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.

The prevalence, virulence, and serogroups of Dichelobacter nodosus and prevalence of Fusobacterium necrophorum in footrot lesions of sheep and cattle in Morocco.

Background and Aim: Footrot is a contagious disease of ruminants leading to severe economic losses. This study aimed to estimate the prevalence, virulence, and serogroups of Dichelobacter nodosus and the prevalence of Fusobacterium necrophorum in footrot lesions of sheep and cattle. Materials and Methods: A total of 106 pathogenic lesion samples were taken from 74 sheep and 32 cattle exhibiting typical footrot lesions and were analyzed for the presence of D. nodosus and F. necrophorum by real-time polymerase chain reaction (PCR). Both virulence and serogroup were estimated for D. nodosus positive samples. Results: Among the 106 samples, 89 were positive by PCR for F. necrophorum, D. nodosus, or both. Dichelobacter nodosus was detected at a rate of 78.3% versus 28.3% for F. necrophorum. Virulent D. nodosus strains were detected in 67.5% of positive samples, with a higher rate in sheep (73.4%) than in cattle (47.4%). Benign D. nodosus strains were detected in 57.8% of samples, with a lower prevalence rate in sheep (50%) than in cattle (84.2%). The positive samples of D. nodosus revealed the presence of three dominant serogroups (D, H, I) and three minor serogroups (G, C, A) by serogroup-specific multiplex PCR. Conclusion: The findings provided information on the prevalence of D. nodosus and F. necrophorum strains in footrot lesions of sheep and cattle in some regions of Morocco, which will be useful for developing an effective autovaccine for the prevention of this disease in cattle and sheep in these regions.

Comparative protection of small ruminants against Mannheimia haemolytica infection by inactivated bacterin and toxoid vaccines.

Background and Aim: Mannheimia haemolytica causes respiratory infection and mortality in sheep and goats, similar to the effects in cattle, which causes major economic damage. Regular vaccinations alongside good management practices remain the most efficient tools for controlling this disease. Indeed, vaccines against pasteurellosis are available, but results on their efficacy have varied. Therefore, this study aimed to evaluate the efficacy of three vaccines against mannheimiosis in small ruminants. Materials and Methods: We evaluated three vaccines developed from a local field isolate based on the inactivated bacterium, its toxoid, and a mixture of bacterin/toxoid, which we then tested on sheep and goats. Selected criteria that were evaluated were safety, antibody response, and protection through a challenge. Post-vaccination monitoring was carried out by enzyme-linked immunosorbent assay. The evaluation was based on antibody responses to vaccination in sheep and goats for both bacteria and leukotoxin. Protection was assessed by clinical and lesion scores after the challenge of vaccinated goats with a pathogenic strain. Results: The three tested vaccines were completely safe, did not cause any adverse reactions, and induced significant antibody titers in immunized animals. Following M. haemolytica challenge, unvaccinated goats showed clinical signs with lesions typical of the disease. Meanwhile, the best protection was obtained with the inactivated combined bacterin/toxoid vaccine. Conclusion: This study highlighted the effectiveness of adding a bacterial toxoid in the vaccine as a promising solution for preventing mannheimiosis in small ruminants. Because of the worldwide distribution of M. haemolytica infection, general prophylaxis based on a combined inactivated vaccine could greatly benefit.

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.

Development and Evaluation of a Combined Contagious Bovine Pleuropneumonia (CBPP) and Lumpy Skin Disease (LSD) Live Vaccine.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP). Lumpy skin disease (LSD) is a viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD and CBPP are both transboundary diseases spreading in the same areas of Africa and Asia. A combination vaccine to control CBPP and LSD offers significant value to small-scale livestock keepers as a single administration. Access to a bivalent vaccine may improve vaccination rates for both pathogens. In the present study, we evaluated the LSDV/CBPP live combined vaccine by testing the generation of virus neutralizing antibodies, immunogenicity, and safety on target species. In-vitro assessment of the Mycoplasma effect on LSDV growth in cell culture was evaluated by infectious virus titration and qPCR during 3 serial passages, whereas in-vivo interference was assessed through the antibody response to vaccination. This combined Mmm/LSDV vaccine could be used to protect cattle against both diseases with a single vaccination in the endemic countries. There were no adverse reactions detected in this study and inoculated cattle produced high levels of specific antibodies starting from day 7 post-vaccination, suggesting that this combination vaccine is both safe and effective.

Draft Genome Sequence of Pasteurella multocida Serotype A Strain MOR19, Isolated in Morocco.

Pasteurella multocida causes pneumonia in large ruminants. In this study, we determined the genome sequence of the capsular serotype A Pasteurella multocida strain MOR19, isolated from a calf that died from acute pneumonia.

