non-specific antiviral state in the animal or to specifically inhibit a part of the viral life cycle. Certain antiviral cytokines have been shown to promote rapid protection against FMD; however, the effects of different immune-modulators appear to vary across species in ways and for reasons that are not yet understood. Major barriers to the effective incorporation of biotherapeutics into control strategies are cost, limited understanding of their effect on subsequent immune responses to vaccines and uncertainty about their potential impact if used for disease containment. Recent research has highlighted the importance of environmental contamination in FMDV transmission. Effective disinfectants for FMDV have long been available, but research is being conducted to further develop methods for quantitatively evaluating their performance under field, or near-field, conditions. During outbreaks in South Korea in 2010 there was public concern about potential environmental contamination after the mass use of disinfectant and mass burial of culled stock; this should be considered during outbreak contingency planning.

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 3 - Vaccines.

This study assessed research knowledge gaps in the field of FMDV (foot-and-mouth disease virus) vaccines. The study took the form of a literature review (2011-15) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD vaccine research. Vaccines play a vital role in FMD control, used both to limit the spread of the virus during epidemics in FMD-free countries and as the mainstay of disease management in endemic regions, particularly where sanitary controls are difficult to apply. Improvements in the performance or cost-effectiveness of FMD vaccines will allow more widespread and efficient disease control. FMD vaccines have changed little in recent decades, typically produced by inactivation of whole virus, the quantity and stability of the intact viral capsids in the final preparation being key for immunogenicity. However, these are exciting times and several promising novel FMD vaccine candidates have recently been developed. This includes the first FMD vaccine licensed for manufacture and use in the USA; this adenovirus-vectored FMD vaccine causes in vivo expression of viral capsids in vaccinated animals. Another promising vaccine candidate comprises stabilized empty FMDV capsids produced in vitro in a baculovirus expression system. Recombinant technologies are also being developed to improve otherwise conventionally produced inactivated vaccines, for example, by creating a chimeric vaccine virus to increase capsid stability and by inserting sequences into the vaccine virus for desired antigen expression. Other important areas of ongoing research include enhanced adjuvants, vaccine quality control procedures and predicting vaccine protection from immune correlates, thus reducing dependency on animal challenge studies. Globally, the degree of independent vaccine evaluation is highly variable, and this is essential for vaccine quality. Previously neglected, the importance of evaluating vaccination programme effectiveness and impact is increasingly being recognized.

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 6 - Immunology.

This study assessed gaps and priorities for FMDV (foot-and-mouth disease virus) research in the field of immunology. The study took the form of a literature review (2011-15) combined with research updates collected in 2014 from 33 institutes from across the world. Findings were used to identify priority areas for future FMD research. Improved understanding of FMDV immunology facilitates the development of vaccines, adjuvants and diagnostic tests, and will allow better assessment and prediction of vaccine potency and match, with reduced use of animals, particularly large animals, in experimental studies. Continued characterization of the immune systems of several FMD host species has underpinned substantial advances in knowledge of their interaction with FMDV. Recent studies have shed light on the mechanisms underlying formation of the bovine B- and T-cell response; there is also a greater understanding of the significance of non-neutralizing antibodies during FMDV infection and the interactions of antibody-bound virus with immune cells. This knowledge is directly relevant to vaccine development, as well as understanding protection and cross-protection. Despite ongoing research, significant knowledge gaps remain in the areas of neonatal and mucosal immunity. The impact of maternally derived antibody upon the neonate's ability to respond to FMD vaccination has received some attention, but few firm conclusions can be drawn at this stage, and little is known of the cellular response of young animals in general. The mucosal immune system of FMDV-susceptible species requires continued characterization, especially if the potential of mucosal vaccine-delivery systems is to be realized for FMD immunization.

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 4 - Diagnostics.

This study assessed knowledge gaps in foot-and-mouth disease (FMD) research in the field of diagnostics. The study took the form of a literature review (2011-15) combined with research updates collected in 2014 from 33 institutes from around the world. Findings were used to identify priority areas for future FMD research. Molecular and genetic technologies, including sequencing, are developing at an increasing rate both in terms of capability and affordability. These advances potentiate progress in many other fields of research, from vaccine development to epidemiology. The development of RT-LAMP represents an important breakthrough allowing greater use and access to molecular diagnostics. It is now possible to determine virus serotype using PCR, although only for certain virus pools, continued progress is needed to cover the global spectrum of FMD viruses. Progress has also been made in the development of pen-side rapid diagnostics, some with the ability to determine serotype. However, further advances in pen-side serotype or strain determination would benefit both FMD-free countries and endemic countries with limited access to well-resourced laboratories. Novel sampling methods that show promise include air sampling and baited ropes, the latter may aid sampling in wildlife and swine. Studies of infrared thermography for the early detection of FMD have not been encouraging, although investigations are ongoing. Multiplex tests have been developed that are able to simultaneously screen for multiple pathogens with similar clinical signs. Crucial for assessing FMDV freedom, tests exist to detect animals that have been infected with FMDV regardless of vaccination status; however, limitations exist, particularly when testing previously vaccinated animals. Novel vaccines are being developed with complementary DIVA tests for this purpose. Research is also needed to improve the current imprecise approaches to FMD vaccine matching. The development of simple, affordable tests increases access to FMD diagnostics, greatly benefiting regions with limited laboratory capacity.

