The evolution and phylodynamics of serotype A and SAT2 foot-and-mouth disease viruses in endemic regions of Africa.

Foot-and-mouth disease (FMD) is a major livestock disease with direct clinical impacts as well as indirect trade implications. Control through vaccination and stamping-out has successfully reduced or eradicated the disease from Europe and large parts of South America. However, sub-Saharan Africa remains endemically affected with 5/7 serotypes currently known to be circulating across the continent. This has significant implications both locally for livestock production and poverty reduction but also globally as it represents a major reservoir of viruses, which could spark new epidemics in disease free countries or vaccination zones. This paper describes the phylodynamics of serotypes A and SAT2 in Africa including recent isolates from Cameroon in Central Africa. We estimated the most recent common ancestor for serotype A was an East African virus from the 1930s (median 1937; HPD 1922-1950) compared to SAT2 which has a much older common ancestor from the early 1700s (median 1709; HPD 1502-1814). Detailed analysis of the different clades shows clearly that different clades are evolving and diffusing across the landscape at different rates with both serotypes having a particularly recent clade that is evolving and spreading more rapidly than other clades within their serotype. However, the lack of detailed sequence data available for Africa seriously limits our understanding of FMD epidemiology across the continent. A comprehensive view of the evolutionary history and dynamics of FMD viruses is essential to understand many basic epidemiological aspects of FMD in Africa such as the scale of persistence and the role of wildlife and thus the opportunities and scale at which vaccination and other controls could be applied. Finally we ask endemic countries to join the OIE/FAO supported regional networks and take advantage of new cheap technologies being rolled out to collect isolates and submit them to the World Reference Laboratory.

Geographical and age-stratified distributions of foot-and-mouth disease virus-seropositive and probang-positive cattle herds in the Adamawa province of Cameroon.

Six of the seven known serotypes of foot-and-mouth disease (FMD) virus occur in Africa. This paper describes the results of a population-based cross-sectional study of the seroprevalence of FMD and the persistence of the virus in cattle herds and associated sheep flocks in the Adamawa province of Cameroon. Antibody titres measured by the virus neutralising test indicated that serotypes O, A and SAT2 viruses had been circulating in the province. The estimates of apparent seroprevalence in cattle herds, based on five juvenile animals (eight to 24 months old) per herd, were 74.8 per cent for serotype SAT2, 30.8 per cent for serotype A and 11.2 per cent for serotype O, indicating recent exposure; the estimates based on animals more than 24 months of age were 91.1 per cent for SAT2, 83.6 per cent for A and 34.2 per cent for serotype O. Epithelial and oropharyngeal samples were collected from cattle and small ruminants, cultured and typed by ELISA; serotypes A and SAT2 were isolated from both types of sample. The herd-level estimate of apparent prevalence of probang-positive herds was 19.5 per cent and the animal-level estimate of apparent prevalence was 3.4 per cent. The geographical distribution of the seropositive herds based on juveniles suggested that recent SAT2 exposure was widespread and particularly high in the more northern and western parts of the province, whereas recent exposure to serotype A was patchy and more concentrated in the south and east. This distribution corresponded very closely with the distribution of herds from which virus was recovered by probang, indicating recent exposure or infection. No serotype O viruses were recovered from cattle, and the distribution of seropositive herds suggested very localised recent exposure. The apparent prevalence of probang-positive animals declined with the age of the animal and the period since the last recorded outbreak in the herd.

Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship.

