Serological and virological responses in mules and donkeys following inoculation with African horse sickness virus serotype 4.

Two groups, comprising 4 donkeys and 4 mules (group 1) and 4 donkeys and 3 mules (group 2), were used to determine the duration of viraemia and to monitor the development of antibodies following inoculation with African horse sickness virus (AHSV). One group of animals was given a single dose of attenuated AHSV serotype 4 (AHSV 4) vaccine. The second group was inoculated with a virulent field strain of AHSV 4. Both groups were subsequently challenged with the virulent field strain of AHSV 4, 51 and 58 days, respectively, after their primary inoculation. Blood and serum samples, collected on alternate days after the primary inoculations and also after subsequent challenge, were assayed for virus and antibodies. Seven of the 8 AHSV vaccinated (group 1) and 7 of the 7 AHSV inoculated (group 2) animals showed humoral antibody responses after primary inoculation. Although no infectious virus could be isolated from either group for the duration of the study, reverse transcription-PCR data obtained for the second group did show the presence of AHSV viral RNA from as early as day 5 in mules and day 9 in donkeys after the primary inoculation. Viral RNA was detected consistently up to day 47 in some animals and intermittently thereafter. There was no evidence of a second viraemia in any of the animals after challenge. The detection of specific antibodies, against AHSV 4 NS3 protein, in all animals confirmed that both donkeys and mules were infected and that the virus had replicated.

The Culicoides vectors of African horse sickness virus in Morocco: distribution and epidemiological implications.

African horse sickness (AHS) is a vector-borne, infectious disease of equids caused by African horse sickness virus. The only proven field vector of the virus is the biting midge Culicoides imicola, although C. obsoletus and C. pulicaris are suspected vectors. In 1994-5 a total of 3887 light trap samples were collected from 22 sites distributed over most of Morocco. Culicoides imicola was found to be very widely distributed with the greatest catches in the low-lying north-western areas (between Tangier and Rabat) and at Marrakech. Culicoides imicola was absent at one site only, near Settat. In general, the catch of C. imicola peaked in late summer and autumn, with a smaller peak in spring. Catches of C. obsoletus were greatest in the north-western provinces of Morocco and in the south, while catches of C. pulicaris were greatest in the north. Although both species were widely distributed, trap catches were much lower than those of C. imicola. Peak catches were in spring or late summer and autumn. In general, the findings for C. imicola correspond well with the seasonal and spatial distribution of disease outbreaks during the 1989-1991 epizootic of AHS in Morocco. It is suggested that C. obsoletus and C. pulicaris were probably of little significance in the epidemiology of AHS in Morocco in 1989-91.

Studies of the mortality rate of Culicoides imicola in Morocco.

Daily mortality rates of female Culicoides imicola were found for eight sites in Morocco in 1994 and for six sites in 1995. The mortality rates were found by operating Pirbright-type light traps for a number of consecutive nights in late summer or autumn and finding the parous rate assuming a feeding interval of 3 to 5 days. The mortality rates were calculated according to established methods. In Morocco the daily mortality rates were found to vary from about 5% per day (Arbaoua, 1994, 1995 and Sidi Moussa 1995) up to 20-25% per day (Berkane, Marrakech, Tangier). In general, estimates of daily mortality rate were consistent between the two years of study. Among sites, daily mortality rate was significantly correlated with the average night-time minimum wind speed but not mean or maximum night-time wind speeds, or with temperature, humidity or saturation deficit. The observed mortality rates suggest that at Arbaoua, were 1,000 flies to become infected with African horse sickness virus, at least 330 would live long enough to take 3 or more infective blood meals on hosts. At Berkane, the survival rate per 1,000 is less than 10. In general, the pattern observed for daily mortality rate, combined with the relative population sizes of C. imicola in Morocco, agree well with the observed distribution of African horse sickness in the country during the 1989-1991 epizootic.

Application of an indirect fluorescent antibody assay for the detection of African horse sickness virus antibodies.

