Diversity of SAT2 Foot-and-Mouth Disease Virus in Sudan: Implication for Diagnosis and Control

doi:10.21203/rs.3.rs-771285/v1

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Diversity of SAT2 Foot-and-Mouth Disease Virus in Sudan: Implication for Diagnosis and Control

https://doi.org/10.21203/rs.3.rs-771285/v1

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Like other East African countries, Sudan experienced circulation of several SAT2 topotypes. In Sudan, topotype XIII and VII of SAT2 virus were recorded. This work meant to evaluate the impact of such diversity on diagnosis and control. A Sudanese SAT2 foot-and-mouth disease virus (FMDV) of topotype VII originated in 2010 showed poor antigenic relationship (r1 value ≈ 0.00) with Sudanese SAT2 FMDV from 2008 when topotype XIII was circulating. After one or three doses of a vaccine derived from SAT2 virus in 2010, heterologous antibody titres with SAT2 virus in 2008 were ≤ 1.2 log 10, not consistent with likely protection, while homologous titres were 1.65 (after one dose) or 1.95 and 2.55 log10 (after 3 doses). SAT2 positive field sera from Sudan in 2016 were not unvaryingly identified by virus neutralization tests (VNT) employing SAT2 viruses from 2010 and 2008. Proportions of positive sera by SAT2 virus from 2010 were always higher than those by viruses from 2008; consistent with the more frequent and recent circulation of topotype VII prior to 2016. Proportions by SAT2 virus from 2010 were 0.68 (±0.1) in one location (n=72), 0.39 (±0.1) in another one (n=94) and 0.52 (±0.1) in the whole test group (n=166). Corresponding values by viruses of 2008 were 0.53 (±0.1), 0.27 (±0.1) and 0.38 (±0.1). In the whole test group, differences were statistically significant (p=.02339). Like post-vaccination sera, field sera (natural immunity) showed no considerable cross neutralization between topotype VII and presumably XIII; almost 45% (43/96) of SAT2 positive field sera were positive to one topotype but not to the other. Experimental and surveillance findings emphasized the implication of SAT2 diversity in Sudan. It is concluded that it is difficult to control SAT2 infection in Sudan using a monovalent vaccine. Beside a prophylactic vaccine from topotype VII, stockpiling of antigens from topotype XIII and rapid genotyping and matching studies are necessary approaches. When more frequent circulation of more than one topotype occurs, retrospective diagnosis by serological surveys could be problematic or imprecise.

Veterinary Epidemiology

Foot-and-mouth disease virus (FMDV)

SAT2

Sudan

East Africa

DiagnosisControl

Foot-and-mouth disease (FMD) is a highly infectious viral disease of cloven hoofed animals; mainly cattle, sheep and goats, and pigs. In attempts for identification of priority diseases by the Food and Agriculture Organization (FAO) and the Office International des Epizooties (OIE), FMD was ranked, globally, as the first and foremost priority (Domenech et al. 2006). It is of devastating economic sequences; not only jeopardizes international trade in animals and animal byproducts but also limits animal production of milk, meat and draft power (Knight-Jones and Rushton 2013). Foot-and-mouth disease virus (FMDV) is a positive sense, single stranded RNA virus of the genus Aphthovirus of the family Picornaviridae (Grubman and Baxt 2004). It is an antigenically variable pathogen of seven immunologically distinct serotypes; O, A, Asia1, C and the three South African Territory (SAT) serotypes SAT1, 2 and 3. Further, mutation within each serotype leads to emergence of intratypic variants what complicates laboratory diagnosis and selection of an appropriate vaccine to control outbreaks in the field (Grubman and Baxt 2004).

