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The African horse sickness virus in Morocco


This project was designed to gain a clear understanding of the controlling epidemiological factors involved in the persistence of African horse sickness virus (AHSV) in Morocco by :
- Identifying the major insect vector(s) and providing detailed information on its/their distribution, seasonal incidence and population densities;
- Elucidating the role of vaccinated equines and non-horse equines in the maintenance of the virus;
- Assessing the stability of the AHS vaccine virus in use in Morocco, subsequent to its passage through vector insects;
- Investigating the environmental conditions (particularly temperature) necessary for AHSV virogenesis to take place in vector insects;
- Developing appropriate epidemiological models for AHS.

Vector surveillance and satellite imagery
- Culicoides imicola is widely distributed throughout Morocco. It is most abundant in the Northwest and at Marrakech; there is an intermediate abundance at Berkane, Errachidia and Taroudant; and the insect is rare at the remaining sites. At only one site (Settat) was C. imicola apparently absent. The species was found at altitudes ranging from 15 - 1275 m and in climatic conditions ranging from subhumid to Saharan. At some sites (Marrakech, Sablons and Sidi Moussa) C. imicola is present in adult phase throughout the year. Sparingly, there was a strong relationship between abundance and the proportion of the year in which adult activity was detected. There was a peak of abundance of C. imicola at most sites in late summer and early autumn. The only exceptions to this rule were in the extreme Northwest where the peak was less pronounced and in June/July at Arbaoua and in March at Tangier.
- Culicoides imicola activity levels were low at mean air temperatures of less than 15°C or above 30°C; and at minimum relative humidities above 40% (probably days of rain). There was also a significant negative correlation between maximum wind speed and the number of C. imicola captured. Sparingly, analyses of activity data suggest that C. imicola is less active on days when it is dryer and more windy than usual but are more active on days when it is moister and calmer than usual. Activity seems to be independent of whether the day is much cooler or hotter than usual (within the constraints of the < 15°C and > 30°C levels mentioned above).
- The potential AHSV vectors, C. obsoletus and C. pulicaris, are both widely distributed in Morocco but are generally less abundant than C. imicola. Greatest densities of C. obsoletus were in the North of Morocco, particularly at Tangier, while those of C. pulicaris were more north-eastern. Both species occur in adult phase throughout the year. Culicoides obsoletus was most abundant from February to June (peak in April); C. pulicaris also showed a peak in April.
- During the 1989-91 epizootic in Morocco, investigations showed that all cases of AHS were reported between the months of July and December and there was a large peak in the number of reported cases in the month of October. Furthermore, outbreaks were relatively rare in the areas of Morocco where C. pulicaris is common. These data suggest that in Morocco neither C. obsoletus or C. pulicaris contributed significantly to the AHS epizootic.
- Analyses of data identified a strong correlation between the mean abundance of C. imicola and NDVImin . Using NDVImin 4 out 5 of the observed C. imicola "abundant" sites and 9 out of 11 of the observed "rare/absent" sites were correctly predicted. Furthermore, using the same technique, 19 out of 26 C. imicola collecting sites in Iberia were also correctly predicted. It is therefore clear that the relationship between NDVImin and C. imicola extends beyond the Moroccan context.

AHSV in the African donkey and mule, and the European donkey
- ELISA and virus neutralisation results show that detectable AHSV antibodies develop in vaccinated mules and donkeys later than in horses. Furthermore, virus isolation procedures failed to detect the presence of a vaccine virus vitraemia in any of 8 vaccinated mules and donkeys. However, it was shown that some AHS vaccinated and protected horses do develop a viraemia on reinfection, with virulent virus even when this is of the same serotype as the vaccine and even in the face of circulating antibody.
- When non-vaccinated mules and donkeys were infected with virulent ASHV4, viraemia detected by virus isolation or by PCR, and extending for as long as 16 dpi in donkeys (measured by virus isolation) and up to 51dpi in mules (measured by PCR) was recorded. During this work, no signs of clinical disease were observed in any of the 16 African donkeys and mules, and 4 European donkeys.

AHS vaccine virus in vector Culicoides
- AHS vaccine virus as used in Morocco was passaged through vector Culicoides via oral and intrathoracic routes. Progeny virus from the first and second insect passages was then inoculated separately into a total of 8 AHS naive horses. None of the experimental horses developed signs of clinical disease. Neither did either of 2 vaccine control horses that had received standard doses of the vaccine. However 1 of the 2 control horses did develop a viraemia due to the vaccine virus that extended from 8 to 17 days post vaccination.

Effect of temperature on AHSV virogenesis in vector Culicoides and Culicoides survival
- Culicoides survival rates at 10, 15 and 20°C were very similar and 80-90% of midges remained alive after 14 days. At 25°C after the same time period, survival was reduced to 40%. The rate of AHS virogenesis in vector Culicoides and the infection rate were also related to temperature. At 25°C a maximum mean virus titre of 104.3 TCID50 per midge was reached by 9dpi and the infection rate was among 60-80%. At 20°C virogenesis was slower and the maximum titre was reached only after 23 days. The infection rate was also reduced to 50-70%. At 15°C there was an overall decline in virus titre with time although in a few individuals virogenesis up to 104.0 TCID50 per midge did still occur. However, the infection rate at this temperature decreased dramatically to 0-15% by 9dpi. At 10°C there was no detectable virogenesis and all midges tested negative by 13dpi. However, when surviving midges that had been maintained at 10°C for 35 days were returned to 25°C for 3 days, the infection rate increased from an apparent zero to 15.5%.

