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 invivo expression of viral capsids in vaccinated animals. Another promising vaccine candidate comprises stabilized empty FMDV capsids produced invitro 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.

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.

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.

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.

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.

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.

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. (C) 2014 Elsevier Ltd.All rights reserved.

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.

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.

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.

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.

Two serological tests for detection of antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, were compared by using field sera collected from sheep and cattle as part of serosurveys in Ghana. Sera selected as either negative or positive by a new polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) were tested by the indirect MAP1-B ELISA. Cutoff values of 14 percent positivity (14 PP) for both ruminant species were obtained for the MAP1-B ELISA by using preseroconversion Ghanaian sera and were compared with previously recommended cutoff values of 29 PP for sheep and 38 PP for cattle. With the 14-PP cutoff, of 151 sheep sera which tested negative by PC-ELISA, 89% were also negative by MAP1-B ELISA, while of 419 sheep sera positive by PC-ELISA, 98% were also positive by MAP1-B ELISA. Of 261 bovine sera negative by PC-ELISA, 82% were also negative by MAP1-B ELISA. Of 511 bovine sera positive by PC-ELISA, only 47% were positive by MAP1-B ELISA; these included 168 sera collected from cattle following first seroconversion as detected by both tests, with 125 of these sera positive by PC-ELISA but only 59 and 5 positive by MAP1-B ELISA with the 14- and 38-PP cutoff levels, respectively. These results indicate that both assays are highly sensitive and specific for detection of E. ruminantium exposure in sheep but that the MAP1-B ELISA lacks sensitivity for postseroconversion bovine sera in comparison to the PC-ELISA. Both tests confirm E. ruminantium seroprevalence of at least 70% in Ghanaian sheep; levels of exposure among Amblyomma variegatum-infested Ghanaian cattle are likely to be higher than the seroprevalence value of 66% obtained with the PC-ELISA.

A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnostic sensitivity and specificity were 86 and 100%, respectively, with a cutoff of 50 PI and 79 and 100% with a cutoff of 70 PI. Cross-reactions with high-titer experimental or field antisera to other Ehrlichia and Anaplasma species were observed at up to 68 PI in cattle and up to 85 PI in sheep, and therefore to exclude these cross-reactions, cutoffs of 70 PI for bovine serology and 85 PI for small-ruminant serology were selected. Application of the PC-ELISA to bovine field sera from South Africa gave a higher proportion of positive results than application of the murine macrophage immunofluorescent antibody test or indirect ELISA, suggesting a better sensitivity for detection of recovered cattle, and results with bovine field sera from Malawi were consistent with the observed endemic state of heartwater and the level of tick control practiced at the sample sites. Reproducibility was high, with average standard deviations intraplate of 1.2 PI and interplate of 0.6 PI. The test format is simple, and the test is economical to perform and has a level of sensitivity for detection of low-titer positive bovine sera that may prove to be of value in epidemiological studies on heartwater.

The Immunoglobulin IgG(1) and IgG(2) isotype immune responses of domestic ruminants and mice to Cowdria. ruminantium live infection or by immunization with inactivated organisms were determined by the enzyme linked immunosorbent assay and Western blotting. Immunization of goats with inactivated elementary bodies (IEBs) led to a predominant IgG(1) isotype response. This indicated that a Th2 response was induced. After challenge, the IgG isotype responses were mixed whereby both IgG(1) and IgG(2) antibodies were detected. Two goats that survived virulent challenge had a predominant IgG(2) isotype response. In cattle live infection by natural challenge or experiment led to a predominant IgG, isotype response. Immunization of cattle with IEBs however led to mixed IgG responses characterized by similar IgG(1) and IgG(2) ratios. In the mouse live infection led to a predominant fgG(2) isotype response. This indicated the mouse developed a true Th-1 type cell mediated immune response when inoculated with live organisms. Immunization with inactivated organisms on the other hand led to a dominant IgG(1) response. It is evident from this work that the immune responses of ruminants and mice to C. ruminantium are different and that using mice as the experimental model for immune responses to Cowdria ruminantium. is not the appropriate.

This study sought to identify the surface exposed antigenic components of Cowdria ruminantium elementary body (EB) by biotin labeling, determine effect of reducing and non-reducing conditions and heat on the mobility of these antigens and their reactivity to antibodies from immunized animals by Western blotting. Six surface exposed antigens of the C. ruminantium EB were identified by biotin labeling with molecular masses of 21kDa, 28kDa, 31kDa, 62kDa, 74kDa and 115kDa and are therefore outer membrane proteins. The effect of reducing agents was to increase the number antigens bands, indicating there may be 2-5 antigenic (polypeptides) on the surface of the EB. On the other hand, heating did not affect the number of antigen bands. The identification of six surface exposed antigens of C. ruminantium and the successful purification of two of these antigens, makes it possible to use them for immunization so as to determine which of them induce protective responses. Keywords: Cowdria ruminantium, Elementary body, biotin-labelling, surface antigens, mobilities, ruminants [IJARD Vol.3 2002: 78-86]

