"Outbreak Study of Foot and Mouth Disease in Relation to Vaccination and Vaccine Efficacy Assessment in Central Ethiopia"

Foot and mouth disease is a devastating and a highly contagious
disease of cloven-hoofed animals as a result of loss of production...

One method of serological monitoring is to identify animals in a vaccinated cattle herd that have been infected with FMD virus from vaccinated animals. Differentiating infected from vaccinated animals (DIVA) shows differences in the antibody responses produced in vaccinated animals compared to those animals are infected with FMD virus. Foot and mouth disease vaccines are purified to contain structural protein viral capsids from which majority of the viral non-structural proteins have been removed. On the other hand, the viral non-structural proteins (NSP) are expressed to elicit matching immune responses. Reduced antibody responses occurred when multiple protein conjugated vaccines sharing common epitopes have been administered simultaneously. Determining whether the current vaccines commonly used in the field could correlate with protection against new outbreaks in vaccinated and non-vaccinated animals is important.
The failure of vaccination could be due to differences between the circulating field strains and the strains included in the vaccine. Vaccine efficacy is obtained from the relative risk of infection or disease in the vaccinated animals compared to the unvaccinated individuals. When most of the population is vaccinated, some cases could not be necessarily due to vaccine failures. Cases could be excluded from the study or can be considered unvaccinated if the interval between the last dose of vaccine and the onset of symptoms is shorter than the incubation period as vaccination could lead to reduced infection in the population. Determining vaccine strain that is antigenically matched to the field strain which can provide protection against outbreaks in the field is needed.

Introduction
Foot and mouth disease is a devastating and a highly contagious disease of cloven-hoofed animals as a result of loss of production; cost of treatments and it is the main barrier to trade of animal and animal products at local and international markets [1]. It is a virus of the genus Aphtho-virus in the family of Picronaviridae of which seven immunologically distinct serotypes (O, SAT2, SAT1, A, C, Asia 1 and SAT 3) are circulating in the wide field but multiple subtype strains have been evolved within each serotypes. Immunization with one serotype doesn't confer protection against other serotypes and fails to protect other subtypes of the same strains and this variation causes failure of the prevention and control options as infection or [2]. FMD is endemic in Ethiopia and outbreaks have been reported in different parts of the country throughout the year.
Besides this, [5] indicated that serotype O is the most prevalent strain worldwide. FMDV produced extensive vesicular lesions on the lips, tongue, gums, dental pad, feet and udder [6]. The disease is characterized by low mortality in adult cattle and massive mortality in young calves due to acute Myocarditis [7]. Authors such as [8] also reported FMD outbreak resulted in 70% to 90 % milk reduction in lactating animals. The incident of new strains and uncontrolled movement are some of the risk factors of FMD outbreak in Ethiopia [9]. Vaccination is considered as a main tool to prevent animal diseases such as Foot and mouth disease. But outbreaks of FMD have been reported after vaccination [10]. This is due to low humoral immune responses against FMDV by the locally produced vaccines and the failure of vaccine due to differences between the circulating field strains and the strains included in the vaccines [11].
Besides this, the virus could re-assort with the field strains and end up with emergence of new strain of the virus. As the duration of FMD vaccine protection is short lived, animals require revaccination twice a year. Any shift between FMDV serotypes may cause more severe outbreaks even in vaccinated animals [8]. The vaccines are DIVA (differentiate infected from vaccinated animal) capable, meaning that certain testing can distinguish between naturally infected and vaccinated animals. However, a study on the specific serotype of virus that causes outbreak is limited, so, identification of the virus strain from outbreak is very important to apply emergency vaccines [12]. Post vaccination outbreak study is important to detect newly emerging variants despite the fact that the existence of other serotypes or new virus subtypes might cause outbreak. Lack of cross protection between FMD virus serotypes as well as incomplete protection between some subtypes of FMDV affect the application of vaccine in the field. against which vaccines of the same serotype will fail to fully protect [13]. In order to achieve better control of the disease in endemic areas, it is important to investigate pre and post vaccination outbreaks to monitor the current variants of FMD virus in the field and explore the efficacy of vaccine strains which are used to combat the circulating viruses. Therefore, this study was aimed to evaluate post vaccination outbreak and explore the efficacy of FMD vaccine against circulating field strains.

