Evaluation of a Combined Live Attenuated Vaccine against Lumpy Skin Disease, Contagious Bovine Pleuropneumonia and Rift Valley Fever.

The use of effective vaccines is among the most important strategies for the prevention and progressive control of transboundary infectious animal diseases. However, the use of vaccine is often impeded by the cost, a lack of cold chains and other factors. In resource-limited countries in Africa, one approach to improve coverage and reduce cost is to vaccinate against multiple diseases using combined vaccines. Therefore, the objective of this study was to evaluate a combined vaccine for the prevention and control of Lumpy Skin Disease (LSD), Contagious Bovine Pleuropneumonia (CBPP) and Rift Valley fever (RVF). The LSD and CBPP were formulated as a combined vaccine, and the RVF was formulated separately as live attenuated vaccines. These consisted of a Mycoplasma MmmSC T1/44 strain that was propagated in Hayflick-modified medium, RVF virus vaccine, C13T strain prepared in African green monkey cells (Vero), and the LSDV Neethling vaccine strain prepared in primary testis cells. The vaccines were tested for safety via the subcutaneous route in both young calves and pregnant heifers with no side effect, abortion or teratogenicity. The vaccination of calves induced seroconversions for all three vaccines starting from day 7 post-vaccination (PV), with rates of 50% for LSD, 70% for CBPP and 100% for RVF, or rates similar to those obtained with monovalent vaccines. The challenge of cattle vaccinated with the LSD/CBPP and the RVF vaccine afforded full protection against virulent strains of LSDV and RVFV. A satisfactory level of protection against a CBPP challenge was observed, with 50% of protection at 6 months and 81% at 13 months PV. A mass vaccination trial was performed in four regions of Burkina Faso that confirmed safety and specific antibody responses induced by the vaccines. The multivalent LSD/CBPP+RVF vaccine provides a novel and beneficial approach to the control of the three diseases through one intervention and, therefore, reduces the cost and improves vaccination coverage.

Poxvirus sensitivity of a novel diploid sheep embryonic heart cell line.

Lumpy skin disease virus (LSDV), camelpox virus (CPV), and orf virus (ORFV) are members of the family Poxviridae. These viruses are usually isolated or produced in embryonated eggs or primary cells because continuous cell lines are less sensitive to infection. Disadvantages of the use of eggs or primary cells include limited availability, potential endogenous contaminants, and a limited ability to perform multiple passages. In this study, we developed a diploid cell culture from sheep embryonic hearts (EHs) and demonstrated its high proliferative and long-term storage capacities. In addition, we demonstrated its sensitivity to representatives of three genera of the family Poxviridae: Capripoxvirus (LSDV), Orthopoxvirus (CPV), and Parapoxvirus (ORFV). The cell culture had a doubling time of 24 h and reached 40 passages with satisfactory yield. This is comparable to that observed in primary lamb testis (LT) cells at passage 5 (P5). After infection, each poxvirus titer was 7.0-7.6 log TCID50/mL for up to five passages and approximately 6.8, 6.4, and 5.6 for the three viruses at P6-P25, P30, and P40, respectively. The sensitivity of sheep EH cells to poxvirus infection did not decrease after long-term storage in liquid nitrogen and was higher than that of primary LT cells, which are used for capripoxvirus and parapoxvirus detection and growth, and Vero cells, which are used for orthopoxvirus detection and growth. Thus, EH diploid cells are useful for poxvirus isolation and production without embryonated eggs or primary cells.

Pathogenicity and molecular characterization of Mycoplasma bovis isolate from calves in Morocco.

Bovine respiratory disease caused by Mycoplasma bovis (M. bovis) represents a major health problem for cattle worldwide that causes considerable financial losses. This study reports for the first time the molecular and pathogenic characterization of a strain of M. bovis isolated from a dead local calf with respiratory symptoms in Morocco. M. bovis was isolated from lung tissue, purified by cloning, and subtyped using MLST analysis. Experimental infection was conducted in naïve calves to evaluate pathogenicity. The isolate was identified as a new subtype ST-204 that shares similarities with the 2019-2021 Spanish strains (ST-169, ST-170, ST-171) and the 2018 Algeria isolate (ST-4). Experimental infection resulted in fever and respiratory symptoms with serous nasal discharge. At postmortem, lung lesions of congestion and hepatization were observed with lymph node enlargement and foci of necrosis. The study confirms the high pathogenicity of the isolate and the important role of M. bovis in bovine respiratory disease.

