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.

First Report of Low Pathogenic Avian Influenza Subtype H9N2 in African Houbara Bustards (Chlamydotis undulata undulata) and Gamebirds in Morocco: Clinico-Pathological Findings, Molecular Characterization, and Associated Coinfections.

We report in this paper the first detection of low pathogenic avian influenza (LPAI) subtype H9N2 in houbara bustards and in gamebirds in Morocco. Starting in 2019, an increase in mortality rates related to respiratory distress was recorded in these species. Necropsy of the specimens revealed fibrinous sinusitis and tracheitis with intra-bronchial fibrin casts, which are consistent with H9N2 infection in chickens; therefore, implication of the virus in these outbreaks was strongly suspected. Consequently, between January 2020 and June 2023, birds with respiratory signs were necropsied for pathological lesions, tissue samples were examined by histopathology, and samples of trachea, lungs, and cecal tonsils were analyzed using quantitative real-time PCR for the detection of H9N2 virus. In addition, the sequencing of isolates was performed and lastly differential diagnosis with other respiratory pathogens was carried out. During the study period, 93 samples were collected from suspected H9N2 outbreaks, of which 30 tested positive for H9N2 virus: 23 Houbara bustards, 4 partridges, 2 quails, and 1 pheasant. Moreover, sequencing of the HA gene of the virus showed 97.33% nucleotide identity with strains reported previously in broilers in Morocco in 2017 and in 2022. Phylogenetic analysis grouped the Moroccan partridge isolates in the same cluster as viruses isolated in Morocco between 2016 and 2022, Algeria (2017), Burkina Faso (2017), Nigeria (2019), and Togo (2020). Additionally, 10 house sparrows from the premises of these birds were examined for the presence of H9N2 virus, revealing a 30% positivity rate. In conclusion, LPAIV H9N2 is circulating in houbara bustards and gamebirds in Morocco, and house sparrows might be a possible source of the infection. To our knowledge, this is the first report of LPAI H9N2 in the African species of houbara bustards worldwide and in gamebirds in Morocco.

Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens.

Our knowledge of the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could influence host physiology through yet unknown phenomena of potential interest to the fields of health and food production. However, the assembly processes of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in five countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain core viruses were not shared by all host individuals in a given country, and their infection rates fluctuated between countries and years. Moreover, the distribution of coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome influence on mosquito physiology.

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.

Attenuation and genetic characteristics of a Moroccan strain of Camel pox virus.

Camel pox (CML) is a widespread infectious viral disease of camels that causes huge economic losses to the camel industry. In this study, a local strain of Camel pox virus (CMLV) was attenuated by 175 serial passages in Vero cells and the residual pathogenicity and infectivity were tested in naïve camels at 120, 150 and 175 passage levels. Also, the safety and immunogenicity of the 175th passage were evaluated in camels using a dose of 104.0 Tissue Culture Dose 50% (TCID50) and monitored for up to one-year post vaccination (pv) for neutralizing antibody. Seroconversion was noted at day 14 pv with neutralizing antibody titers ranging from 0.5 and 1.6 logs over the one-year of the study. Among 8 camels inoculated with the P175 strain, 4 were challenged at 12-month pv with 105.7 TCID50/ml of the original virulent CMLV and complete protection was recorded in all animals. Whole genome sequencing detected six mutations in the original CMLV strain that were not present in the attenuated 175th passage of this strain. Overall, the findings of this study indicated that the 175th passage of the CMLV was attenuated, safe and afforded protection to camels against virulent CMLV, and is therefore, a promising vaccine candidate for the prevention of CML in camels.

AIMSurv: First pan-European harmonized surveillance of Aedes invasive mosquito species of relevance for human vector-borne diseases.

Human and animal vector-borne diseases, particularly mosquito-borne diseases, are emerging or re-emerging worldwide. Six Aedes invasive mosquito (AIM) species were introduced to Europe since the 1970s: Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus. Here, we report the results of AIMSurv2020, the first pan-European surveillance effort for AIMs. Implemented by 42 volunteer teams from 24 countries. And presented in the form of a dataset named "AIMSurv Aedes Invasive Mosquito species harmonized surveillance in Europe. AIM-COST Action. Project ID: CA17108". AIMSurv2020 harmonizes field surveillance methodologies for sampling different AIMs life stages, frequency and minimum length of sampling period, and data reporting. Data include minimum requirements for sample types and recommended requirements for those teams with more resources. Data are published as a Darwin Core archive in the Global Biodiversity Information Facility- Spain, comprising a core file with 19,130 records (EventID) and an occurrences file with 19,743 records (OccurrenceID). AIM species recorded in AIMSurv2020 were Ae. albopictus, Ae. japonicus and Ae. koreicus, as well as native mosquito species.

