Serological response of commercial dairy cattle to inactivated foot-and-mouth disease vaccine ( type-O & A ) in Nigeria

An observational study was conducted in a peri-urban dairy establishment in Jos South, Plateau State Nigeria to determine immune response of dairy cattle to commercial inactivated foot-and-mouth disease vaccine serotypes (O and A). Thirty seven Friesian cattle aged ≥2years old with their crosses (15 selected prevaccination and 22 selected 21 days post-vaccination) were investigated for immune response to vaccination with an inactivated trivalent FMD vaccine containing serotypes O, A and SAT 2). Sera collected on day 0 prevaccination and 21 days post-vaccination was tested for structural protein antibodies to FMD serotypes O and A using the Solid Phase Competitive ELISA assay. The mean OD value for serum end point titre of FMD serotype O pre-vaccination was 11.64% with 6.67% (95%CI: 0.33 – 28.73) of the selected cattle being seropositive, at 21 days post-vaccination the mean OD value in selected cattle was 52.83% with 68.18% (95%CI: 46.95 – 84.89) of the selected cattle seropositive. For the FMD serotype A, 26.67% (95%CI: 9.10 – 52.53) of the selected cattle were seropositive pre-vaccination with a mean OD value of 29.21% and by 21 days postvaccination, 72.73% (95%CI: 51.67 – 88.13) of the selected cattle were seropositive with a mean OD value of 61.70%. Serological response to vaccination improved in most selected cattle by 21 days post-vaccination. This study result has indicated that commercial inactivated FMD vaccines used for the prophylactic control of FMD in commercial dairy farm in Nigeria provoked immune response after a single shot.


Introduction
In Foot-and-mouth disease (FMD) endemic countries, as a prophylactic measure to prevent against outbreaks in susceptible cloven-hoofed farm animals, routine vaccination with inactivated foot-and-mouth disease virus (FMDV) vaccines is recommended (Doel, 2003).However, in most cases clear policies for prophylactic vaccination of farm animals do not exist.According to the Office International des Epizooties (OIE), FMD vaccines used for the prophylactic control of FMD in endemic settings should be vaccines of standard potency formulated to contain sufficient antigen and appropriate adjuvant to ensure that they meet the minimum potency level required (recommended as 3 PD 50 [50% protective dose in vaccinated candidates]) (Elnekave et al., 2013).Administering at least 2 doses one month apart when cattle are vaccinated for the first time against FMD with such vaccine is recommended, however due to cost and logistical reasons this is not routinely practised in most endemic countries (Knight-Jones et al., 2014a).FMD structural protein antibody levels are strongly correlated with protection in vaccinated cattle (Reeve et al., 2011;Robiolo et al., 2010).Therefore, when testing FMD protection from antibody titre, the serological test used should be correlated with protection (OIE, 2013), because when the test antigen used is different from the one evaluated in the study, the extent of the protection may be uncertain.Globally, it has been reported that large scale FMD vaccines are being applied to control FMD in clovenhoofed livestock with over 2 billion doses being used annually (Knight-Jones et al., 2014a).However, despite the sustained efforts in the progressive control of FMD, little is done to evaluate the field performance of FMD vaccines (Knight-Jones et al., 2014a).About 2-5% of cattle globally have been reported to be affected by FMD each year with incidence in other cloven-hoofed species reported slightly lower (Knight-Jones et al., 2013).Losses associated with export of livestock commodities have been reported to be of huge importance to countries with developed livestock industries, which has placed a trade barrier restricting economic growth of such countries (Rich and Perry, 2011;Knight-Jones et al., 2013).In Nigeria, FMD virus is endemic with outbreaks occurring seasonally in most pastoral settings and established farm holdings.To date FMD serotypes O, A, SAT 1 and SAT 2 have been reported in sedentary cattle since the first report of FMD in Nigeria in 1924 (Libeau, 1960;Fasina et al., 2013;Lazarus et al., 2012).Recently between 2009 -2014, outbreaks of FMD serotypes O, A and SAT 2 have been reported in Jos South, a municipal area which is the second administrative unit for a state structure in Nigeria (FAO, 2010a;FAO, 2010b, FAO, 2014a, FAO, 2014b, FAO, 2014c).Although cattle are not routinely vaccinated for FMD in the country, some established dairy farms vaccinate cattle to prevent against seasonal outbreaks.However, the field effectiveness of such vaccines during outbreak is hardly evaluated.As observed in human vaccination campaign programmes, where seroconversion is often studied using pre and post-vaccination sera to access the vaccine response, a similar survey using sera collected post-vaccination are common in livestock (Knight-Jones et al., 2014c), in which case the proportion of livestock with antibody titre above a specific threshold associated with protection is then determined (Robiolo et al., 2010).In this study we present a preliminary report of serological response of Holstein-Friesian cattle and their crosses vaccinated with inactivated trivalent (O, A and SAT 2) FMD vaccines to antigen for serotypes O and A only.