Duration of Immunity Induced after Vaccination of Cattle with a Live Attenuated or Inactivated Lumpy Skin Disease Virus Vaccine.

Vaccines have proven themselves as an efficient way to control and eradicate lumpy skin disease (LSD). In addition to the safety and efficacy aspects, it is important to know the duration for which the vaccines confer protective immunity, as this impacts the design of an efficient control and eradication program. We evaluated the duration of immunity induced by a live attenuated vaccine (LSDV LAV) and an inactivated vaccine (LSDV Inac), both based on LSDV. Cattle were vaccinated and challenged after 6, 12 and 18 months for LSDV LAV or after 6 and 12 months for the LSDV Inac. The LSDV LAV elicited a strong immune response and protection for up to 18 months, as no clinical signs or viremia could be observed after a viral LSDV challenge in any of the vaccinated animals. A good immune response and protection were similarly seen for the LSDV Inac after 6 months. However, two animals developed clinical signs and viremia when challenged after 12 months. In conclusion, our data support the annual booster vaccination when using the live attenuated vaccine, as recommended by the manufacturer, which could potentially even be prolonged. In contrast, a bi-annual vaccination seems necessary when using the inactivated vaccine.

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.

Evaluation of ELISA and VNT for sheeppox virus antibody detection and development of an immunoenzymatic quantitative method.

Vaccination against sheep pox (SPV) is the most efficient tool to control spread of the disease and virus neutralization test (VNT) is the gold standard for vaccination monitoring. In the presented study, we evaluated the use of ELISA and VNT for quantification of SPV humoral response post vaccination. Results confirmed that VNT is more sensitive since ELISA did not detect 22% of positive tested sera, and VNT weak positive sera were either negative or doubtful by ELISA. The most sensitive cells to perform VNT were ESH-L instead of Lamb primary cells. We also investigated immunoperoxidase IPMA and immunofluorescence IFA assays for detection of SPV specific antibodies and IPMA showed higher antibody titers comparatively to IFA. VNT using ESH-L cells with immune-enzymatic revelation provide specific quantitative SPV antibody titers, easier to read in shorter incubation time.

Investigation of Post Vaccination Reactions of Two Live Attenuated Vaccines against Lumpy Skin Disease of Cattle.

Lumpy skin disease virus (LSDV) causes an economically important disease in cattle. The only method for successful control is early diagnosis and efficient vaccination. Adverse effects of vaccination such as local inflammation at the injection site and localized or generalized skin lesions in some vaccinated animals have been reported with live vaccines. The aim of this work was to compare the safety of two lumpy skin disease (LSD) vaccine strains, Kenyan (Kn) Sheep and Goat Pox (KSGP O-240) and LSDV Neethling (Nt) strain, and to determine the etiology of the post-vaccination (pv) reactions observed in cattle. Experimental cattle were vaccinated under controlled conditions with Nt- and KSGP O-240-based vaccines, using two different doses, and animals were observed for 3 months for any adverse reactions. Three out of 45 cattle vaccinated with LSDV Nt strain (6.7%) and three out of 24 cattle vaccinated with Kn strain (12.5%) presented LSD-like skin nodules, providing evidence that the post-vaccination lesions may not be strain-dependent. Lesions appeared 1-3 weeks after vaccination and were localized in the neck or covering the whole body. Animals recovered after 3 weeks. There is a positive correlation between the vaccine dose and the appearance of skin lesions in vaccinated animals; at the 105 dose, 12% of the animals reacted versus 3.7% at the 104 dose. Both strains induced solid immunity when protection was measured by neutralizing antibody seroconversion.

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.

Comparative sensitivity study of primary cells, vero, OA3.Ts and ESH-L cell lines to lumpy skin disease, sheeppox, and goatpox viruses detection and growth.

