First molecular evidence of potential Culicoides vectors implicated in bluetongue virus transmission in Morocco.

BACKGROUND: Bluetongue is a non-contagious viral disease that affects both domestic and wild ruminants. It is transmitted primarily by small hematophagous Diptera belonging to the genus Culicoides (Diptera: Ceratopogonidae). The current study represents the first molecular investigation into the potential role of Culicoides imicola, Culicoides paolae, Culicoides newsteadi, Culicoides spp., and Culicoides circumscriptus as bluetongue virus (BTV) vectors in Morocco. Additionally, the study aimed to evaluate the vectorial activity of midges during the survey seasons. METHODS: Parous females of these species were captured from several regions of Morocco (6 out of 12) from 2018 to 2021 using Onderstepoort Veterinary Institute (OVI) traps. A total of 2003 parous female specimens were grouped into 55 batches. The midge body of each batch was dissected into three regions (head, thorax, and abdomen), and these regions were analyzed separately using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: BTV RNA was detected in 45 out of the 55 batches tested, indicating a positivity rate of 81.8%. The RT-qPCR-positive pools of the studied Culicoides species exhibited high levels of BTV positivity in each body part (head, thorax, and abdomen), confirming the successful replication of the virus within midge bodies. The BTV circulation was substantial across all three survey seasons (spring, summer, and autumn). High infection rates, calculated using the minimum infection rate (MIR) and maximum likelihood estimation (MLE), were observed during the collection seasons, particularly in autumn and spring, and for all investigated Culicoides species, most notably for C. imicola and C. newsteadi. These increased infection rates underscore the significant risk of Culicoides transmitting the BTV in Morocco. CONCLUSIONS: The detection of BTV positivity in Culicoides spp. (lacking wing spots that allow their differentiation according to morphological identification keys) suggested that other Culicoides species are competent for BTV transmission in Morocco. The study results indicated, for the first time at the molecular level, that C. imicola and C. newsteadi are the primary potential vectors of BTV in Morocco and that C. paolae and C. circumscriptus are strongly implicated in the propagation of bluetongue at the national level.

Serological and Molecular Characterization of Small Ruminant Lentiviruses in Morocco.

Recent studies that investigated the origins of SRLV strains offered new insights into their distribution among domestic ruminants. The aim of the study was to investigate SRLV circulation in Morocco. A total of 51 farms were selected in different geographical locations and tested by screening and genotyping ELISA. Whole blood was used for DNA extraction and nested gag PCR. The sample size allowed for an estimation of prevalence lower than 20% (CI 95%). Surprisingly, a large proportion of screening-positive samples were not correctly serotyped. Sanger and NGS amplicon sequencing approaches allowed us to obtain new sequences even from difficult-to-amplify samples. The serological data support the evidence of an intrinsic difficulty of SRLV to spread, likely due to management practices. The low rate of success by genotyping ELISA led us to suppose that divergent strains might have escaped from diagnostic tools, as partially confirmed by the evidence of an A subtype carrying a mismatch in serotyping epitope. The sequence analysis revealed the circulation of novel B and recombinant A/B subtypes. This study highlights the importance of monitoring viral sequences and their evolution to develop specific diagnostic tests, particularly in countries where control measures are in place.

Serological and molecular prevalence study of bluetongue virus in small domestic ruminants in Morocco.

Bluetongue is an arthropod-borne viral disease transmitted by Culicoides biting midges, affecting domestic and wild ruminants. The current study aims to assess the seroprevalence of the bluetongue virus (BTV) and confirm its active circulation among sheep and goats populations in Morocco, as well as study the risk factors associated with BTV infection. To this end, a total of 1651 samples were randomly collected from 1376 sheep and 275 goats in eight (out of 12) regions of the country between March 2018 and July 2021.These samples were primarily tested using competitive ELISA (c-ELISA). Subsequently, 65% of c-ELISA positives (n = 452) were analyzed by real-time reverse transcription-polymerase chain reaction (RT-qPCR). The results revealed an overall BTV seroprevalence in small ruminants in Morocco of 41.7%, including 42.6% in sheep and 37.5% in goats. The RT-qPCR results showed that the overall BTV viropositivity rate was 46.7%, including 48.1% in sheep and 41.8% in goats. These viro-serological rates varied significantly by age, sex, and breed of the tested animals, husbandry method, season, and geographic origin. This indicates that these parameters constitute risk factors for BTV transmission routes in Morocco. The findings also indicate that goats play a role as reservoirs in maintaining the BTV in Morocco. It appears from this study that bluetongue is endemic in Morocco. The environmental and climate conditions as well as the husbandry methods adopted in the country are particularly favorable for the virus transmission throughout the country.