Seroprevalence of Equine Herpesvirus 1 (EHV-1) and Equine Herpesvirus 4 (EHV-4) in the Northern Moroccan Horse Populations.

This study reports the first equine herpesvirus-1 (EHV-1) and equine herpesvirus-4 (EHV-4) seroprevalence investigation in horse populations of Morocco in 24 years. It also aims to determine antibody titers in horses vaccinated under field conditions with a monovalent EHV-1 vaccine. Blood samples were collected from 405 horses, including 163 unvaccinated and 242 vaccinated animals. They were tested using a commercial type-specific enzyme-linked immunosorbent assay (ELISA) and a virus neutralization test (VNT). Overall, 12.8% unvaccinated, and 21.8% vaccinated horses were positive for EHV-1. All samples were positive for EHV-4 when tested with the type-specific ELISA. In the vaccinated group, the VNT revealed a mean antibody titer of 1:49 for EHV-1 and 1:45 for EHV-4. The present study demonstrates that EHV-1 and EHV-4 are endemic in the horse populations in the north of Morocco, with prevalence differences between regions. Furthermore, horses vaccinated with a monovalent EHV-1 vaccine had low antibodies titers. This study highlights the necessity to establish and/or support efficient biosecurity strategies based on sound management of horses and characterize further and potentially improve the efficiency of the EHV vaccines and vaccination protocol used in the field.

Draft Genome Sequence of the Capripoxvirus Vaccine Strain KSGP 0240, Reisolated from Cattle.

Control of lumpy skin disease in cattle is based on vaccination with live attenuated vaccines. The Kenyan strain KSGP 0240 is commonly used to vaccinate ruminants against capripox infections, but the conferred protection is still controversial. In this study, we report the draft genome sequence of the vaccine strain KSGP 0240, reisolated from cattle.

Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Genome Sequence of Mannheimia haemolytica MHA.Sh.MOR19 Serotype 1, a Moroccan Sheep Isolate.

Mannheimia haemolytica is the principle bacterial pathogen in ruminants associated with respiratory disease. Here, we report the draft genome sequence of the Mannheimia haemolytica MHA.Sh.MOR19 strain that was recently isolated in the northwest of Morocco from the lung of a lamb that died from pneumonia. The genome size is 2,434,458 bp.

Capripoxvirus Infections in Ruminants: A Review.

Lumpy skin disease, sheeppox, and goatpox are notifiable diseases of cattle, sheep, and goats, respectively, caused by viruses of the Capripoxvirus genus. They are responsible for both direct and indirect financial losses. These losses arise through animal mortality, morbidity cost of vaccinations, and constraints to animals and animal products' trade. Control and eradication of capripoxviruses depend on early detection of outbreaks, vector control, strict animal movement, and vaccination which remains the most effective means of control. To date, live attenuated vaccines are widely used; however, conferred protection remains controversial. Many vaccines have been associated with adverse reactions and incomplete protection in sheep, goats, and cattle. Many combination- and recombinant-based vaccines have also been developed. Here, we review capripoxvirus infections and the immunity conferred against capripoxviruses by their respective vaccines for each ruminant species. We also review their related cross protection to heterologous infections.

An Evaluation of Three Different Primary Equine Influenza Vaccination Intervals in Foals.

In order to evaluate the effect of three different primary vaccination intervals on EI vaccine response, 21 unvaccinated thoroughbred foals were randomly divided into three groups of 7 and vaccinated with three different intervals of primary immunization (i.e., with 1, 2 or 3 months intervals between V1 and V2, respectively). The antibody response was measured for up to 1 year after the third immunization V3 (administered 6 months after V2) by single radial hemolysis (SRH) assay. All weanlings had seroconverted and exceeded the clinical protection threshold 2 weeks after V2 and 1 month after V3 until the end of the study. Significant differences were measured at the peak of immunity after V2 and for the duration of the immunity gap between V2 and V3. The group with one month primary vaccination interval had a lower immunity peak after V2 (158.05 ± 6.63 mm2) and a wider immunity gap between V2 and V3 (18 weeks) when compared with other groups (i.e., 174.72 ± 6.86 mm2 and 16 weeks for a two months interval, 221.45 ± 14.48 mm2 and 12 weeks for a 3-month interval). The advantage observed in the group with 1 month primary vaccination interval, which induces an earlier protective immunity, is counterbalance with a lower peak of immunity and a wider immunity gap after V2, when compared with foals vaccinated with 2- and 3-month intervals.