Protection against challenge infection of FMD is associated with neutralizing antibody response (Pay and Hingley 1987, 1992; Hamblin et al. 1986; McCullough et al. 1992). Accordingly, cross protection test (Periera 1978) and then virus neutralization reaction (Rweyemamu 1984) were used to identify intratypic antigenic differences of FMD virus. The major neutralization site(s)/antigenic determinant (s) of FMDV is located within the G-H loop of the VP1 (Strohmair et al. 1982; Crowther et al. 1993; Borrego et al. 1995; Mateu et al. 1995) which is one of the four structural proteins, VP1 to -4, that form the icosahedral capsid of the virion (Grubman and Baxt 2004). Thus, neutralization and antigenic diversity as measured by virus neutralization test (VNT) are functions of this site which is also responsible for cell receptors recognition (Acharya et al. 1990). The capsid proteins, including VP1, are encoded within the P1 region of the open reading frame (ORF) of the FMDV genome (Grubman and Baxt 2004). In similar manner to cross protection test and virus neutralization reaction in showing intratypic antigenic differences of FMDV, comparative sequence analysis of the P1 gene is used to measure genetic diversity of FMDV. Comparative sequence analysis of the P1 gene has exposed the circulation of groups of genotypes with, usually, less than 15% nucleotide difference within geographical boundaries and used the term topotypes to describe them (Samuel and Knowles 2001). The two approaches, neutralization reaction and comparative sequence analysis of the P1 gene, could reveal the emergence of a novel variant. Nonetheless, the impact of specific amino acid changes in nucleic acid on antigenicity are not well determined and care must be taken when extrapolating between nucleotide differences and antigenic homology (Paton et al. 2005).

In Africa, beside the diversity of the circulating field strains, epidemiological patterns of FMD show additional unique characteristics. Most importantly, the three SAT serotypes are unique to the continent; mainly in sub-Saharan Africa. Of them, SAT2 is the one responsible for most of the SAT outbreaks in domestic animals (Bastos et al. 2003; Hall et al. 2013). It is also the one that has spread from sub-Saharan Africa to North Africa and the Middle East in several different occasions; in 1990, 2000, 2003 (Bastos et al. 2003; Hall et al. 2013) and apparently in 3 separate events in one year, in 2012 (Ahmed et al. 2012; Hall et al. 2013). It stayed in record in Egypt in North Africa for 5 years, between 2014 and 2018 (Soltan et al. 2019; http://www.wrlfmd.org.). In addition, the SAT2 virus is particularly diverse; it is subclassified into 14 topotypes (Bastos et al. 2003; Sahle et al. 2007) with 80% nucleotide identity instead of 85% (Knowles and Samuel 2003). Eight of these topotypes were detected in East Africa compared to four in southern African region and two in West Africa (Bastos et al. 2003; Sahle et al. 2007). Four countries in East Africa experienced circulation of more than one SAT2 topotype; Kenya, Ethiopia, Uganda and Sudan while Kenya, in addition, showed the highest within-country heterogenous SAT2 strains (Bastos et al. 2003; Ayelet et al. 2009; Habiela et al. 2010; http://www.wrlfmd.org). Bastos et al (2003) expected that no single set of serotype specific primers would be suitable for detection of all SAT2 field variants and that control of SAT2 infection by vaccination would be extremely difficult. Implication of genetic diversity on control of SAT viruses in sub-Saharan Africa was investigated in virus samples representing the period from 1974 to 2002 that included isolates from East Africa (Maree et al. 2011). Other reports investigated the impact of genetic diversity of SAT viruses on control in South Africa (Sirdar et al. 2019; Blingaut et al. 2020). To our knowledge, no such effort was devoted recently to East Africa. In this work, retrospective diagnosis of SAT2 infection in Sudan, an East African country, by serological surveys was evaluated and cross protection between recent Sudanese SAT2 isolates was investigated. In Sudan, topotype VII of SAT2 virus was reported in 2007, 2010, 2013, 2014 and 2017 and topotype XIII in 1977 and 2008 (Habiela et al. 2010; http://www.wrlfmd.org). The latter topotype was reported only in Sudan and Ethiopia (Lycett et al. 2019).