An 813-bp fragment of the groEL gene of several Ehrlichia (Cytoecetes) phagocytophila-like organisms of British, Dutch, and North American origin was amplified by nested PCR from genomic DNA derived from sheep, goats, cattle, horses, and humans. Restriction fragment length polymorphisms (RFLP), as predicted from sequence data among granulocytic ehrlichiae, were demonstrated by digestion of amplicons with MboII and HaeIII enzymes. Granulocytic Ehrlichia of European origin could be readily distinguished from North American isolates, and European Ehrlichia from ruminant origin could be distinguished from a granulocytic sample from a horse from Scotland. The latter had a banding pattern identical to that predicted from the sequence in the database for an equine isolate from Switzerland and a human case of granulocytic ehrlichiosis in Slovenia. E. phagocytophila-like amplicons from four blood stabilates derived from sheep, one isolate obtained from a horse from Scotland and a sample derived from cattle from England were subsequently cloned and sequenced. Sequence analysis revealed 5 nucleotide differences on average when compared to E. equi. Interestingly and confirming the RFLP results, samples derived from horses in Scotland and Switzerland appeared almost identical to a human isolate in Slovenia that also differed from the human sequences in the database. Previous research showed that granulocytic Ehrlichia isolated in humans, dogs and horses in Europe and the United States were almost indistinguishable at 16S rDNA level. The use of groEL gene revealed further differences. This study indicates that the emerging cases of granulocytic ehrlichiosis in hosts other than ruminants in Europe are more closely related to E, phagocytophila than to E. equi and the HGE agent in North America. It is expected that additional studies of less conserved genes will help to clarify the classification of species in the E. phagocytophila genogroup. The use of MboII and other restriction enzymes could be used as a means of rapid diagnosis of granulocytic Ehrlichia of the E. phagocytophila genogroup, and as a tool in risk analysis studies.

Heartwater is a severe disease of cattle, sheep and goats caused by the rickettsia Cowdria ruminantium and transmitted by ixodid ticks of the genus Amblyomma. Cowdria isolates made as blood stabilates by infection of susceptible sheep with organisms derived from Amblyomma ticks were assessed for their virulence and other vital profiles. One millilitre of blood stabilate was inoculated into each naive Corriedale sheep. The sheep were monitored daily by rectal temperature and for clinical signs and examined for post mortem lesions when they died. Mice were inoculated with 0.2 ml of fresh febrile blood collected at the time of high fever, monitored on a daily basis for clinical signs and post mortems were carried out when the mice died. The serological response of the sheep was determined using the competitive-ELISA test while for the mice indirect-ELISA was used. Mice were sacrificed 28 days minimum post inoculation; organ homogenates were prepared and used to infect a susceptible sheep. Demonstration of colonies of the organism in sheep brain and neutrophil cultures was used as confirmation of infection with the agent for each isolate. The analysis of clinical and post mortem parameters showed that the isolates were different from each other in a qualitative and quantitative way. Heartwater isolates could be classified according to variables in sheep and Balb/c mice. Two isolates infected and killed mice while ail but one caused fatal infections in sheep. These characteristics could be a reflection of their genetic divergence, which might be traced in the DNA sequence of the organisms. This is the second lime Cowdria isolates have been made in Kenya but the first time Kenyan isolates from different geographical regions of the country have been compared with each other. The study reveals more about Cowdria organisms and the disease they cause. It also brings to light the magnitude of a potential health problem should farming practices shift from current routines in a fundamental way in this country. The significance of differences between these isolates will be mentioned with regard to cross immunity.

ABSTRACT 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.

Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, has been cultivated in two tick cell lines, the Ixodes scapular is line IDE8 and an Amblyomma variegatum line AVL/CTVM13. In Giemsa-stained preparations, polymorphic rickettsiae are seen intracellularly within cytoplasmic vacuoles and extracellularly, liberated from damaged cells. Some organisms appear similar to the large ring and horseshoe forms seen in vivo in brain capillaries, others resemble immature morulae seen in mammalian endothelial cell cultures, but the typically spherical elementary body stage produced in endothelial cells has not been observed. Transmission electron micrographs of C. ruminantium-infected tick cells reveal membrane-lined vacuoles containing between 2 and 100 rickettsial organisms 0.4-2.0 mum in size. The organisms have double membranes and are polymorphic, reticulated or occasionally electron-dense in appearance, and are apparently undergoing binary fission, resembling the stages described in nymphal' and adult Ambiyomma gut epithelium and salivary glands. Some rickettsial colonies contain a homogeneous electron-dense inclusion body, or tiny vesicles in the space between the organisms. In AVL/CTVM13 cells some vacuoles appear to contain subgroups of rickettsiae within a second double membrane.

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.