Experimental infection of indigenous North African goats with goatpox virus.

BACKGROUND: Goatpox is a viral disease caused by infection with goatpox virus (GTPV) of the genus Capripoxvirus, Poxviridae family. Capripoxviruses cause serious disease to livestock and contribute to huge economic losses. Goatpox and sheeppox are endemic to Africa, particularly north of the Equator, the Middle East and many parts of Asia. GTPV and sheeppox virus are considered host-specific; however, both strains can cause clinical disease in either goats or sheep with more severe disease in the homologous species and mild or sub-clinical infection in the other. Goatpox has never been reported in Morocco, Algeria or Tunisia despite the huge population of goats living in proximity with sheep in those countries. To evaluate the susceptibility and pathogenicity of indigenous North African goats to GTPV infection, we experimentally inoculated eight locally bred goats with a virulent Vietnamese isolate of GTPV. Two uninfected goats were kept as controls. Clinical examination was carried out daily and blood was sampled for virology and for investigating the antibody response. After necropsy, tissues were collected and assessed for viral DNA using real-time PCR. RESULTS: Following the experimental infection, all inoculated goats displayed clinical signs characteristic of goatpox including varying degrees of hyperthermia, loss of appetite, inactivity and cutaneous lesions. The infection severely affected three of the infected animals while moderate to mild disease was noticed in the remaining goats. A high antibody response was developed. High viral DNA loads were detected in skin crusts and nodules, and subcutaneous tissue at the injection site with cycle threshold (Ct) values ranging from 14.6 to 22.9, while lower viral loads were found in liver and lung (Ct = 35.7 and 35.1). The results confirmed subcutaneous tropism of the virus. CONCLUSION: Clinical signs of goatpox were reproduced in indigenous North African goats and confirmed a high susceptibility of the North African goat breed to GTPV infection. A clinical scoring system is proposed that can be applied in GTPV vaccine efficacy studies.

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.

Complete genome analysis and time scale evolution of very virulent infectious bursal disease viruses isolated from recent outbreaks in Morocco.

Emerging of very virulent infectious bursal disease virus (vvIBDV) genotype in poultry flocks in Morocco were characterized. VP2 sequence analysis showed that the strains of Moroccan vvIBDV genotypes clustered separately from classic and vaccine strains reference of IBDV. The full-length genome of four Moroccan vvIBDV strains was determined, in order to get a more exhaustive molecular characterization allowing to conduct the evolution time scale and speculations on their origin. In a phylogenetic tree, nucleotide sequences of segment A and B formed a common branch with those vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of deduced amino acid sequences segment B, confirmed the presence of the conserved TDN tripeptide found in all of the vvIBDV genotype and revealed the presence of 2 substitutions I472L and E688D specific for the vvIBDV Moroccan isolates. The deduced amino acid sequences of segment A genes showed the presence of the "signature" typical of the vvIBDV genotype and revealed the presence of 7 aa substitutions specific for the vvIBDV Moroccan strains. The evolution rate for IBDV VP2 gene was estimated at 5.875 × 10-4 substitutions/site/year. The estimation of the time to most common recent ancestor of Moroccan vvIBDV based on the VP2 sequences available was 31 years, corresponding to 3 years earlier than the first vvIBDV case detection in layers in the country.

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.

Diversity of Dromedary Camel Coronavirus HKU23 in African Camels Revealed Multiple Recombination Events among Closely Related Betacoronaviruses of the Subgenus Embecovirus.