Materials and Methods
The study was conducted in the dairy farms of Adea berga and Welmera areas on pre and post vaccination outbreak of Foot and Mouth Disease (FMD) in crossbred animals. During this period, a total number of 90 crossbred of cattle affected with FMD were included in this study. Foot and mouth disease outbreak was defined as the occurrence of new cases in the dairy farms [14]. Adea berga district is located in Oromia regional State, West shewa zone which is about 70 km from Addis Ababa. The total area of woreda is 798.35 square kilometers. It is located at 9o 12' to 9o 37' latitude and 38o17' to 38o 36' longitude. The altitude of the area ranges from 1400 to 3500 meters above sea level. The annual average of rainfall ranges from 918 mm to 1368 mm while the minimum and maximum temperature reaches 10 °C and 25 °C respectively. The farming system of the area is mixed type where crop production and livestock rearing are done side by side [15]. Welmera district is 40 km away from the capital city, Addis Ababa and the area is situated at 9o04'-9o13' N latitude and 38o29'-38o39' E longitude.
The average altitude of the area ranges from 2200-2500 meter above sea level. The district is known by crop-livestock mixed farming system is a common practice. The rainfall pattern of the district is bimodal, with a short rainy period from February to April and a long rainy season from mid-June to September. The annual temperature and rainfall ranges from 18 °C to 24 °C and 1000 to 1100 mm, respectively.

Foot and Mouth Disease Infected Dairy Cows Kept by Farmers
Cattle that had manifested clinical signs of the disease and death cases in the farms were included in the study. Contributing factors such as age, and sex categories were considered during outbreak assessment.

Vaccination, Outbreak and Vaccine Efficacy Assessments
The farms were subsequently monitored for any disease incidence and development of clinical symptoms with special emphasis to mouth cavity and inter-digital space lesions [16]. The Vaccine assessment was performed in response to vaccination, and its protection level.

Data Analysis
The data generated from outbreak data and other field assessments were recorded and coded using Microsoft Excel spreadsheet and analyzed using STATA version 13 for Windows

Outbreak of FMD in Vaccinated and Non-Vaccinated Animals
Within the non-vaccinated young stock and females, the mortality proportion was (0.5%) and (0.43%) respectively while within the vaccinated groups, the mortality proportion in females was (0.94%) ( Table 3).

Post-vaccination Assessment of Immunity at Population Level
The overall population immunity is the proportion of animals with immunity in the whole population susceptible to infection, or vaccine that has been targeted for FMD control. This immunity occurs when large part of the population becomes immune to a virus through vaccination or infection. This is resulted from vaccine coverage and the proportion of animals responded to immunization, other sources of immunity, namely infection, earlier vaccination or maternally derived antibodies. In areas where FMD control is common and endemic, significant levels of post-infection immunity is ranged from 15-30% or beyond.

Post Vaccination Reaction
Out of 30 cattle considered for immunity assessment, a subset of 24 animals were appropriate for vaccination in the population.

Immunological Study
Foot and mouth disease virus is diagnosed by specific antibody response [17]. Serological tests are used to detect the immunity of animals exposed to foot and mouth disease virus (FMDv) or to elicit matching immune response that can be diagnosed by serological principles [18].

Randomized Control Trial
The protective efficacy of vaccine and its adverse effect is evaluated by double blind randomized controlled trial (RCT). It remains the golden standard for the initial evaluation of the safety and efficacy of vaccine. Once RCT of a vaccine has been conducted and the vaccine has been shown to be efficacious, further trials can be conducted after the vaccine has been introduced into the field use. The groups or herd being evaluated or compared could be individuals or the population of animals [19].

Observational and Clinical Studies
Observational studies, direct (cohort) and indirect (casecontrol), is used to determine whether the persisting disease is the result of vaccine failure or failure to vaccinate. The allocation is nonrandom and there is potential for bias and confounding which could be controlled. The case control approach has been widely used to assess disease risks associated with non-interventional exposures, but with rare cases in the context of vaccine evaluation [20]. It is used to determine whether the outbreak of disease was due to vaccine efficacy or other cases and used to identify causes of the new cases.