Animals
Holstein Friesian cattle and their crosses belonging to a private commercial dairy farm in Jos South, Plateau State, Nigeria were used for this study.The dairy farm accommodated over a thousand cattle with their calves.From history, there was no introduction of cattle from other localities into the dairy farm and the farm is situated at the outskirt of a local community.As a routine prophylactic programme, all animals on the farm are vaccinated against FMDV serotypes; A, O and SAT 2 annually.

Vaccination and sample collection
During August, 2012, 300 cattle on the farm were vaccinated with alhydrogel-saponin adjuvanted inactivated FMD vaccine against FMD types O, A and SAT 2 (Aftovax®, Merial Animal Health Ltd/Botswana Vaccine Institute Gaborone).Cattle were individually injected with 3 ml of the vaccine subcutaneously in the neck region using a hypodermic syringe system.Prior to vaccination, 15 cattle aged ≥2years old were randomly selected and whole blood collected from the jugular vein, using plain evacuated tubes to determine the antibodies status prior to vaccination.Blood was allowed to clot at ambient temperature in the field and transported to the laboratory where it was centrifuged at 1450 x g for 10 min and serum collected and stored at -20°C until testing.After 21 days post-vaccination, 22 cattle were randomly selected and bled to determine seroconversion and immune response to vaccination.

Serological test
Samples were analysed for FMD-specific antibodies using a Solid-phase Competitive ELISA (SPCE) as previously described elsewhere (Grazioli et al., 2008) for serotypes A and O at the Foot-and-Mouth Disease Laboratory, National Veterinary Research Institute, Vom.The assays were performed using the antibodies FMDV ELISA kits for serotypes A and O produced by IZSLER Biotechnology Laboratory, Brescia-Italy.
Briefly, 96 wells pre-coated with FMDV antigens captured by FMD serotypes A and O specific MAb flatbottomed plates were used.Four dilutions at 1/10, 1/30, 1/90 and 1/270 were performed for the sera.Without washing, the conjugate (Horse radish peroxidase) was added and incubated at room temperature for 1 hour.The plates were then washed, after which the substrate/chromogen solution (Tetra-methyl-benzidine) was added and kept in the dark for 20 minutes.The reaction was later stopped by the addition of a stop solution and the plates were read on a MultiSkan® spectrophotometer ELISA plate reader (Thermo Scientific, USA) at 450 nm wavelength.Serum end-point titre was expressed as the highest dilution producing 50% inhibition, with serum having end point titre ≥50% being classified as positive.

Statistical analysis
The data was entered into Microsoft Excel® and exported in R software (version 3.0.2) for analysis.The percentage of seropositive cattle for FMD serotypes O and A were determined and the exact 95% confidence intervals (CI) were calculated using the Pearson-Klopper method.The mean serum end point titre (optical density [OD]) for FMD serotype O and A pre-and post-vaccination were calculated.Differences in the mean OD for each serotype, pre-and post-vaccination, with their 95% CI were calculated using the t-test.

