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.

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 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.

Using geostatistics to better understand the epidemiology of animal rabies in Morocco: what is the contribution of the predictive value?

This study aims to characterize the spatial distribution of animal rabies in Morocco in order to provide appropriate control approaches. Descriptive analyses of the epidemiological data show that the number of reported canine rabies cases greatly underestimates the true incidence of the disease. Underreporting subsequently affects the coherence of its spatial distribution. To perform accurate geographic distribution mapping of the disease based on interpolation methods, a data set was created using data between 2000 and 2018 to compare the derived disease cases with known true values in order to identify disease clusters. The subsequent interpolation was conducted using Ordinary Kriging regression methods and the semi variogram to focus on short distances and reduce uncertainty. The estimated clusters of rabies were evaluated using a cross validation step which revealed predicted cases close to the true values. To improve the precision of analysis, the authors displayed georeferenced dog and human rabies cases reported during the last three years, demonstrating reliable results that correspond to the estimated cluster areas similar to the true disease incidence on the field. This work highlights a strong correlation between infrastructure projects (i.e. railways, roads, facilities) and rabies epizootics for several specific locations. This study is the first attempt to use geostatistics to build upon the understanding of animal rabies in Morocco and shed light on the most appropriate strategies to sustainably reduce and mitigate the risk of rabies. There has been little literature on the use of kriging methods in animal health research. Thus, this study also aimed to explore a novel method in the veterinary sciences to establish kriging as a valid and coherent analysis tool to identify the extent to which the geostatistic area can objectively support understanding on animal rabies and saw it as being highly instrumental in coping with gaps in the data.

Evaluation of integrated control of three dog transmitted zoonoses: Rabies, visceral leishmaniasis and cystic echinococcosis, in Morocco.

The present study is a component of a major European research project, ICONZ (Integrated Control of Neglected Zoonoses). The objective of this component was to implement, and then evaluate an Integrated Control Intervention (ICI) against three dog transmitted zoonoses, namely rabies, visceral leishmaniasis, and cystic echinococcosis. This was undertaken in Sidi Kacem Province (northwestern Morocco) where 22 control and 22 treated douars (villages) were randomly allocated to two control and one evaluation interventions over a period of 13 months. Across the 44 douars, an overall total of 6922 dogs were registered, 4519 were vaccinated against rabies, and 2885 persons attended the complementary health education campaigns; whereas, within the 22 treated douars, 466 dogs received anti-sandfly collars (deltamethrin-impregnated) and 2487 were dewormed (praziquantel). Evaluation of the ICI, revealed that (i) a canine rabies vaccination coverage of 65.0% was achieved, (ii) the use of deltamethrin-impregnated collars for dogs provided highly significant protection (p = 0.01) against leishmaniasis infection of more than 44%, and up to 100% for dogs that had kept the collar until the end of the intervention, (iii) despite a non-significant difference (p>0.05), dog-deworming with praziquantel yielded a reduction in the rate of Echinococcus granulosus infection, and (iv) health education was successful in improving respondents' knowledge; However, the target communities remained unconvinced of the necessity of changing some of their risky behaviours. Lastly, the estimated total cost of the global intervention, including its research components, was US$ 143,050, of which 67% was for disease control work with clear evidence of significant economies of scale due to targeting three diseases together. It appears from this study that the integrated control approach against the three zoonoses was effective on both economic and logistical levels.

Sheep pestivirus in Morocco: sero-epidemiological and molecular study.

The present study is the first to investigate Border disease caused by the sheep pestivirus (SPV) in sheep herds in Morocco. Sero-epidemiological investigations were carried out in six regions of the Kingdom, known as important in terms of sheep breeding. A total of 760 blood samples were collected including aborted ewes from 28 randomly selected farms. The samples were analysed, for the determination of anti-pestivirus antibodies, using indirect ELISA technique. Next, reverse transcriptase PCR (RT-PCR) was conducted on serologically negative samples to identify possible persistently infected (PI) animals, through detection of specific RNA fragment. The results revealed an overall SPV seroprevalence in studied areas of 28.9%. The difference in seroprevalence between the six investigated regions was not statistically significant (p>0.05) and varied slightly from 20.9% to 37.5%. Furthermore, 93% of investigated farms were affected with an average seroprevalence of 22.7% (with a variation of 1%-74%). RT-PCR results were all negative, indicating the absence of PI animals in the tested samples. Nevertheless, the present study revealed that SPV is endemic in Morocco.

Risk factors for MERS coronavirus infection in dromedary camels in Burkina Faso, Ethiopia, and Morocco, 2015.

Understanding Middle East respiratory syndrome coronavirus (MERS-CoV) transmission in dromedary camels is important, as they consitute a source of zoonotic infection to humans. To identify risk factors for MERS-CoV infection in camels bred in diverse conditions in Burkina Faso, Ethiopia and Morocco, blood samples and nasal swabs were sampled in February-March 2015. A relatively high MERS-CoV RNA rate was detected in Ethiopia (up to 15.7%; 95% confidence interval (CI): 8.2-28.0), followed by Burkina Faso (up to 12.2%; 95% CI: 7-20.4) and Morocco (up to 7.6%; 95% CI: 1.9-26.1). The RNA detection rate was higher in camels bred for milk or meat than in camels for transport (p = 0.01) as well as in younger camels (p = 0.06). High seropositivity rates (up to 100%; 95% CI: 100-100 and 99.4%; 95% CI: 95.4-99.9) were found in Morocco and Ethiopia, followed by Burkina Faso (up to 84.6%; 95% CI: 77.2-89.9). Seropositivity rates were higher in large/medium herds (≥51 camels) than small herds (p = 0.061), in camels raised for meat or milk than for transport (p = 0.01), and in nomadic or sedentary herds than in herds with a mix of these lifestyles (p < 0.005).

Use of FTA® card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa.

Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA® card technology. The DNA samples were taken by applying the FTA® paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA® samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries.

Knowledge, attitudes and practices with regard to the presence, transmission, impact, and control of cystic echinococcosis in Sidi Kacem Province, Morocco.

BACKGROUND: This study is a component of a large research project on five major neglected zoonotic diseases (NZDs) including cystic echinococcosis and was undertaken in the Province of Sidi Kacem over a period of four years (April 2009-March 2013). METHODS: Questionnaires were administered at community level in a total of 27 communes and visits were made to all of the 10 abattoirs situated in the Province, to collect qualitative data on determinants of transmission for disease in humans and animals. More specifically, community knowledge, attitudes and practices related to cystic echinococcosis were assessed, as well as the extent to which local customs and behaviours may promote transmission. Abattoir infrastructure and practices, and their role in perpetuating disease transmission were also critically evaluated. RESULTS: The results show that only 50 % of people have heard of the disease, and of those, only 21 % are aware of the dog's role in disease transmission. Sixty-seven per cent of respondents stated that dogs are fed ruminant organs deemed unfit for human consumption. Owned dogs have access to the family home, including the kitchen, in 39 % of households. The extent of this close proximity between humans and animals is even more pertinent when one considers that dogs are omnipresent in the community, with an average of 1.8 dogs owned per household. The unrestricted access of dogs to abattoirs is a huge issue, which further promotes disease transmission. CONCLUSION: This study would suggest that the high prevalence of cystic echinococcosis in humans and animals in Morocco is largely due to three factors: 1) abundance of dogs 2) engagement in risky behaviour of the local population and 3) poor abattoir infrastructure and practices. This has serious implications in terms of the socio-economic impact of the disease, especially for rural poor communities.