Viruses:

SAT2 FMDVs used in this work were, all, local Sudanese isolates designated SAT2-Kh 1/08, SAT2-Kh 2/08 and SAT2-Nk 1/010 what is referring to its serotype, geographical origin within Sudan, year and order of isolation from that origin. The first two isolates were isolated from Khartoum in 2008 (Raouf et al. 2010) while the third virus was isolated from North Kordfan State in 2010. Viruses were isolated in calf kidney cell (CKC) culture (SAT2-Kh 1/08 and SAT2-Kh 2/08) or baby hamster kidney (BHK21) cell culture (SAT2-Nk 1/010) and all were adapted to grow in BHK21 cell culture through 16-22 serial passages. The character of adaptation was defined as the virus growth that produces complete cytopathic effects (CPE) in 24 hours and effect absolute determination of titre. Isolates were serotyped and re-serotyped using reference ELISAs; the indirect sandwich ELISA kit (Pirbright, UK) for FMDV antigen detection (Roeder and Le Blanc Smith 1987) and an updated version of the FMDV serotyping ELISA (IZSLER, Brescia, Italy and Pirbright, UK) (Grazioli et al. 2020), including detection of serotypes SAT1 and SAT2. SAT2-Nk 1/010 was genotyped at the World Reference Laboratory (WRL) for FMD, with the identity of SUD/4/2010, as topotype VII strain Alx-12 (http://www.wrlfmd.org/). While in 2008 topotype XIII was shown to be circulating in Sudan at Gazeera State neighbouring to Khartoum (Habiela et al. 2010).

Field serum samples:

Field sera comprised 166 bovine sera, from Khartoum (72) and the Red Sea (94) States, proved to be positive to antibodies against nonstructural proteins (NSPs) of FMDV. Sera were collected during a programme "Surveillance of Trade Sensitive Disease" (STSD) in 2016 for the study of anti-NSPs activity in Sudanese cattle (Anon 2016); tested using the commercial ID ELISA, recently evaluated (Browning et al. 2020), and kept at – 20 °C till used. Sera had been collected using simple random sampling technique (SRS) from apparently healthy cattle, older than one year with no history of vaccination against FMD. Serum samples from Khartoum State showed anti-NSPs activity of 86.1% (360/418) and from the Red Sea showed an activity of 59.9% (247/412) (Anon 2016). Prior to collection of field serum samples, in the last 10 years, SAT2 infection in Khartoum was identified in 4 occasions; in 2007, 2013 and 2014 with topotype VII (http://www.wrlfmd.org/) and in 2008 with an unidentified topotype (Raouf et al. 2010). The history of SAT2 infection in the Red Sea was unknown.

SAT2 vaccine material:

SAT2 vaccine material was derived from SAT2-Nk 1/010. The virus was grown in BHK21 cell cultures in large Roux bottles, harvested 18 hours later by freezing of the whole culture and clarified by simple centrifugation at 2000 rpm for 10 minutes. Inactivation was carried out using 2-bromo-ethylamine HBr (BEA) in two cycles each of 24 hours at cold room temperature (26 ºC) according to the method described by Bahnemann (1990). The inactivated antigen was adjuvated with 25% (V/V) of aluminium hydroxide 2% gel (Barteling 2002) and saponin (AppliCHem) at the rate of 1.66 mg/ml of the final vaccine blend.

Animal inoculation experiment:

Five yearling calves of cross breeds, screened negative to antibodies of NSPs of FMDV and to antibodies of SP of SAT2 FMDV, were used. Animals were screened using the commercial ID ELISA kit (Browning et al. 2020) and VNT (OIE 2018) employing SAT2-Nk 1/010. Two animals (No. 81 and 83) were primed with 3 ml of the antigen, two animals (No. 82 and 85) with 0.5 ml and one animal (No. 89) was left as in contact control. Vaccinated animals were boosted with two doses of the antigen, each of 3 ml, 6 and 21 weeks (animal No. 81, 83 and 82) or 4 and 21 weeks (No. 85) later. The in contact control animal was vaccinated with 3 ml of the antigen at the end of the experiment. All animals were bled at regular intervals and at 2 weeks following the last vaccine dose. All inoculated animals and the in contact control were screened for anti-NSPs activity at regular intervals and at the end of the experiment and demonstrated no activity.