Global Foot-and-Mouth Disease Research Update and Gap Analysis: 7 - Pathogenesis and Molecular Biology.

We assessed research knowledge gaps in the fields of FMDV (foot-and-mouth disease virus) pathogenesis and molecular biology by performing a literature review (2011-15) and collecting research updates (2014) from 33 institutes from across the world. Findings were used to identify priority areas for future research. There have been important advances in FMDV pathogenesis; FMDV remains in lymph nodes of many recovered animals that otherwise do not appear persistently infected, even in species previously not associated with the carrier state. Whether virus retention helps maintain host immunity and/or virus survival is not known. Studies of FMDV pathogenesis in wildlife have provided insights into disease epidemiology, in endemic and epidemic settings. Many aspects of FMDV infection and virus entry remain unknown; however, at the cellular level, we know that expression level and availability of integrins (that permit viral entry), rate of clearance of infected cells and strength of anti-viral type I IFN (interferon) response are key determinants of tissue tropism. Extending findings to improved understanding of transmission requires a standardized approach and adoption of natural routes of infection during experimental study. There has been recognition of the importance of autophagosomes for FMDV entry into the cytoplasm following cell surface receptor binding, and that distinct internal cellular membranes are exploited for viral replication and immune evasion. New roles for viral proteins in blocking type I IFN production and downstream signalling have been identified facilitating research in anti-viral therapeutics. We know more about how infection affects cell protein expression, and research into molecular determinants of capsid stability has aided the development of stable vaccines. We have an expanding knowledge of viral and host molecular determinates of virulence and infectiousness, and of how phylogenetics may be used to estimate vaccine match and strain distribution. With ongoing advances, these areas could translate into significantly improved disease control.

Mass vaccination, immunity and coverage: modelling population protection against foot-and-mouth disease in Turkish cattle.

Foot-and-mouth disease (FMD) in Turkey is controlled using biannual mass vaccination of cattle. However, vaccine protection is undermined by population turnover and declining immunity. A dynamic model of the Turkish cattle population was created. Assuming biannual mass vaccination with a single-dose primary course, vaccine history was calculated for the simulated population (number of doses and time since last vaccination). This was used to estimate population immunity. Six months after the last round of vaccination almost half the cattle aged < 24 months remain unvaccinated. Only 50% of all cattle would have received > 1 vaccine dose in their life with the last dose given ≤ 6 months ago. Five months after the last round of vaccination two-thirds of cattle would have low antibody titres (< 70% protection threshold). Giving a two-dose primary vaccination course reduces the proportion of 6-12 month old cattle with low titres by 20-30%. Biannual mass vaccination of cattle leaves significant immunity gaps and over-reliance on vaccine protection should be avoided. Using more effective vaccines and vaccination strategies will increase population immunity, however, the extent to which FMD can be controlled by vaccination alone without effective biosecurity remains uncertain.

Randomised field trial to evaluate serological response after foot-and-mouth disease vaccination in Turkey.

Despite years of biannual mass vaccination of cattle, foot-and-mouth disease (FMD) remains uncontrolled in Anatolian Turkey. To evaluate protection after mass vaccination we measured post-vaccination antibodies in a cohort of cattle (serotypes O, A and Asia-1). To obtain results reflecting typical field protection, participants were randomly sampled from across Central and Western Turkey after routine vaccination. Giving two-doses one month apart is recommended when cattle are first vaccinated against FMD. However, due to cost and logistics, this is not routinely performed in Turkey, and elsewhere. Nested within the cohort, we conducted a randomised trial comparing post-vaccination antibodies after a single-dose versus a two-dose primary vaccination course. Four to five months after vaccination, only a third of single-vaccinated cattle had antibody levels above a threshold associated with protection. A third never reached this threshold, even at peak response one month after vaccination. It was not until animals had received three vaccine doses in their lifetime, vaccinating every six months, that most (64% to 86% depending on serotype) maintained antibody levels above this threshold. By this time cattle would be >20 months old with almost half the population below this age. Consequently, many vaccinated animals will be unprotected for much of the year. Compared to a single-dose, a primary vaccination course of two-doses greatly improved the level and duration of immunity. We concluded that the FMD vaccination programme in Anatolian Turkey did not produce the high levels of immunity required. Higher potency vaccines are now used throughout Turkey, with a two-dose primary course in certain areas. Monitoring post-vaccination serology is an important component of evaluation for FMD vaccination programmes. However, consideration must be given to which antigens are present in the test, the vaccine and the field virus. Differences between these antigens affect the relationship between antibody titre and protection.