Filarial nematodes are important and widespread parasites of animals and humans. We have been using the African bovine parasite Onchocerca ochengi as a chemotherapeutic model for O. volvulus, the causal organism of 'river blindness' in humans, for which there is no safe and effective drug lethal to adult worms. Here we report that the antibiotic, oxytetracycline is macrofilaricidal against O. ochengi. In a controlled trial in Cameroon, all adult worms (as well as microfilariae) were killed, and O. ochengi intradermal nodules resolved, by nine months' post-treatment in cattle treated intermittently for six months. Adult worms removed from concurrent controls remained fully viable and reproductively active. By serial electron-microscopic examination, the macrofilaricidal effects were related to the elimination of intracellular micro-organisms, initially abundant. Analysis of a fragment of the 16S rRNA gene from the O. ochengi micro-organisms confirmed them to be Wolbachia organisms of the order Rickettsiales, and showed that the sequence differed in only one nucleotide in 858 from the homologous sequence of the Wolbachia organisms of O. volvulus. These data are, to our knowledge, the first to show that antibiotic therapy can be lethal to adult filariae. They suggest that tetracycline therapy is likely to be macrofilaricidal against O. volvulus infections in humans and, since similar Wolbachia organisms occur in a number of other filarial nematodes, against those infections too. In that the elimination of Wolbachia preceded the resolution of the filarial infections, they suggest that in O. ochengi at least, the Wolbachia organisms play an essential role in the biology and metabolism of the filarial worm.

Cattle protected from onchocerciasis by ivermectin are highly susceptible to infection after drug withdrawal.

Ivermectin administration is now the major tool in the control of human onchocerciasis (caused by Onchocerca volvulus) based on its suppression of microfilariae and hence the prevention of disease. However, in Africa, transmission is not eliminated and treated populations continue to be exposed to infective larval (L(3)) challenge, albeit at reduced levels. We have investigated whether protective immunity might develop under such conditions using the analogous host-parasite system Onchocerca ochengi in cattle, based on our previous findings in cattle exposed to challenge, that in vivo ivermectin attenuates the development of adult infections and that irradiation-attenuated L(3) induce significant protection. In a two-phase prospective study over 4 years, groups of cattle were exposed to severe natural challenge. In the first phase, 38/40 animals treated either with ivermectin or with moxidectin at either monthly or 3-monthly intervals had not developed detectable infections after 22 months of exposure whereas, in a non-treated control group (n = 14) nodule prevalence was 78.6% and the geometric mean (range) nodule load was 4.8 (0-33). In the second phase, all drug treatments were withdrawn, a new control group (n = 8) introduced, and exposure continued at the same site. After 24 months, all groups had developed patent infections, with geometric mean (range) nodule loads of 17.4 (4-99), 38.4 (10-111), 50.7 (26-86), 14.3 (0-69) and 14.7 (0-55) for the control, monthly-ivermectin, 3-monthly ivermectin, monthly moxidectin and 3-monthly moxidectin groups, respectively. There was no evidence of protection-indeed the 3-monthly ivermectin group was significantly (P < 0.05) hyper-susceptible. In addition, microfilarial densities and the rate of increase in microfilarial load were significantly higher (P < 0.05) in the ivermectin-treated groups than in control animals. These results have important implications for ivermectin-based control of human onchocerciasis and suggest that humans exposed to ongoing transmission in endemic areas whilst receiving ivermectin are unlikely to develop immunity and will be highly susceptible should drug distribution cease.

Evaluation of Culicoides insignis (Diptera: Ceratopogonidae) as a vector of bluetongue virus.

Based upon epidemiological evidence, Culicoides insignis Lutz is a probable biological vector of bluetongue viruses (BTV) in South Florida, the Caribbean Region and Central America. The vector potential of this species for BTV was evaluated in the laboratory in a series of experiments using insects caught in the field. Although there was great variation in the percentage of flies that fed from any one catch, it was demonstrated that C. insignis became infected after membrane feeding on a mixture of blood and virus. The infection rates ranged from 20 to 62.5%. Following intrathoracic inoculation, BTV replicated to high titres in C. insignis. Such flies were also shown to be capable of transmitting BTV to susceptible sheep and embryonated chicken eggs. This series of experiments provides the first conclusive evidence that C. insignis is a biological vector of bluetongue virus. This is the first proven vector of BTV in the neotropics.

