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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Thermal Stability Study of Five Newcastle Disease Attenuated Vaccine Strains.

Newcastle disease (ND) is a big concern throughout the world because of the devastating losses that can occur with commercial and backyard poultry. The major problem in many countries is the loss of the vaccine's effectiveness due to inadequate use or storage conditions, particularly in hot climates. In the present study, stability of the five, most-used NDV vaccine strains (I-2, LaSota, B1, Clone 30 [C30], and VG-GA) was tested comparatively at different storage temperatures (4 and 37 C for the freeze-dried form and 4, 24, 37, and 45 C for the freeze-dried vaccine reconstituted in diluents). The vaccine stability was evaluated by the cumulative infectious titer drop and the theoretical shelf life at particular temperatures. Results showed that I-2 and LaSota are the most stable vaccine strains compared to B1, C30, and VG-GA; they registered the lowest titer drops and the longest shelf life whether at cool, high, or room temperatures and for both freeze-dried and reconstituted vaccines.