Discussion
Vaccination of dairy cattle with a trivalent FMD vaccine (type O, A, and SAT 2) is practiced in some selected dairy farms in Nigeria.However, several previous reports have indicated that imported commercial FMD vaccines have performed sub-optimally under field situation in Nigeria (Nicholls et al., 1983).In this study, we attempted to select a subset of vaccinated population of cattle in order to evaluate immune response to FMD vaccine, testing for serotypes O and A only.To our knowledge, this is about the first time an attempt was made to investigate immune response to commercial FMD vaccines containing serotypes O and A in Nigeria.In a previous study involving FMD serotypes SAT 1 and SAT 2 isolated from Nigeria, it was demonstrated that cattle responded satisfactorily to vaccination irrespective of age, breed, or simultaneous vaccination with Contagious Bovine Pleuro-pneumonia (CBPP) vaccine (Nicholls et al., 1983).Although it is a known fact that FMD vaccines need to be adequately matched to the field virus to ensure sufficient protection against a challenge with a field virus, little is done in most developing countries to match vaccines before implementing campaign programmes.This may not however, be unconnected with the fact that FMD vaccines are not always readily available and farmers would have to make do with what they have.In most cases, this ends up with outbreaks occurring even after animals have been vaccinated against FMD.This study has demonstrated that the current vaccination programme practiced on the farm does not provide sufficient protection of the herd against FMD should an active outbreak occur within the inter-vaccination interval.In most cases, it is believed that once animals are vaccinated against a disease they are adequately protected.However, with the increased incidence of disease in vaccinated population the need for postvaccination monitoring is being highlighted.Also contrary to the standard recommendation by the manufacturers that cattle in endemic settings should be vaccinated at least twice within a period of 2-8 weeks and thereafter every 4-6 months depending upon the epidemiological situation within the region, most regions in developing countries adopt a single course of an annual mass vaccination campaign proceeding periods of suspected outbreaks.
In this study few animals had evidence of pre-existing antibodies to the serotypes being tested for prevaccination which might be as a result of the rapid decline of antibodies to the previous vaccination as reported previously for alhydrogel-saponin adjuvanted vaccines (Hunter, 1996;Hunter, 1998;Cloete et al., 2008).However, by 21-days post-vaccination there was a marked improvement in immune response of vaccinated cattle for both serotypes O and A, which is indicative of seroconversion to vaccination.It has been reported that vaccinated livestock respond rapidly to the first dose of vaccine and produce peak antibody titres between 14 and 28 days depending on the vaccine composition (Doel, 2003).Immune response against FMDV includes circulating humoral antibody has been shown to correlate with protection (Reeve et al., 2011;Robiolo et al., 2010;Mackowiak et al., 1962;van Bekkun, 1969;Pay and Hingley, 1987;McCullough et al 1992a;McCullough et al., 1992b).Therefore serological evidence of FMD antibodies in vaccinated cattle in the absence of circulating field virus might be an indicator of protection against challenge with a homologous virus.This study is limited due to the number of cattle sampled pre and post-vaccination period which might underscore the level of the herd immunity.Another limitation to the study is the inability to test for serotype SAT 2 antigen in the serum of vaccinated cattle which would have provided a better understanding of the immune response to the SAT 2 antigen contained in the vaccine.Since we have not been able to monitor the herd longitudinally for duration of immunity post-vaccination, it will not be possible to demonstrate the duration of antibodies response that persists in vaccinated cattle which will be a guide to designing an effective vaccination regimen.Therefore, we recommend that a detailed and structured study should be designed with the aim of investigating immune response and duration of immunity in Nigerian cattle to commercial vaccines containing relevant FMD serotypes representative of the region.Efforts should also be intensified to develop vaccines using indigenous isolates targeting representative field viruses in Nigeria.Designing an effective vaccination programme with a quality vaccine against FMD will be a welcome development for Nigeria at this moment when the global oil price is declining and the national population is increasing with high demand for animal proteins.

Conclusions
In this study, we have demonstrated that a commercial FMD vaccine with a standard antigen payload used according to the manufacturer's instruction for the inoculation of dairy cattle in Nigeria produced measurable level of anti-FMD antibodies against serotypes O and A by 21 days post-vaccination.

Figure 1 .
Figure 1.Boxplots showing the mean optical density (OD) values for FMD serotype O and A prevaccination and 21 days post-vaccination.Boxplots shows the median value (horizontal line), interquartile range (box) and range (whiskers).