Retrospective evaluation of foot-and-mouth disease vaccine effectiveness in Turkey.

Foot-and-mouth disease (FMD) is present in much of Turkey and its control is largely based on vaccination. The arrival of the FMD Asia-1 serotype in Turkey in 2011 caused particular concern, spreading rapidly westwards across the country towards the FMD free European Union. With no prior natural immunity, control of spread would rely heavily on vaccination. Unlike human vaccines, field protection is rarely evaluated directly for FMD vaccines. Between September 2011 and July 2012 we performed four retrospective outbreak investigations to assess the vaccine effectiveness (VE) of FMD Asia-1 vaccines in Turkey. Vaccine effectiveness is defined as the reduction in risk in vaccinated compared to unvaccinated individuals with similar virus exposure in the field. The four investigations included 12 villages and 1230 cattle >4 months of age. One investigation assessed the FMD Asia-1 Shamir vaccine, the other three evaluated the recently introduced FMD Asia-1 TUR 11 vaccine made using a field isolate of the FMD Asia-1 Sindh-08 lineage that had recently entered Turkey. After adjustment for confounding, the TUR 11 vaccine provided moderate protection against both clinical disease VE=69% [95% CI: 50%-81%] and infection VE=63% [95% CI: 29%-81%]. However, protection was variable with some herds with high vaccine coverage still experiencing high disease incidence. Some of this variability will be the result of the variation in virus challenge and immunity that occurs under field conditions. In the outbreak investigated there was no evidence that the Asia-1 Shamir vaccine provided adequate protection against clinical FMD with an incidence of 89% in single vaccinated cattle and 69% in those vaccinated two to five times. Based on these effectiveness estimates, vaccination alone is unlikely to produce the high levels of herd immunity needed to control FMD without additional control measures.

Molecular characterization of foot-and-mouth disease viruses collected from Sudan.

The aim of this study was to characterize foot-and-mouth disease (FMD) viruses collected between 2004 and 2008 from Sudan, a country where FMD is endemic. Using virus isolation and antigen ELISA, three FMD virus serotypes (O, A and SAT2) were detected in 24 samples that were submitted to the FAO World Reference Laboratory for FMD. Pan-serotypic real-time RT-PCR assays targeting the 5' untranslated region (5'UTR) and 3D genes of FMD virus were also used to contribute to the laboratory diagnosis of these cases. The lack of concordant results between the real-time RT-PCR assays for three serotype O viruses was attributed to four nucleotide mismatches in the 5'UTR PCR primer and probe sites (three substitutions for the sense-primer and one in the TaqMan(®) probe region). Taken together, the laboratory results showed that recent FMD outbreaks that occurred during 2008 in northern and central Sudan were caused by serotypes O and SAT2, while serotype A was last detected in 2006. Phylogenetic analyses of VP1 sequences from these viruses were used to determine the relationships with 23 older viruses from Sudan and other viruses from West and East Africa. For serotype O, closest genetic identities were between concurrent and historical Sudanese isolates, indicating that within-country circulation is an important mechanism by which FMD is maintained year-on-year in Sudan. A similar pattern was also evident for serotype A and SAT2 viruses; however, these lineages also contained recent representative FMD viral isolates from other countries in the region suggesting that long-distance animal movement can also contribute to FMD dispersal across sub-Saharan Africa. These findings provide the first molecular description of FMD viruses that are circulating in Sudan, and highlight that further sampling of representative viruses from the region is required before the complex epidemiology of FMD in sub-Saharan Africa can be fully understood.

Recent spread of a new strain (A-Iran-05) of foot-and-mouth disease virus type A in the Middle East.