Genetic recombination has frequently been observed in coronaviruses. Here, we sequenced multiple complete genomes of dromedary camel coronavirus HKU23 (DcCoV-HKU23) from Nigeria, Morocco, and Ethiopia and identified several genomic positions indicative of cross-species virus recombination events among other betacoronaviruses of the subgenus Embecovirus (clade A beta-CoVs). Recombinant fragments of a rabbit coronavirus (RbCoV-HKU14) were identified at the hemagglutinin esterase gene position. Homolog fragments of a rodent CoV were also observed at 8.9-kDa open reading frame 4a at the 3' end of the spike gene. The patterns of recombination differed geographically across the African region, highlighting a mosaic structure of DcCoV-HKU23 genomes circulating in dromedaries. Our results highlighted active recombination of coronaviruses circulating in dromedaries and are also relevant to the emergence and evolution of other betacoronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV).IMPORTANCE Genetic recombination is often demonstrated in coronaviruses and can result in host range expansion or alteration in tissue tropism. Here, we showed interspecies events of recombination of an endemic dromedary camel coronavirus, HKU23, with other clade A betacoronaviruses. Our results supported the possibility that the zoonotic pathogen MERS-CoV, which also cocirculates in the same camel species, may have undergone similar recombination events facilitating its emergence or may do so in its future evolution.

Molecular characterization and phylogenetic analysis of very virulent infectious bursal disease virus circulating in Morocco during 2016-2017.

Very virulent infectious bursal disease virus (vvIBDV), the cause of significant economic losses in many poultry-producing areas, has been present in Morocco since 1991. In spite of the introduction of vaccination, disease outbreaks are frequently observed. To ascertain if vaccines failure may be due to the emergence of new strains, the aim of this study was to perform for the first time the molecular characterization of vvIBDV strains circulating in Morocco by focusing on the hypervariable region (HVR) of the VP2 protein, which is frequently used for molecular epidemiology and phylogenetic studies. Field samples of haemorrhagic bursae of Fabricius were collected for molecular characterization in different parts of the country during 2016-2017 from 48 chicken flocks showing symptoms of disease. In a phylogenetic tree, nucleotide sequences containing the VP2 HVR of 13 samples that were positive for vvIBDV formed a common branch with those of vvIBDV references strains published in GenBank, but they clearly grouped into a distinct subcluster. An alignment of the deduced amino acid sequences, in addition to confirming the presence of the "signature" typical of the vvIBDV HVR, also revealed the presence of substitutions in hydrophilic loops that are known to be involved in the elicitation of neutralizing antibodies. One of these substitutions is unique to the Moroccan isolates. These results represent the first molecular characterization of vvIBDV isolates in Morocco and may indicate that one of the causes of vaccine ineffectiveness is antigenic drift.

Impact of Mixed Equine Influenza Vaccination on Correlate of Protection in Horses.

To evaluate the humoral immune response to mixed Equine Influenza vaccination, a common practice in the field, an experimental study was carried out on 42 unvaccinated thoroughbred weanling foals divided into six groups of seven. Three groups were vaccinated using a non-mixed protocol (Equilis® Prequenza-Te, Proteqflu-Te® or Calvenza-03®) and three other groups were vaccinated using a mix of the three vaccines mentioned previously. Each weanling underwent a primary EI vaccination schedule composed of two primary immunisations (V1 and V2) four weeks apart followed by a third boost immunisation (V3) six months later. Antibody responses were monitored until one-year post-V3 by single radial haemolysis (SRH). The results showed similar antibody responses for all groups using mixed EI vaccination and the group exclusively vaccinated with Equilis® Prequenza-TE, which were significantly higher than the other two groups vaccinated with Proteqflu-TE® and Calvenza-03®. All weanlings (100%) failed to seroconvert after V1 and 21% (9/42) still had low or no SRH antibody titres two weeks post-V2. All weanlings had seroconverted and exceeded the clinical protection threshold one month after V3. The poor response to vaccination was primarily observed in groups exclusively vaccinated with Proteqflu-Te® and Calvenza-03®. A large window of susceptibility (3⁻4.5-month duration) usually called immunity gap was observed after V2 and prior to V3 for all groups. The SRH antibody level was maintained above the clinical protection threshold for three months post-V3 for the groups exclusively vaccinated with Proteqflu-Te® and Calvenza-03®, and six months to one year for groups using mixed EI vaccination or exclusively vaccinated with Equilis® Prequenza-Te. This study demonstrates for the first time that the mix of EI vaccines during the primary vaccination schedule has no detrimental impact on the correlate of protection against EIV infection.