As new vaccines are introduced, booster doses are added and the timings of doses changed, clinical evaluation of vaccine efficacy is
important. The analysis in case control studies employ odds ratios, conditional or unconditional logistic regression, if other variables need to be controlled [21]. Vaccine efficacy and effectiveness (VE) are explained as one minus some measure of Relative Risk (RR) in the vaccinated group compared with unvaccinated group that is VE = 1 -RR. The percentage reduction in the disease condition among vaccinated subjects is attributed to vaccination [22]. animals that were exposed to similar level of virus using the, VE= RU-RV/RU, where RU is the incidence risk or rate in the unvaccinated population, and RV is the incidence in those vaccinated.

Susceptibility, Infection and Progression
Therefore, VE (Percentage) is 1 -RV/RU in the field studies.
When most of the population is vaccinated, rare cases emerge, and this could not be necessarily indicative of a declining vaccine effectiveness or efficacy. Cases can be excluded from the study or can be considered unvaccinated if the interval between the last dose of vaccine and the onset of symptoms is shorter than the incubation period. Reduced infectiousness could play a role in the transmission dynamics in populations that are nearly 100% vaccinated [20].

Vaccine Matching and Vaccine Strain Selection
The immunity to FMDV is serotype specific, and even within serotypes cross protection between strains is incomplete. Vaccine strain selection is the method of incorporating one or more vaccine strains that are able to induce protective immunity against infection from one or more circulating virus strains. A highly potent vaccine may cross-protect against a wide range of divergent strains and give relatively long-lasting immunity after a single dose. In contrast, a vaccine with a low potency will induce an antigenically narrow and short-lived protection. The protective level of induced immunity depends upon potency of the vaccine, the antigenic match between the vaccine strain and the field strain and the vaccination schedule [24]. Antibody titers to some live virus may be lower when administered in a combination vaccine than when administered separately. Poor antibody responses is associated with multiple protein conjugated vaccines sharing common epitopes that have been administered simultaneously [26]. necrosis of cardiac muscle and cardiac failure [29]. More than 50% of ruminants that recover from illness and those that are vaccinated and have been exposed to virus can carry virus particles in the pharyngeal region up to 3.5 years in cattle and 9 months in sheep [30]. The oral and pedal lesions observed in the infected and dead cattle were typical of FMD infection.

Multiple Strains and Combination Vaccines
Vaccination is an important component that could reduce the burden of FMD and block circulation of the virus. The disease is mainly controlled by vaccination and is used to reduce morbidity and mortality [23]. The level of neutralizing antibodies that correlate with protection are based on experimental challenge studies giving high levels of virus at a single time point through inoculation routes.
Natural exposure is made likely through multiple routes, at variable levels and for variable periods of time under field conditions and different levels of antibody may correlate with protection. This property is important to consider potent vaccination strategies in addition to other factors such as the antigen payload [31]. In seronegative animals, maternally derived antibodies interfere with the response to FMD vaccines [32]. Vaccination is considered as main tool to prevent animals from FMD. But outbreaks of FMD have been reported after vaccination [10]. This was also observed in the findings that vaccinated animals infected (0.94%) by the disease.
The finding also coincided with [11] who reported locally produced vaccines induce very low humoral immune responses against FMDV infection. The failure of vaccination may be due to differences between the circulating field strains and the strains included in the vaccine [8,10]. However, a shift of between FMDV serotypes may cause more severe outbreaks even in vaccinated animals. According to a study conducted by [8]   for serological determination, protection must be correlated with a specific assay [29]. The challenge virus is homologous to the vaccine and test strain and protection in the field differs as the virus challenge is prolonged, although evaluation during field challenge is recommended [35][36][37].

Conclusion and Recommendations
Foot and mouth disease is a highly contagious acute vesicular viral disease that affects cloven-hoofed animals and is mainly controlled by vaccination. The circulation of the virus in susceptible livestock imposes severe restrictions on the movement and trade of animals and derived products, causing serious economic loss to the affected countries. In FMD endemic areas vaccination of susceptible populations is widely used as a major control measure. Commercial vaccine formulations that contain more than one virus strain is recommended to induce immune responses. Therefore, foot and mouth disease control should be instituted based on the availability of potent and master vaccines against the circulating field strains.