Determining the serological relationship between Sudanese SAT2 FMD virus isolates:

Antigenic relationships between Sudanese SAT2 FMDVs were determined by driving the r1 value using the two-dimensional virus micro-neutralization test (2D-VNT) described by Rwemamu et al. (1978). Pooled 21 day post-vaccination sera from two animals vaccinated with 3 ml priming dose were employed in the 2D-VNT. The r1 value represented the ratio between antibody titre against the heterologous virus (SAT2-Kh 1/08 or SAT2-Kh 2/08)/the antibody titre against the homologous virus (SAT2-Nk 1/010). Antibody titres were calculated from regression data as the log10 reciprocal antibody dilution required for 50% neutralization of 100 tissue culture infective dose 50 (log10 SN50/100 TCID50). Values of r1 greater or equal to 0.3 indicate close antigenic relationship so that a vaccine containing the homologous virus or a similar one is likely to confer protection against both strains. While values less than 0.3 indicate poor antigenic relationship and that a vaccine containing the homologous virus or a similar one is unlikely to confer protection against the heterologous virus or a similar one.

For performing the test, a checkerboard titration of the vaccine and test virus was carried out against the inactivated (56 ºC for 30 minutes) vaccine serum in flat bottomed 96 well microtitre coaster plate from column 1 to 8. Each virus dilution was tested into two rows while column 9 and 10 represented a back-titration of virus and column 11 and 12 represented cell control. The test was carried out in 50 μl volume. The vaccine serum was diluted in a twofold dilution series and viruses in a half log dilution series. Virus and serum diluents were GMEM containing 0.0487% Na HCO3 (W/V), 10% TPB (V/V) and 10% tris buffer (V/V). Outgrowth media contained in addition 10% (V/V) fetal calf serum (Sigma). After incubation of the serum/virus mixture for one hour at room temperature, 50 μl of a suspension of BHK21 cells was added to each well. Plates were sealed and incubated at 37 ºC with a source of humidity. Cytopathic effects (CPE) were assessed microscopically 48-72 hours later and by staining of plates with 0.1% crystal violet in 10% formol-saline. The titre of the test and vaccine virus, from the back-titration, and the antibody titres of the vaccine serum against the vaccine and test virus for each virus dose used, calculated according to the method of Kärber (1931), were used to generate the regression data.

Evaluation of cross protection between SAT2-Nk 1/010 and SAT2-Kh 1/08:

Cross protection between the two isolates was evaluated indirectly by antibody titration against SAT2-Kh 1/08 after vaccination of the target species with SAT2-Nk 1/010. The quantitative VN microtest (OIE 2018) in BHK-21 cells, using flat bottomed 96 well microtitre coaster plates, was applied. In absence of information on correlation between homologous or heterologous protection and antibody response, cut-off titres of 1/50 (10^1.7) was considered protective (McCullough et al. 1992). Serum titres were calculated according to the method of Kärber (1931).

Homologous and heterologous antibody titres of test sera were determined simultaneously. Test sera included post-vaccination sera from the five vaccinated calves collected 2 weeks following the 3^rd vaccine dose (four calves) or following the primary dose (one calf). All sera were inactivated at 56 ºC for 30 minutes. Pre-titrated stock virus of SAT2-Nk 1/010 and SAT-Kh 1/08 were diluted to contain 100 TCID50/50 μl. Starting from a 1/8 dilution, sera were diluted in a twofold dilution series across the coaster plate using 50 μl volume and two columns per serum. Virus and serum diluent were as described previously for the 2D-VNT. After addition of virus, plates were incubated for one hour at room temperature. At the end of incubation time 50 μl volume of BHK21 cells suspended in the previously described outgrowth media was added to each well. Plates were sealed with adhesive tape and incubated at 37 ºC with a source of humidity. Each run of test included virus titration and virus and cell control. Plates were read microscopically 48-72 hours later, fixed and stained as described for the 2D-VNT.

Screening of field sera for antibodies against SAT2 infection using Sudanese SAT2 isolates:

A screening format of VNT as described recently (Alfouz et al. 2021) was applied. It was similar to the quantitative VN microtest (OIE 2018) except that sera were tested at final dilutions of 1/32 (10^-1.5) and 1/64 (10^-1.8). The adopted procedure decreased the test workload and span the cut-off value of 1/45 (10^-1.65) and of 1/32 (10^-1.5) suggested for serosurveillances and for individual sera respectively by the OIE manual (2018). The latter cut-off was adopted for discrimination of positive sera in this work. Control sera included fetal calf sera (FCS) (Sigma) free from antibodies against FMDV and known positive field bovine sera for SAT2 FMD virus antibody (Alfouz et al. 2021). All sera were tested against SAT2-Nk 1/010 and SAT2-Kh 1/08.