Lumpy skin disease virus (LSDV), sheeppox virus (SPPV) and goatpox (GTPV) virus have been usually grown on primary cells for diagnosis, production and titration purposes. The use of primary cells present several inconvenient, heavy preparation, heterogeneous cell population, non-reproducible viral titration and presence of potential endogenous contaminants. Therefore investigating sensitivity of candidate continuous cell lines is needed. In this study, we compared the above Capripox viruses (CaPVs) sensitivity of primary cells of four origin (heart, skin, testis and kidney), with three cell lines (Vero, OA3.Ts and ESH-L). We tested sensitivity for virus isolation, replication cycle and titration, revealed by cytopathic effect (CPE), immunoenzymatic staining and immunofluorescence. Our results show that ESH-L cells and primary fetal heart cells present the highest sensitivity for CaPVs growth and detection. Vero cells can replicate those viruses but without showing any CPE while the titer obtained on OA3.Ts is lower than primary and ESH-L cells. ESH-L cells are an effective alternative to primary cells use for growing Capripoxviruses and their diagnosis.

Production of small ruminant morbillivirus, rift valley fever virus and lumpy skin disease virus in CelCradle™ -500A bioreactors.

BACKGROUND: Animal vaccination is an important way to stop the spread of diseases causing immense damage to livestock and economic losses and the potential transmission to humans. Therefore effective method for vaccine production using simple and inexpensive bioprocessing solutions is very essential. Conventional culture systems currently in use, tend to be uneconomic in terms of labor and time involved. Besides, they offer a limited surface area for growth of cells. In this study, the CelCradle™-500A was evaluated as an alternative to replace conventional culture systems in use such as Cell factories for the production of viral vaccines against small ruminant morbillivirus (PPR), rift valley fever virus (RVF) and lumpy skin disease virus (LSD). RESULTS: Two types of cells Vero and primary Lamb Testis cells were used to produce these viruses. The study was done in 2 phases as a) optimization of cell growth and b) virus cultivation. Vero cells could be grown to significantly higher cell densities of 3.04 × 109 using the CelCradle™-500A with a shorter doubling time as compared to 9.45 × 108 cells in Cell factories. This represents a 19 fold increase in cell numbers as compared to seeding vs only 3.7 fold in Cell factories. LT cells achieved modestly higher cell densities of 6.7 × 108 as compared to 6.3 × 108 in Cell factories. The fold change in densities for these cells was 3 fold in the CelCradle™-500A vs 2.5 fold in Cell factories. The titers in the conventional system and the bioreactor were not significantly different. However, the Cell-specific virus yield for rift valley fever virus and lumpy skin disease virus are higher (25 virions/cell for rift valley fever virus, and 21.9 virions/cell for lumpy skin disease virus versus 19.9 virions/cell for rift valley fever virus and 10 virions/cell for lumpy skin disease virus). CONCLUSIONS: This work represents a novel study for primary lamb testis cell culture in CellCradle™-500A bioreactors. In addition, on account of the high cell densities obtained and the linear scalability the titers could be further optimized using other culture process such us perfusion.

Experimental infection of dromedary camels with virulent virus of Peste des Petits Ruminants.

Peste des Petits Ruminants Virus (PPRV) causes a severe contagious disease of sheep and goats and has spread extensively in last years through Asia and Africa. PPRV, known to infect exclusively small ruminants, has been recently reported in camels in Iran and Sudan. Reported clinical symptoms are similar to those observed in small ruminants, fatality rate still unknown. However most of the authors reported seropositive camels without clinical signs. Camel sensitivity to PPRV is still controversial and more investigation need to be performed. In this study, we tested camel susceptibility by an experimental infection using a virulent PPRV strain belonging to lineage IV. Young dromedary camels were infected intravenously and observed one month for clinical symptoms. Viraemia and virus secretion charge in swabs were evaluated by PCR. Seroconversion was assessed by ELISA and virus neutralisation test. Infected animals did not manifest any clinical symptoms of the disease and no virus was detected in secretions. Seroconversion was observed from day 14 post infection.

Susceptibility of Moroccan sheep and goat breeds to peste des petits ruminants virus.