Vector competence parameters of Culicoides variipennis (Diptera: Ceratopogonidae) for bluetongue virus serotype 2.

Culicoides variipennis (Coquillett), the only proven vector of bluetongue virus (BLU) in the western hemisphere, was evaluated as a vector of bluetongue virus serotype 2 (BLU 2). This serotype was isolated from sentinel cattle in south Florida at a site devoid of C. variipennis. Culicoides variipennis readily fed on a mixture of defibrinated blood and BLU 2 through chicken skin membrane. An infection rate of 46.2% was obtained. A growth curve of virus titers recovered from orally infected flies showed a linear relationship between the virus titers and the period of extrinsic incubation. Culicoides variipennis also became infected when inoculated intrathoracically with BLU 2. Peak titers were higher and more rapidly attained in inoculated flies when compared with orally infected flies. Infected C. variipennis also transmitted BLU 2 to sheep via bite. These results demonstrate that C. variipennis is a potential biological vector of BLU 2 in the laboratory. The implication of this on the epidemiology of BLU 2 in the United States is that BLU 2 should have become more widespread in ruminants in the United States. The fact that this has not occurred during the past 10 yr is discussed.

Risk factors for herdsman-reported foot-and-mouth disease in the Adamawa Province of Cameroon.

We analysed responses from 147 Fulani herdsmen to a questionnaire about cattle herd-level risk factors for foot-and-mouth disease (FMD) in the previous year. The study used a cross-sectional design with a stratified, two-stage random sample of cattle herds in the Adamawa Province of Cameroon. The questionnaire was pre-tested at a local cattle market before a final version was translated into Foulfoulde (the local Fulani dialect). Variables were screened using a univariable analysis and logistic multiple-regression models were developed in a forward-selection process. Fifty-eight percent (50-65; 90% CIs) of herdsmen reported FMD in their herd in the previous 12 months. Important risk factors for FMD in the previous 12 months included going on transhumance (OR=2.6), buying cattle from markets (OR=2.2), mixing of herds at watering points (OR=2.4), feeding cotton-seed cake (OR=3.3), buffalo near the herd (OR=2.2) and administrative division. For the subset of herds that went on transhumance, coming in contact with an FMDV-diseased herd while on transhumance was the strongest factor (OR=16).

Viral haemagglutination of glutaraldehyde-fixed sheep erythrocytes.

Sheep erythrocytes stabilized by treatment with glutaraldehyde were used successfully in haemagglutination tests with Newcastle disease, and equine influenza viruses. The fixation enabled storage of the cells for at least 21 days without altering their agglutinable properties. Stability was best when the cells were stored at -114 degrees C in the vapour phase of liquid nitrogen.

Foot and mouth disease and livestock husbandry practices in the Adamawa Province of Cameroon.

Foot and mouth disease (FMD) is a highly contagious viral disease of even-toed ungulates and is endemic in most of the tropics. A cross-sectional study using a stratified, two-stage random sample design was undertaken in the Adamawa Province of Cameroon. The objectives were to measure the reported herd-level prevalence of FMD and a range of husbandry practices important for its transmission. The owner-reported prevalence for the previous 12 months was 57.9% (50.4-65.4%), although there was a significant variation across the Province. During the previous dry season, 46.5% (38.6-54.4%) of herds had gone on transhumance. Herds had high numbers of contacts with other herds while on transhumance (98.6%), at pasture (95.8%) and at night (74.4%), with medians of 7-10, 4-6 and 1-3 daily contacts, respectively. The high level of endemic FMD and potential for disease spread presents a significant challenge for control and eradication. Locally sustainable methods need to be developed upon which larger regional control programmes could be built in the future.

Comparison of two 3ABC enzyme-linked immunosorbent assays for diagnosis of multiple-serotype foot-and-mouth disease in a cattle population in an area of endemicity.