Biological and molecular characterization of a sheep pathogen isolate of Mannheimia haemolytica and leukotoxin production kinetics.

BACKGROUND AND AIM: Mannheimia haemolytica (Mha) is a common agent of pneumonia in ruminants globally, causing economic losses by morbidity, mortality, and treatment costs. Infection by Mha is often associated with or promoted by respiratory viral pathogens and environmental conditions. Infections due to Mha have rarely been described in small ruminants. This study reports the biological and molecular characteristics of a new Moroccan Mha isolate from small ruminants presenting typical respiratory symptoms. We also studied the cultural parameters, growth kinetics, and Lkt excretion of the isolate and its pathogenicity on laboratory animals and small ruminants. MATERIALS AND METHODS: Suspected pasteurellosis cases in sheep and goat flocks in Morocco were investigated. A local strain of Mha was isolated and identified using biochemical and molecular methods. Polymerase chain reaction-targeting specific genes were used for serotyping and phylogenetic analyses; further, leukotoxin production, cytotoxicity, and pathogenicity of the isolate in mice, goats, and sheep were investigated. RESULTS: Phylogeny analysis revealed 98.76% sequence identity with the USA isolate of 2013; the strain growth with a cycle of 9-10 h with leukotoxin secretion was detected by NETosis and quantified by cytotoxicity and mortality of mice. Goat and sheep infections cause hyperthermia, with characteristic postmortem lesions in the trachea and lung. CONCLUSION: A local isolate of Mha from sheep that died of pneumonia was characterized for the 1st time in North Africa using biological and molecular methods. Although growth on appropriate culture media is accompanied by intense leukotoxin secretion, experimental infections of sheep and goats cause hyperthermia and typical lesions of pneumonia.

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.

Development of an inactivated combined vaccine for protection of cattle against lumpy skin disease and bluetongue viruses.

Lumpy Skin Disease (LSD) and Bluetongue (BT) are the main ruminants viral vector-borne diseases. LSD is endemic in Africa and has recently emerged in Europe and central Asia as a major threat to cattle industry. BT caused great economic damage in Europe during the last decade with a continuous spread to other countries. To control these diseases, vaccination is the only economically viable tool. For LSD, only live-attenuated vaccines (LAVs) are commercially available, whilst for BT both LAVs and inactivated vaccines are available with a limited number of serotypes. In this study, we developed an inactivated, oil adjuvanted bivalent vaccine against both diseases based on LSDV Neethling strain and BTV4. The vaccine was tested for safety and immunogenicity on cattle during a one-year period. Post-vaccination monitoring was carried out by VNT and ELISA. The vaccine was completely safe and elicited high neutralizing antibodies starting from the first week following the second injection up to one year. Furthermore, a significant correlation (R = 0.9040) was observed when comparing VNT and competitive ELISA in BTV4 serological response. Following BTV4 challenge, none of vaccinated and unvaccinated cattle were registered clinical signs, however vaccinated cattle showed full protection from viraemia. In summary, this study highlights the effectiveness of this combined vaccine as a promising solution for both LSD and BT control. It also puts an emphasis on the need for the development of other multivalent inactivated vaccines, which could be greatly beneficial for improving vaccination coverage in endemic countries and prophylaxis of vector-borne diseases.

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.

Experimental evaluation of the cross-protection between Sheeppox and bovine Lumpy skin vaccines.

The Capripoxvirus genus includes three agents: Sheeppox virus, Goatpox virus and Lumpy skin disease virus. Related diseases are of economic importance and present a major constraint to animals and animal products trade in addition to mortality and morbidity. Attenuated vaccines against these diseases are available, but afforded cross-protection is controversial in each specie. In this study, groups of sheep, goats and cattle were vaccinated with Romania SPPV vaccine and challenged with corresponding virulent strains. Sheep and cattle were also vaccinated with Neethling LSDV vaccine and challenged with both virulent SPPV and LSDV strains. Animals were monitored by clinical observation, rectal temperature as well as serological response. The study showed that sheep and goats vaccinated with Romania SPPV vaccine were fully protected against challenge with virulent SPPV and GTPV strains, respectively. However, small ruminants vaccinated with LSDV Neethling vaccine showed only partial protection against challenge with virulent SPPV strain. Cattle showed also only partial protection when vaccinated with Romania SPPV and were fully protected with Neethling LSDV vaccine. This study showed that SPPV and GTPV vaccines are closely related with cross-protection, while LSDV protects only cattle against the corresponding disease, which suggests that vaccination against LSDV should be carried out with homologous strain.

Development and Evaluation of an Inactivated Lumpy Skin Disease Vaccine for Cattle.