BACKGROUND: Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants in Asia and Africa. In 2008, a PPR outbreak was reported for the first time in Morocco and a mass vaccination campaign allowed control of the disease. In this study, the susceptibility of four Moroccan local breeds of small ruminants to PPR virus was investigated by experimental infections. The objective was to make recommendations for improved epidemiological surveillance in Morocco by evaluating the susceptibility of the dominant Moroccan small ruminant breeds. Three parameters were studied: hyperthermia, clinical scoring and virus excretion. The outcome was compared to Alpine goats, which are considered one of the most sensitive breeds. RESULTS: The study showed that the local goat breed was the most sensitive breed with a susceptibility rate of 67%, followed by Timahdit, Beni Guil and Sardi sheep with 48, 29 and 26%, respectively. Serological testing including enzyme-linked immunosorbent assay and viral neutralization showed that the Timahdit breed developed a stronger antibody response compared to the other breeds. Although the clinical signs observed in the sheep were mild, evidence of viral excretion was detected by means of a polymerase chain reaction assay. CONCLUSIONS: It is recommended that effective surveillance should focus on susceptible breeds complemented with serological surveillance of the sheep population.

Multiple alignment comparison of the non-structural genes of three strains of equine influenza viruses (H3N8) isolated in Morocco.

BACKGROUND: Three equine influenza viruses, A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004(H3N8), and A/equine/Essaouira/3/2004(H3N8), were isolated from different Equidae during local respiratory disease outbreaks in Morocco in 1997 and 2004. Their non-structural (NS) genes were amplified and sequenced. RESULTS: The results show high homology of NS nucleotide sequences of A/equine/Nador/1/1997 with European strains (i.e., A/equine/newmarket/2/93 and A/equine/Grobois/1/1998) and clustered into the European lineage. However, NS gene of A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) strains indicated high homology with equine influenza strains that had circulated before 1990 (A/equine/Fontainbleu/1/1979(H3N8), which belonged to a pre-divergent phase Amino acid sequence comparison of the NS1 protein with reference strain A/equine/Miami/1963(H3N8) shows that the A/equine/Nador/1/1997(H3N8) strain has 12 substitutions at the residues D/24/N, R/44/K, S/48/I, R/67/Q, A/86/V, E/139/K, A/112/T, E/186/K, L/185/F, A/223/E, S/213/T and S/228/P. In both A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) strains, the NS1 sequences present one common mutation at the residue: S/228/P. CONCLUSION: It seems that all of these substitutions are not produced at the key residues of the RNA-binding domain (RBD) and the effector domain (ED). Consequently, we can suppose that they will not affect the potency of inhibition of cellular defences, and the virulence of the Moroccan equine strains will be maintained.

Cleavage site and Ectodomain of HA2 sub-unit sequence of three equine influenza virus isolated in Morocco.

BACKGROUND: The equine influenza (EI) is an infectious and contagious disease of the upper respiratory tract of horses. Two outbreaks were notified in Morocco during 1997 and 2004 respectively in Nador and Essaouira. The aims of the present study concern the amino acids sequences comparison with reference strain A/equine/Miami/1963(H3N8) of the HA2 subunit including the cleavage site of three equine influenza viruses (H3N8) isolated in Morocco: A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004 (H3N8) and A/equine/Essaouira/3/2004 (H3N8). RESULTS: The obtained results demonstrated that the substitutions were located at Ectodomain (ED) and transmembrane domain (TD), and they have only one arginine in cleavage site (HA1-PEKQI-R329-GI-HA2). In the Ectodomain, the mutation N/1542/T deleted the NGT glycosylation site at position 154 for both strains A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8). Except for mutation D/1602/Y of the A/equine/Nador/1/1997(H3N8) strain, the other mutations were involved in non conserved sites. While the transmembrane domain (TM) of the strain A/equine/Essaouira/3/2004(H3N8) exhibits a substitution at residue C/1992/F. For the A/equine/Nador/1/1997(H3N8) strain the HA2 shows a mutation at residue M/2072/L. Three Moroccan strains reveals a common substitution at the residue E/2112/Q located between transmembrane domain TM and the cytoplasmic domain (CD). CONCLUSION: The given nature virulence of three Moroccan strains, the identified and reported mutations certainly played a permissive role of infection viral process.

Phylodynamics and human-mediated dispersal of a zoonotic virus.

Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.