The development of a serological test for foot-and-mouth disease virus (FMDV) which is quick and easy to use, which can identify all seven serotypes, and which can differentiate vaccinated from convalescing or potential virus carriers would be a major advance in the epidemiological toolkit for FMDV. The nonstructural polyprotein 3ABC has recently been proposed as such an antigen, and a number of diagnostic tests are being developed. This paper evaluates the performance of two FMDV tests for antibodies to nonstructural proteins in an unvaccinated cattle population from a region of Cameroon with endemic multiple-serotype FMD. The CHEKIT-FMD-3ABC bo-ov (CHEKIT) enzyme-linked immunosorbent assay (ELISA) (Bommeli Diagnostics/Intervet) is a commercially available test that was compared with a competitive 3ABC ELISA (C-ELISA) developed in Denmark. The tests were compared with the virus neutralization test as the "gold standard." Diagnostic sensitivity and specificity were examined over a range of test cutoffs by using receiver operating characteristic curves, which allowed comparison of the overall performance of each test. The results indicated that the CHEKIT ELISA kit was 23% sensitive and 98% specific and the Danish C-ELISA was 71% sensitive and 90% specific at the recommended cutoff. These results have important implications if the tests are to be used to screen herds or individual cattle in surveillance programs, at border crossings for import-export clearance, or following emergency vaccination in an outbreak situation.

Molecular epidemiology of foot-and-mouth disease viruses in the Adamawa province of Cameroon.

Foot-and-mouth disease virus (FMDV) causes a highly contagious viral disease of even-toed ungulates and is one of the most important economic diseases of livestock. Most studies of FMDV are done in countries where control measures are being implemented. In contrast, in areas such as sub-Saharan Africa, where FMDV is endemic and new strains are likely to emerge, there are only sporadic submissions to the World Reference Laboratory, Pirbright, United Kingdom. This paper describes the molecular epidemiology of FMDV in the Adamawa province of Cameroon based on a population sample of cattle herds. Serotypes SAT2 and A were isolated in the cross-sectional study. SAT2 isolates were all similar, with phylogenetic distances of <6%, and were most closely related to published sequences of isolates from Eritrea and Saudi Arabia. Serotype A isolates were more variable, with phylogenetic distances of 0 to 11%, and were most closely related to historic isolates from Cameroon. Use of a population-based sample gives a representative sample of virus diversity and will improve our understanding of the evolution of FMDV and its epidemiology. A supplementary study of pigs passing through the railhead collection yard at Ngaoundere detected a serotype O virus. A third pilot longitudinal study monitored viral persistence in three cattle herds over 12 months, and serotype O and A viruses were recovered from a herd 12 months after it was first recorded as being infected with SAT2 virus. The pig type O isolate was not closely related to that recovered from the cattle, suggesting that the pigs had not introduced the O virus into the cattle herds.

Susceptibility of the Namchi and Kapsiki cattle of Cameroon to trypanosome infection.

Two indigenous Cameroonian taurine cattle breeds (Namchi and Kapsiki) were evaluated for trypanosusceptibility following inoculation with Trypanosoma congolense. The degree of zebu ancestry in the experimental animals was assessed using 6 microsatellite markers which are known to have certain unique alleles which are diagnostic of Bos indicus genetic input. Their response to the infection was compared to that of known trypanotolerant (N'Dama) and trypanosusceptible (Ngaoundere Gudali) cattle. The Namchi and the N'Dama controlled the development and severity of anaemia and parasitaemia better than the Kapsiki and the Gudali. For these parameters, there was no significant difference between the N'Dama and Namachi nor between the Kapsiki and Gudali. Similarly, weight loss showed significant breed variation. The N'Dama lost the least weight and the Kapsiki the most. Zebu introgression in the Namchi was comparable to that in the N'Dama while that of the Kapsiki breed was higher, indicating a high level of cross breeding. From the results, the Namchi are considered trypanotolerant while the Kapsiki are trypanosusceptible. The potential exploitation of the indigenous Namchi cattle is discussed.