This report describes the characterization of a new genotype of foot-and-mouth disease virus (FMDV) type A responsible for recent FMD outbreaks in the Middle East. Initially identified in samples collected in 2003 from Iran, during 2005 and 2006 this FMDV lineage (proposed to be named A-Iran-05) spread into Saudi Arabia and Jordan and then further west into Turkey reaching European Thrace in January 2007. Most recently A-Iran-05 has been found in Bahrain. To the east of Iran, it has been recognized in Afghanistan (2004-07) and Pakistan (2006-07). Throughout the region, this lineage is now the predominant genotype of FMDV serotype A sampled, and has appeared to have replaced the A-Iran-96 and A-Iran-99 strains which were previously encountered. In August 2007, a new A-Iran-05 sub-lineage (which we have called A-Iran-05(ARD-07)) was identified in Ardahan, Turkey, close to the border with Georgia. This new sub-lineage appeared to predominate in Turkey in 2008, but has, so far, not been identified in any other country. Vaccine matching tests revealed that the A-Iran-05 viruses are antigenically different to A-Iran-96 and more like A(22). These findings emphasize the importance of undertaking continued surveillance in the Middle East and Central Asia in order to detect and monitor the emergence and spread of new FMDV strains.

Incursions of foot-and-mouth disease virus into Europe between 1985 and 2006.

Foot-and-mouth disease (FMD) is one of the biggest threats to animal health in European countries. In the last 22 years (1985-2006), FMD has occurred 37 times in 14 European countries. Serotype O was most frequently involved in these outbreaks followed by A, C and Asia 1. Sometimes, epidemics were very limited and at other times, they were the cause of devastating economic losses. In most cases (22/37), the origin of the outbreaks could not be determined. For some of these outbreaks, however, routes of introduction and spread were identified through epidemiological inquiries. Moreover, in some cases, the origin of the virus was also traced by phylogenetic analysis of the partial or complete sequences of VP1 genes. Lessons learned from the outbreaks are still useful as most of the same risk factors persist. However, efforts made by FMD-free countries to help those where the disease is endemic are a valuable strategy for the reduction of the global risk. The present and the future potential sources of FMD infection need to be identified to best focus European efforts.

Evaluation of laboratory tests for SAT serotypes of foot-and-mouth disease virus with specimens collected from convalescent cattle in Zimbabwe.

During a field study in Zimbabwe, clinical specimens were collected from 403 cattle in six herds, in which the history of foot-and-mouth disease (FMD) vaccination and infection appeared to be known with some certainty. Five herds had reported outbreaks of disease one to five months previously but clinical FMD had not been observed in the sixth herd. A trivalent vaccine (South African Territories [SAT] types 1, 2 and 3) had been used in some of the herds at various times either before and/or after the recent outbreaks of FMD. The primary aim of this study was to evaluate the performance of serological tests for the detection of SAT-type FMD virus infection, particularly elisas for antibodies to non-structural proteins (NSPs) of FMD virus and solid phase competition ELISAS (SPCEs) for serotypes SAT1 and SAT2. Secondary aims were to examine NSP seroconversion rates in cattle that had been exposed to infection and to compare virus detection rates by virus isolation and real-time reverse transcriptase-PCR (rtRT-PCR) tests on both oesophagopharyngeal fluids and nasopharyngeal brush swabbings. In addition, the hooves of sampled animals were examined for growth arrest lines as clinical evidence of FMD convalescence. Laboratory tests provided evidence of FMD virus infection in all six herds; SAT2 viruses were isolated from oesophagopharyngeal fluids collected from two herds in northern Zimbabwe, and SAT1 viruses were isolated from three herds in southern Zimbabwe. Optimised rtRT-PCR was more sensitive than virus isolation at detecting FMD virus persistence and when the results of the two methods were combined for oesophagopharyngeal fluids, between 12 and 35 per cent of the cattle sampled in the convalescent herds were deemed to be carriers. In contrast, nasopharyngeal swabs yielded only two virus-positive specimens. The overall seroprevalence in the five affected herds varied with the different NSPS from 56 per cent to 75 per cent, compared with 81 per cent and 91 per cent by homologous SPCE and virus neutralisation tests respectively. However, if serological test results were considered only for the cattle in which persistent infection with FMD virus had been demonstrated, 70 to 90 per cent scored seropositive in the different NSPs.

Porcine enteric spirochete infections in the UK: surveillance data and preliminary investigation of atypical isolates.