Statistical analysis:

For comparison of the performance of Sudanese SAT2 isolates in screening of field sera for SAT2 antibodies, proportion positive and prevalence rates by each strain and by both strains in each sub-population and in the whole tested population were calculated. Proportion positive were determined by dividing the number of positive serum samples identified by that strain (or by both strains) in the sub-population or in the whole population by the total number of samples tested in the sub-population or in the whole population. Prevalence rates were calculated using serial testing approach (Fletcher and Fletcher 2005; Thrusfield 2007). Prevalence rates in each sub-popualtion (Khartoum or the Red Sea) were calculated by multiplying the obtained proportion positive value in the sub-population by the seroprevalence by the ID ELISA in that sub-population which was provided by the STSD (Anon 2016). For calculation of the prevalence rate in the whole test sample (from Khartoum and the Red Sea), the denominator was calculated as follow:

(Number of samples tested in Khartoum x 100/seroprevalence by the ID ELISA in Khartoum) + (Number of samples tested in the Red Sea x 100/seroprevalence by the ID ELISA in the Red Sea).

Then the prevalence by each strain or by both strains was calculated by dividing the total number of positive samples identified by that strain (or by both strains) by the obtained denominator and multiplying the result by100.

Calculated proportion positive and prevalence rates were compared by driving 95% confidence interval (C. I.) measures and P-values. The 95% C. I. were derived using the formula: P± 1.96√p(1-p)/n (Thrusfield 2007); where P is the estimated proportion or prevalence, n is the number of samples tested and 1.96 is the appropriate multiplier for the selected level of confidence. When C. I. values did not overlap then the statistics will always be statistically significantly different (Knezevic 2008). P-values were calculated using chi-squared test available at the Statistical Packages for Social Sciences (SPSS) at (www.sociostatistics.com); if p < 0.05, then results were significantly different.

Antigenic relationship and cross protection between Sudanese "SAT2" isolates:

Using the two-dimensional VNT that employed 21 day sera post vaccination with SAT2-Nk 1/010 (table 1), no neutralization for SAT2-Kh 1/08 and SAT2-Kh 2/08 was observed at 100 TCID₅₀. Results suggested that SAT2-Kh 1/08 and SAT2 Kh 2/08 are antigenically different from SAT2 Nk 1/010 and that a vaccine containing the latter isolate is unlikely to protect against SAT2-Kh 1/08 and SAT2-Kh 2/08.

Table 1: Antigenic relationship of Sudanese SAT2 viruses

Serum	Hetrologous virus strain*	Regression slope		Strain relationship**
Serum	Hetrologous virus strain*	Homologous	Heterologous	Strain relationship**
SAT2-Nk 1/010	SAT2-Kh 1/08	-0.374	-1.425	-0.01
SAT2-Nk 1/010	SAT2-Kh 2/08	-.87	-1.139	-0.05

*The designation for the homologous virus is the same as that for the serum in each case.

**r1 value

Cross protection between a vaccine containing SAT2-Nk 1/010 and SAT2-Kh 1/08 was examined indirectly using post vaccination sera collected 2 weeks following one vaccine dose and 2 weeks following an additional two booster doses (table 2). In every case, heterologous titres were less than or equal to 1.2 log 10; well below the likely threshold of heterologous protection of 1.7 log 10. Simultaneously, homologous titres following one dose of the vaccine were greater than or equal to 1.65 log 10 or greater than or equal to 2 log 10 following 3 doses of the vaccine.

Table 2: Cross protection between SAT2-Nk 1/010 and SAT2-Kh 1/08

Animal No.	Vaccination History*	Log 10 neutralization titres 2 weeks post vaccination
Animal No.	Vaccination History*	Homologous titres	Heterologous titres
81	3 doses; before 23, 17 and lastly 2 weeks before bleeding	≥ 2.55	<1.2
83	//	1.95	1.2
82	//	2.4	1.2
85	3 doses; before 23, 19 and lastly 2 weeks before bleeding	2.4	<1.2
89	One dose; 2 weeks before bleeding	≥ 1.65	<1.2