Epidemiological modelling
- A simulation model was developed to investigate what factors affect the likelihood of an epizootic after the introduction of AHSV into an area. Epizootics were most likely to occur in late summer or autumn and were usually rapid with the time from first to last case being < 4 weeks. If no control measures were taken, most of the horses in a herd were infected by the virus following introduction. The model also predicted a very low prevalence of infected midges even at the peak of an epizootic.

- With respect to vaccination strategies, protecting donkeys as well as horses increased the effectiveness of vaccination. Prevention of 50% of epizootics required > 75% coverage of horses and donkeys, or > 90% of horses alone. Protection after the introduction of the virus was rarely successful in preventing outbreaks though it reduced the number of animals affected during an outbreak. If horses alone were protected, the number of donkeys was the most significant factor determining the level of protection needed to prevent an epizootic. If both hosts were protected, the abundance of other hosts as a source for vector bloodmeals was the most significant factor.

Training in Culicoides biology and identification
- Moroccan participants and interested parties were provided with a comprehensive training course comprising both theoretical and practical components and involving both laboratory and field based studies.

Conclusions and additional comments

Vector surveillance and satellite imagery
- Culicoides imicola is widespread in Morocco and is present in adult phase in some areas throughout the year. The distribution and population peaks of this species coincide in time and space with the recorded outbreaks of AHS (1989-91) while those of the potential AHSV vectors, C. obsoletus and C. pulicaris, do not. It is therefore likely that C. imicola is the only major AHSV vector in Morocco and that C. obsoletus and C. pulicaris made no significant contribution to the AHS epizootics. It is also likely that AHSV overwintered in Morocco by cycling between surviving adult C. imicola and non-horse equines, and possibly vaccinated horses (which animals do not usually exhibit clinical disease).
- Culicoides imicola adults were active > 15°C and < 30°C but within these limits other factors were of greater significance than temperature. Wind speed and humidity are important factors in determining activity levels on a given night. Analyses revealed that the effects of these factors may be a function of their "relative" level (relative to the normal for that time of year) rather than their absolute level.
- The satellite-derived variable NDVImin was found to be a better predictor of the distribution of C. imicola in Morocco than any of the ground-measured variables. This variable was also able to correctly predict the presence or absence of C. imicola at 19 out of 26 sites in Iberia. A map of the NDVImin of Morocco and Iberia can therefore act directly as a preliminary "risk map" for the presence/absence, or abundance of C. imicola in the region. Similar maps for other regions (i.e. Italy, Greece) may provide the basis for detailed ground surveys searching for the vector.

AHSV in the African donkey and mule, and the European donkey
- The results suggest that African donkeys and mules, and European donkeys rarely exhibit obvious clinical signs of AHSV infection. However, all of these non-horse equines are capable of developing a viraemia that may extend well beyond the duration expected in horses. These animals may therefore provide a covert source of virus for vector insects in the field.

AHS vaccine virus in vector Culicoides
- Clinical disease was not observed when AHS type 4 vaccine virus was passaged through vector insects and then inoculated into susceptible horses. To date there is therefore no evidence to suggest that the AHS vaccine viruses as used in Morocco are able to revert to virulence following passage through vector insects.

AHSV vaccinated horses
- Experimental results suggest that some AHSV vaccinated and protected horses may be able to develop a viraemia on reinfection, with virulent virus of the same serotype and even in the face of circulating antibody. Such animals may continue to play a part in the epidemiology of the disease by providing a source of virus for vector insects.

Effect of temperature on AHSV virogenesis in vector Culicoides and Culicoides survival
- The rate of AHSV virogenesis in vector insects is related to temperature as is infection rate and Culicoides survival rate. At low temperatures (- 10°C) AHSV does not replicate in vector Culicoides and apparent infection rates rapidly decrease. However, at these low temperatures the life span of the insect is significantly extended, potentially to as long as 90 days (Boorman 1990). Our results suggest that though AHSV may not replicate in vector Culicoides at low temperatures it is able to persist in them at undetectable levels for considerable periods of time (> 35 days) and then commence replication should temperatures rise. This situation could provide an overwintering mechanism for AHSV in the absence of vertebrate involvement.

Epidemiological modelling
- Midge population size, the recovery rate in horses and time of the year when virus is introduced were the most significant factors in determining whether or not an epizootic of AHS occurred. The uncertainty in inter-bloodmeal interval, removal rate (recovery and mortality) of infectious horses, midge population size and transmission rates were significant factors in the size of an epizootic. These factors should therefore be priorities for empirical research and should be considered in the design of control strategies in areas considered to be "at risk" of virus introduction.

Vaccination strategies
- Protecting donkeys (non-horse equines) as well as horses significantly increased the effectiveness of vaccination. Protection after virus introduction was rarely successful in preventing outbreaks though it did reduce their severity. Should both horses and donkeys be protected then the abundance of other hosts for vector bloodmeals was a significant factor. Thus the prevention of epizootics by vaccination is easier in areas where there few donkeys or where they are also vaccinated or where there are fewer vectors or where other hosts are also available for vector bloodmeals (to increase the apparent iinter-bloodmeal interval by diverting some vector bites to insusceptible hosts).


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Ash Road, Nr Woking
United Kingdom

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