An indirect fluorescent antibody (IFA) technique was used to screen and quantify antibodies against African horse sickness virus (AHSV) in equine sera. Results obtained with the IFA assay were compared directly with those obtained with standard complement fixation (CF) and virus neutralisation (VN) tests using horse sera from experimental studies and samples from the field. Positive fluorescent antibody titres were detected from as early as 7 days after primary vaccination and persisted for at least six months. The IFA technique offers a clear advantage over CF tests, where the antibodies are often of shorter duration and where sera from donkeys and mules are frequently anticomplementary. The sensitivity and specificity of the IFA test compared with the VN test were 98% and 83.3%, respectively. The IFA test is rapid, relatively easy to perform and inexpensive, and can be recommended as an alternative assay for screening different species of equidae in AHSV control and surveillance programmes.

Use of climatic data and satellite imagery to model the abundance of Culicoides imicola, the vector of African horse sickness virus, in Morocco.

African horse sickness (AHS) is a vector-borne, infectious disease of equids caused by African horse sickness virus (AHSV). The only proven field vector of the virus is the biting midge Culicoides imicola. Following a recent epizootic (1989-91) of AHS in Morocco, light traps and automatic weather stations were operated for 2 years at twenty-two sites distributed over much of the country. The annually-averaged mean daily trap catch of C. imicola at these sites was negatively correlated with wind speed, and positively correlated with the average and mean annual minimum NDVI (Normalized Difference Vegetation Index, a remotely sensed measure of vegetation activity). There were no significant correlations between the mean daily trap catch and air temperature, soil temperature, relative humidity, saturation deficit, rainfall, altitude or the mean annual maximum or range of NDVI. The best two-variable model, which combined WindspeedMnAvMn (the average daily minimum wind speed of the least windy month) and NDVImin (the average annual minimum NDVI) as predictors, explained over 50% of the variance in the annually-averaged mean daily trap catch of C. imicola. There was a significant, positive correlation between minimum wind speed at night and the daily mortality rate of adult female C. imicola and it is suggested that the relationship between wind speed and the abundance of C. imicola arises from effects on adult mortality or dispersal. Considering several climatic variables, in North Africa NDVImin was most significantly correlated with total annual rainfall. It is suggested that the relationship between NDVImin and the abundance of C. imicola arises from the impact of soil moisture on both. It is proposed that areas of Morocco with higher levels of soil moisture in late summer or autumn provide more, larger and/or more enduring breeding sites for C. imicola, as well as supporting more photosynthetically active vegetation and hence having higher NDVI.

The spatial and seasonal distribution of African horse sickness and its potential Culicoides vectors in Morocco.

African horse sickness (AHS) is a vector-borne, infectious disease of equines that is caused by African horse sickness virus (AHSV). The only proven field vector is the biting midge Culicoides imicola, although C. obsoletus and C. pulicaris are suspected vectors. There was a recent epizootic of AHS in Iberia (1987-90) and Morocco (1989-91). In 1994-45 a total of 3887 light trap samples were taken from twenty-two sites distributed over most of Morocco. Culicoides imicola was found to be very widely dispersed, with the greatest catches in the low-lying northwestern areas (between Tangier and Rabat) and at Marrakech. Culicoides imicola was absent at one site only, near Settat. Culicoides imicola was found at altitudes ranging from 4 to 1275 m and in climatic conditions ranging from subhumid to saharan. In general, the catch of C.imicola peaked in late summer and autumn, with a smaller peak in spring. In areas where the insect appears most abundant at least one adult C.imicola per night may be caught in a light trap at all times of year, thus providing a possible means of viral overwintering. Culicoides obsoletus and C.pulicaris are also widely distributed in Morocco but trap catches were much lower than for C.imicola. Peak catches occurred in spring, and late summer and autumn. Other frequently caught species were C.circumscriptus, C.newsteadi, C.puncticollis and members of the odibilis subgenus. In general, the findings for C.imicola correspond well with the distribution of disease outbreaks during the epizootic. Although disease outbreaks were widespread in the country, the greatest number of reported cases was in the northwest (1989-90); in 1991 there were also significant numbers in Marrakech province. No cases were reported in a large area to the west of the Atlas mountains (including Settat) despite the presence of a large equine population. It is likely that during the epizootic the virus overwintered in the northwest (1989) and in Marrakech province (1990). Disease outbreaks occurred from July to December, with a peak from September to November. An unexplained phenomenon is the large number of reported cases of AHS in mules in Chefchaouen province in 1990, despite the apparent low abundance of C.imicola at a site at Chefchaouen. It is argued that C.obsoletus and C.pulicaris were probably of little significance to the epidemiology of AHS in Morocco in 1989-91.