*Animals No. 82 and 85 received, each, a priming dose of 0.5 ml instead of 3 ml

Performance of Sudanese "SAT2" isolates in VNT:

In all tested samples, 53 were found to be positive by both strains (SAT2-Nk 1/010 and SAT2-Kh 1/08), 33 by SAT2-Nk 1/010 only and 10 sera by SAT2-Kh 1/08 only. In Khartoum, 33 sera were detected by both strains, 16 by SAT2-Nk 1/010 only and 5 by SAT2-Kh 1/08 only while comparable figures in the Red Sea State were 20, 17 and 5. Proportions of positive sera and calculated seroprevalences by either of the two strains and by the two strains are shown in table (3). Proportions of positive sera and calculated seroprevalences by SAT2-Nk 1/010 were consistently higher than those by SAT2-Kh 1/08 (table 3). However, only in the whole tested lot, the statistic was significant (table 4, 5 and 6). Consistently, proportions of positive sera and calculated seroprevalences by both strains were similar to those found by SAT2-Nk 1/010 but statistically significantly higher than those found by SAT2-Kh 1/08 (table 4, 5 and 6).

Table 3: Performance of Sudanese SAT2 isolates in surveillance of SAT2 antibodies in bovine field sera-2016

	Khartoum State			Red Sea State			Total test samples
No. tested	72			94			166
Test virus	SAT2-Kh 1/08	SAT2-Nk 1/010	SAT2-Kh 1/08 and SAT2-Nk 1/010	SAT2-Kh 1/08	SAT2-Nk 1/010	SAT2-Kh 1/08 and SAT2-Nk 1/010	SAT2-Kh 1/08	SAT2-Nk 1/010	SAT2-Kh 1/08 and SAT2-Nk 1/010
No. positive	38	49	54	25	37	42	63	86	96
Proportion of positive sera (95% C.I.)	.5277 (.4124-.643)	.6805 (.5728-.7882)	.75 (.65-.85)	.2659 (.1766-.3552)	.3936 (.2949-.4923)	.4468 (.3463-.5473)	.3795 (.3057-.4533)	.518 (.442-.594)	.5783 (.5032-.6534)
Seroprevalence (95% C.I.)	45.44% (38/84) (34.8%- 56.08%)	58.6% (49/84) (48.07%-69.13%)	64.57% (54/84) (55.71%-75.99%)	15.94% (25/157) (10.22%- 21.66%)	23.59% (37/157) (16.95%-30.23%)	26.78% (42/157) (19.83%-33.67%)	(63/241) 26.14% (20.6%-31.68%)	(86/241) 35.68% (29.64%-41.72%)	(96/241) 39.83% (33.65%-46.01%)

Table (4): Comparison of the performance of Sudanese SAT2 isolates in the whole tested lot using P-values (Chi-square test)

		SAT2-Kh 1/08	SAT2-Nk 1/010
	Seroprevalence (proportion of +ve sera)	26.14% (.3795)	35.68% .518)
SAT2-Nk 1/010	35.68% (.518)	.02339 (.0111)
both	39.83% (.5783)	.0013 (.0002)	.3474 (.2701)

Table (5): Comparison of the performance of Sudanese SAT2 isolates in Khartoum State using P-values (Chi-square test)

		SAT2-Kh 1/08	SAT2-Nk 1/010
	Seroprevalence (proportion of +ve sera)	45.44% (.5277)	58.6% (.6805)
SAT2-Nk 1/010	58.6% (.6805)	.0894 (.0608)
both	64.57% (.75)	.0131 (.0055)	.4283 (.3558)

Table (6): Comparison of the performance of Sudanese SAT2 isolates in the Red Sea State using P-values (Chi-square test)

		SAT2-Kh 1/08	SAT2-Nk 1/010
	Seroprevalence (proportion of +ve sera)	15.94% (.2659)	23.59% (.3936)
SAT2-Nk 1/010	23.59% (.3936)	.0889 (.0626)
both	26.78% (.4468)	.0191 (.0096)	.5155 (.4600)