Investigations into the possible causes of colitis and typhlocolitis were carried out on 98 pig units in the United Kingdom between 1997 and 1999. Brachyspira pilosicoli was identified most commonly, occurring as the suggested primary agent in 18% of the outbreaks but forming part of mixed infections in another 24% of outbreaks. The equivalent figures for other bacterial pathogens were: B. hyodysenteriae, 13% and 16%; Lawsonia intracellularis, 10% and 15%: Salmonella species, 6% and 12%; and Yersinia species, 4% and 10%. Unclassified Brachyspira species of unknown pathogenicity were identified in 12% of outbreaks. The 24 unclassified isolates divided into three groups on the basis of their phenotypic characteristics. In addition, there were 50 atypical Brachyspira species isolates that showed differences between their phenotypic characteristics and genetic identity based on sequence analysis of a section of the 23S rDNA gene. Four representative atypical isolates were found to be pathogenic as a result of an experimental oral challenge study in pigs.

Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line.

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells.

Natural prevalence of infection with Ehrlichia (Cytoecetes) phagocytophila of Ixodes ricinus ticks in Scotland.

Ixodes ricinus nymphs and adults were collected from vegetation and from sheep at four sites in Scotland typical of areas endemic for tick-borne fever in sheep caused by infection with Ehrlichia (Cytoecetes) phagocytophila (Rickettsiales). The great majority of ticks examined was from woodland sites adjacent to sheep farms where there was a high probability of them feeding on roe deer (Capreolus capreolus) in a non-domestic focus of infestation and infection. Ticks were examined for infection by five methods. Batches of ticks were examined either by feeding on susceptible sheep or by feeding on rabbits and then prepared as stabilate which was inoculated into susceptible sheep. The sheep were monitored for clinical signs of tick borne fever. Batches of ticks were examined by polymerase chain reaction for Ehrlichia phagocytophila. Salivary glands were dissected out and stained by the Feulgen method to detect Ehrlichia masses, and were examined by indirect fluorescent antibody test. Each of the methods detected infection in ticks and the prevalence of infection in nymphs with the various methods ranged from >0.25% to 2.0%. Small samples of adults examined by Feulgen staining of salivary glands indicated infection prevalences of 2.1% in males and 1.6% in females. It is considered that these low infection prevalences may be typical of natural foci of infection where deer could be a major host of ticks and E. phagocytophila.

Nitric oxide is produced by Cowdria ruminantium-infected bovine pulmonary endothelial cells in vitro and is stimulated by gamma interferon.

Nitric oxide (NO) is a labile inorganic free radical produced by NO synthase from the substrate L-arginine in various cells and tissues including endothelial cells. A substantial elevation of nitrite levels indicative of NO production occurred in cultures of Cowdria ruminantium-infected bovine pulmonary endothelial cells (BPEC) incubated in medium alone. Exposure of the infected cultures to recombinant bovine gamma interferon (BorIFN-gamma) resulted in more rapid production of NO, reduced viability of C. ruminantium, and induction of endothelial cell death. Significant inhibition of NO production was noted after addition of the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA), indicating that the increase in production occurred via the inducible NO synthase pathway. Reduction in the infectivity of C. ruminantium elementary bodies (EBs) occurred in a dose-dependent manner after incubation with the NO donor molecule S-nitroso-N-acetyl-DL-penicillamine (SNAP) prior to infection of endothelial cells. The level of infection in cultures maintained in SNAP was reduced in a dose-dependent manner with significant negative correlation between the final level of infection on day 7 and the level of SNAP (r = -0.96). It was established that pretreatment and cultivation of C. ruminantium EBs with the NO donor molecule SNAP reduced infectivity to cultures and viability of EBs with the implication that release of NO in vivo following infection of endothelial cells may have an effect upon the multiplication of the agent in the host animal and may be involved in the pathogenesis of heartwater through the effect of this molecule upon circulation.

Detection of peste des petits ruminants virus antigen in conjunctival smears of goats by indirect immunofluorescence.

Peste des petits ruminants virus (PPRV) antigen was detected in conjunctival epithelial cells obtained from goats in the early or late stage of the disease by the use of a specific monoclonal antibody (mAb) to PPRV in an immunofluorescent antibody test (IFAT). The affected goats were sampled during an outbreak of peste des petits ruminants in Eritrea. Syncytia were also observed in some smears, consistent with a morbillivirus infection, but the IFAT was more sensitive than staining for syncytia in the detection of viral antigen, the two tests giving 63 per cent and 40 per cent of animals, respectively, with positive tests. Positive immunofluorescence was observed in samples from goats in the early and late stages of the disease, but was not observed with a specific rinderpest mAb or with conjunctival smears from uninfected animals. It is concluded that preparation of conjunctival smears for staining for syncytia is a simple procedure which can be applied in the field, and by use of a specific mAb PPRV infection can be rapidly confirmed and differentiated from rinderpest.

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein.

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully chracterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.