Lumpy skin disease (LSD) of cattle is caused by a virus within Capripoxvirus genus. It leads to huge economic losses in addition to trade and animal movement limitation. Vaccination is the only economically feasible way to control this vector-borne disease. Only live attenuated vaccines have been used so far and no inactivated vaccine has been developed nor tested in cattle. In this study, we developed an inactivated oily adjuvanted vaccine based on Neethling strain and tested it on cattle. Selected criteria of appreciation were safety, antibody response by Virus Neutralization and protection through challenge. A field trial was also performed in Bulgaria. The vaccine was safe and did not cause any adverse reaction, high level of specific antibodies was obtained starting from day 7 post-vaccination and protection against virulent challenge strain that caused typical disease in control animals was total. Induced protection was similar to that obtained with live vaccine, without any adverse effect. In addition, the field study confirmed safety and efficacy of the vaccine, which did not show any adverse reaction and induced a high level of antibodies for up to one year. General prophylaxis based on inactivated vaccine could be of great benefit in endemic countries or at risk regions.

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.

Pathogenicity and Full Genome Sequencing of the Avian Influenza H9N2 Moroccan Isolate 2016.

In Morocco in early 2016, a low pathogenic avian influenza virus serotype H9N2 caused large economic losses to the poultry industry, with specific clinical symptoms and high mortality rates on infected farms. Subsequent to the H9N2 outbreak, the causal agent was successfully isolated from chicken flocks with high morbidity and mortality rates, propagated on embryonated eggs, and fully sequenced. The phylogenetic analysis suggested that the Moroccan isolate could have derived from the Middle East isolate A/chicken/Dubai/D2506.A/2015. This study was designed to assess the pathogenicity of the Moroccan isolate H9N2 in experimentally infected broiler and specific-pathogen-free (SPF) chickens. At 22 days of age, one broiler and two SPF chicken groups were inoculated by dropping 0.2 ml of the H9N2 isolate (107.5 EID50/ml) in both nostrils and eyes. Clinically inoculated chickens with H9N2 displayed mild lesions, low mortality rates, and an absence of clinical signs. The H9N2 virus was more pathogenic in broiler chickens and produced more severe tissue lesions compared to SPF chickens. The viral shedding was detected up to 6 days postinoculation (pi) in oropharyngeal and cloacal swabs in infected birds with a maximum shedding in the oropharynges of the broiler group. All experimental chickens seroconverted and registered high hemagglutination inhibition titers as early as day 7 pi. The present study indicates that the H9N2 virus isolated from a natural outbreak was of low pathogenicity under experimental conditions. However, under field conditions infection with other pathogens might have aggravated the disease.

Estudio de patogenicidad y secuenciación del genoma completo del aislamiento de virus de la influenza aviar H9N2 de Marruecos del año 2016. En Marruecos, a principios de año 2016, el serotipo H9N2 del virus de la influenza aviar de baja patogenicidad (LPAIV) causó grandes pérdidas económicas en la industria avícola, con signos clínicos específicos y altas tasas de mortalidad en las granjas infectadas. Posterior al brote de H9N2, el agente causal se aisló con éxito de parvadas de pollos con altas tasas de morbilidad y mortalidad, se propagó en huevos embrionados y se secuenció completamente. El análisis filogenético sugirió que el aislado marroquí podría haberse derivado del aislamiento de Medio Oriente (A/pollo/Dubai/D2506.A/2015). Este estudio se diseñó para evaluar la patogenicidad del aislado marroquí H9N2 en pollos de engorde infectados experimentalmente y en pollos libres de patógenos específicos (SPF). A los 22 días de edad, un grupo de pollos de engorde y dos grupos de aves libres de patógenos específicos se inocularon mediante la instilación de 0.2 ml del aislamiento H9N2 (107.5 dosis infectantes de embrión de pollo 50% [EID50] por ml) en ambas fosas nasales y en los ojos. Los pollos clínicamente inoculados con el virus subtipo H9N2 mostraron lesiones leves, bajas tasas de mortalidad y ausencia de signos clínicos. El virus H9N2 fue más patógeno en los pollos de engorde y produjo lesiones tisulares más graves en comparación con las aves libres de patógenos específicos. La excreción viral se detectó hasta seis días después de la inoculación en frotis orofaríngeos y cloacales de aves infectadas con una excreción máxima en la orofarínge del grupo de pollos de engorde. Todos los pollos experimentales seroconvirtieron y registraron altos títulos de inhibición de hemaglutinación tan pronto como en el día siete después de la inoculación. El presente estudio indicó que el aislamiento viral H9N2 de un brote natural fue de baja patogenicidad en condiciones experimentales. Sin embargo, en condiciones de campo, la infección con otros patógenos pudo haber agravado la enfermedad.

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.