In essence, the result presented in this work showed that Sudanese SAT2 isolates in 2008 and 2010 were antigenically poorly related and that SAT2 positive field sera from Sudan were not unvaryingly detectable by VNT employing these isolates. The isolate from 2010 (SAT2-Nk 1/010) is of the topotype VII (http://www.wrlfmd.org/) while the isolates from 2008 (SAT2-Kh 1/08 and SAT2-Kh 2/08) have not been genotyped; yet they are likely to be of topotype XIII. Topotype XIII has been detected circulating in Gazeera State in February/2008 (Habiela et al. 2010); same time of isolation of SAT2 strains from neighbouring Khartoum State (Raouf et al. 2010). The reported antigenic differences of SAT2-Kh 1/08 with an isolate representing topotype VII (SAT2-Nk 1/010) further strengthen this suggestion. Genotypes of SAT2 serotypes are with nucleotide differences of ≥ 20%, more than 15%, (Knowles and Samuel 2003) and are likely to represent distinct antigenic variants (Lycett et al. 2019; Reeve et al. 2016). The two topotypes were detected intermittently in Sudan. Topotype XIII was detected less frequently (two episodes) lastly in 2008 (http://www.wrlfmd.org/; Habiela et al. 2010); eight years before collection of the tested sera in 2016. Topotpye VII was reported, before collection of the tested sera, more frequently (four episodes) in 2007, 2010 2013 and 2014 (Anon 2016; http://www.wrlfmd.org/). Concurrently, using SAT2-Nk 1/010 in VNT to detect SAT2 activity consistently produced statistically similar estimates of seroprevalences to those revealed by using the two strains (the likely nearest estimate to true prevalence). Whereas using SAT2-Kh 1/08 produced statistically significantly lower estimates than those revealed by using the two strains (table 4, 5 and 6). It was fair to conclude that the VNT employed SAT2-Nk 1/010 was more sensitive in detection of SAT2 activity in 2016 in Sudan than the one employed SAT2-Kh 1/08. These findings were in agreement with the OIE recommendation that for optimum sensitivity of serological tests of antibodies against structural proteins of FMDV, the virus used in the test should closely match the strain circulating in the field (OIE 2018).

Control of FMD in likely endemic areas like Sudan relies on prophylactic vaccination and restriction of animal movement. The presented results suggested that a vaccine derived from one local SAT2 isolate representing topotype VII is unlikely to be effective against the other topotype (s) of SAT2 virus circulating in Sudan. These isolates were adapted to grow in BHK21 cells through 16-22 serial passages; the same approach used for development of new FMD vaccines (Mohapatra et al. 2015; Chamberlain et al. 2015; Dill et al. 2018). Further, no inconsistency was observed when they were used for surveillance of SAT2 antibodies in field sera. Therefore, no much influence on their antigenicity upon adaptation could be inferred. Vaccine recommendations of the WRL for FMD for East Africa (Anon 2018) included SAT2/Eritrea from internationally produced vaccines and SAT2/K52/84 from KEVIVAPI Kenya. The former is of topotype VII (Anon 2018) like SAT2-Nk 1/010 and showed a close antigenic relationship (r1 value of 0.58) with Sudanese isolates from 2010 (Anon 2012) while the latter vaccine strain is of topotype IV (Anon 2018) which is different from both topotypes detected circulating in Sudan. Therefore vaccine recommendations for East Africa are also unlikely to be effective against both topotypes of SAT2 virus known to be circulating in Sudan or across SAT2 topotypes circulating in East Africa. That is agreeing with the suggestion of Maree et al. (2011) for the need to develop multiple regions specific or topotype-specific vaccine strains against SAT2 infection. Pronounced antigenic differences, as measured by r1 values, were well known for SAT2 virus (Maree et al. 2011; Sirdar et al. 2019). None of 20 SAT2 virus isolates, from 10 topotypes not including XIII, showed r1 value above 0.39 using convalescent antisera of strain ERI/12/89 from topotype VII, and only 6 of them produced r1 values between 0.2 and 0.39 (Maree et al. 2011). Using multivalent vaccine against SAT2 on prophylactic bases raises the cost of vaccination considerably or is even unjustifiable when occurrence is sporadic as it is likely the case of topotype XIII in Sudan. Since 2008, topotype XIII has not been reported though, between 2008 and 2018, FMDV was serotyped and genotyped annually in Sudan (http://www.wrlfmd.org/). On the other hand, it is justifiable to follow the vaccine recommendation for East Africa and use a strain from topotype VII such as SAT2/Eritrea or SAT2-Nk 1/010 for prophylactic vaccination in Sudan. Topotype VII kept appearing in Sudan; lastly in 2017 (http://www.wrlfmd.org/). Lycett et al. (2019) observed that compared to other SAT2 viruses, strains from clade SAT2-VII seemed to be undergoing faster evolution and faster spatial spread. Instead of administrating multivalent prophylactic vaccines, antigen banks from strains of topotype XIII could be built to mount a rapid emergency response once circulation of topotype XIII is identified. Equally important is that field isolates of serotype SAT2 be promptly genotyped. Genotyping lends more rapid results than serological characterization. Establishing molecular diagnosis of SAT2 infection at the national level is crucial for control of SAT2 infection.

Undetected circulation of topotype XIII cannot be altogether ruled out. In endemic areas, multiple infections by different serotypes may result in protection against further rounds of infection with other serotypes (Doel 2005). Indeed, in this work, following vaccination with topotype VII, protection against another topotype (XIII) in the same serotype seemed unlikely. Yet, natural and vaccine induced immunity to FMDV are different in duration and quality (Doel 2005; Habiela et al. 2014). Doel (2005) observed that it is unreasonable to assume that antibodies in long-term convalescent animals would be qualitatively equivalent to specific serum antibodies from animals 21-28 days after vaccination which are used to correlate protection and neutralization titres. Therefore, mild clinical or subclinical infections with topotype XIII were not completely unlikely in the field. However, the heterotypic immune response and the subsequent partial or complete protection against infection are correlated with cross neutralization reactions (Hedger et al. 1982). In this work, surveillance of field sera by VNT using viruses belonging to topotypes VII and presumably XIII showed no evidence of considerable such cross neutralization reactions. Evidently, SAT2-Nk 1/010 showed more sensitivity in detection of SAT2 activity in the field sera than SAT2-Kh 1/08 (table 4, 5 and 6). Only 55% (53/96) of all SAT2 positive sera in this work were positive by strains from 2008 (presumably topotype XIII) and 2010 (topotype VII). This fraction increased to 61% (33/54) in Khartoum where prevalence of anti-NSPs antibodies reached 86% and more multiple infections were expected. It decreased to 48% (20/42) in the Red Sea where anti-NSPs activity was around 60% and less multiple infections were expected. To put these findings in a perspective, in Eritrea where no serotype C was reported (Paton et al. 2021), 31% (59/189) of anti-NSPs positive bovine sera in 2009 were showing the highest titre of neutralizing antibodies to serotype C (Tekleghiorghisin et al. 2013). The apparent lack of considerable cross neutralization between topotypes of SAT2 virus indicate that when more frequent circulation of more than one topotype occur, retrospective diagnosis by serological surveys to determine prevalence of SAT2 infection could be problematic or imprecise. Recently, in Sudan in a survey to determine prevalence of SAT2 antibodies by VNT, Alfouz et al. (2021) employed isolates from 2008 (SAT2-Kh 1/08) and 2010 (SAT2-Nk 1/010) outbreaks.

Acknowledgement

The Director CVRL, late Dr. Yousif Hussien, encouraged enthusiastically this work. Efforts of the technical staff at the Department of FMD-CVRL were indispensible.

Funding

This work was funded and supported by the Government of Sudan (GoS).

Compliance with ethical standards

Competing interests

The authors declare no competing interests.

Code availability

'Not applicable'

Author’s contributions

Yazeed A/Raouf carried out some of the laboratory work, analyzed data and wrote most of the manuscript. Inas Ibrahim carried out part of the laboratory work and wrote parts of the materials and methods section.

Ethics approval

Animal species used were indicated for the type of the study. Minimum numbers required to maintain the scientific quality of the experiment were included. Throughout the experiment good animal welfare was provided and all experimental procedures were approved by the Ethics Scientific Committee of CVRL-Sudan (1/A/1/824)

Consent to participate

'Not applicable'

Consent for publication

'Not applicable'

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Diversity of SAT2 Foot-and-Mouth Disease Virus in Sudan: Implication for Diagnosis and Control

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