Serological responses in relation to vaccination and infection in Zimbabwe cattle following outbreaks of FMD

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1 Appendix 17 Serological responses in relation to vaccination and infection in Zimbabwe cattle following outbreaks of FMD Donal Sammin 2 *, David Paton 1 *, Geoff Hutchings 1, Nigel Ferris 1, Scott Reid 1, Andrew Shaw 1, Nick Knowles 1, Jean-Francois Valarcher 1,Satya Parida 1, Catherine Holmes 1, Debi Gibson 1, Mandy Corteyn 1, Rosa Fernandez 1, Pip Hamblin 1 1 FAO World Reference Laboratory for FMD, Institute for Animal Health, Ash Road, Pirbright, Surrey, GU24 0NF, UK 2 Animal Health Service, FAO Headquarters, Viale delle Terme di Caracalla, Rome, Italy Abstract Objectives: The aim of this study was to evaluate the performance of serological tests in the detection of SAT-type FMDV infection in cattle, particularly ELISAs for detection of antibodies to non-structural proteins of FMDV (NSPE) and solid phase competition ELISAs (SPCE). Secondary aims were: to compare virus detection rates by virus isolation and RT-PCR tests on both oesophago-pharyngeal (OP) fluids and nasopharyngeal (NP) brush swabbings; to evaluate salivary IgA responses and to examine NSP seroconversion rates in vaccinated cattle that had been exposed to infection. Materials and Methods: Epidemiological information and clinical specimens were collected from six cattle herds in two different regions of Zimbabwe (404 cattle). One herd was thought not to have been infected, whilst the other five had had outbreaks of disease 1-5 months previously. A trivalent SAT 1, 2 and 3 vaccine had been used in some of these herds at various times either before and/or after the recent outbreaks of FMD. Sampled animals were clinically inspected at the time of sampling for the presence/absence of hoof lesions as clinical evidence of FMD convalesence. All specimens were collected over a ten day period and shipped to the Pirbright Laboratory to be tested for: (1) FMDV-specific serum antibody (by NSPEs, SPCEs and virus neutralisation); (2) the presence of virus and viral RNA in OP fluids and NP swabs and (3) salivary FMDV-specific IgA. Results: SAT 2 viruses were isolated in cell culture from OP fluids collected at two outbreak locations in Mashonaland provinces (Northern Zimbabwe) whereas SAT 1 viruses were isolated from three FMDaffected herds in Masvingo province (Southern Zimbabwe). Combining the results for OP fluids of virus isolation and RT-PCR, the prevalence of persistently infected animals ranged from 14-38%. In contrast, NP swabs yielded only two virus positive. The overall seroprevalence varied with different NSPEs from 48% to 67% whilst 70% and 82% serpositivity was apparent by homologous SPCE and VNT respectively. Approximately 75% to 90% of carriers scored seropositive in different NSPEs. Salivary IgA results are awaited. Discussion: Two different serotypes were involved in FMD outbreaks in Zimbawe in 2003/2004. Infection with either type could be readily detected by available tests for virus or antibody, but NP swabs were not a very sensitive method for virus detection. Introduction: Recent outbreaks of SAT-type FMD in Zimbabwe provided an opportunity to carry out a field study,collecting clinical specimens to evaluate different laboratory tests which had not so far been fully assessed with such serotypes. Tests for antibodies to non-structural proteins of FMDV (NSPE) have been developed to detect antibodies induced by infection, but not vaccination. They target antibodies to antigens that are highly conserved amongst FMDV serotypes, but the SAT viruses are the most distantly related to other FMDV serotypes and therefore the sensitivity of NSPE for detecting antibodies induced by SAT types of FMDV should be evaluated. Since it has been shown that the sensitivity of NSPE for detecting infected animals may be lower in previously vaccinated cattle, herds with a history of vaccination followed by infection were specifically sought for this study. A particular interest was to investigate the sensitivity of NSPE for the detection of FMDV carriers amongst vaccinated and subsequently infected cattle. Therefore, full virological work-up was essential on all of the sampled cattle. The solid phase competition ELISA (SPCE) is being introduced as a replacement for the liquid phase blocking ELISA (LPBE) due to the higher specificity of the SPCE (Paiba et al., 2004). SPCE is serotype- 108

2 specific, so it has been necessary to develop SAT-specific tests, but there has been a shortage of sera containing SAT-induced antibodies with which to evaluate the performance of these assays. Other aims of the study were: (1) To compare the sensitivities of RT-PCR methods and virus isolation in detection of SAT viruses in oesophago-pharyngeal (OP or probang ). (2) To investigate if long nasopharyngeal brush swabs could provide suitable for detection of FMDV carriers. (3) To determine the prevalence of FMDV carriers in vaccinated and subsequently infected herds. (4) To evaluate an assay for detection of salivary IgA as a means of identifying FMDV carriers. Materials and Methods: Sample collection and shipment All necessary sample buffers, sampling tubes and probang rods were shipped to Zimbabwe from the WRL FMD, along with packaging to enable the materials to be returned safely and in compliance with IATA regulations. Herd Selection The criteria for herd selection were: (i) that clinical FMD had occurred between 1 and 6 months previously and (ii) that convalescent or in-contact animals could be identified for sampling. Sampling protocol The following protocol was applied at each sampling location: (i) relevant epidemiological information was gathered (ii) the identity of each sampled animal was recorded; dentition was examined to estimate age and the hooves were examined for evidence of linear breaks. (iii) specimens were collected in the following order: 50 ml of blood was taken from the jugular vein; two different types of swab were used to collect saliva; a guarded brush swab was passed via the nares to collect nasopharyngeal mucus/cells and fluid from the oropharynx and oesophagus was collected with a probang cup. (iv) a field laboratory was established at each sampling location to process specimens on-site; saliva was expressed from swabs; specimens collected from the pharynx (OP fluid and nasopharyngeal swabbings) were divided into an aliquot for virus isolation and one to which a Lysis/Binding Buffer (Roche) was added for RT-PCR (v) all specimens were labeled and with the exception of blood, which was allowed to clot at ambient temperature, were immediately stored on ice. Further processing of specimens prior to shipment On return from the field, specimens were further processed at the Central Veterinary Laboratory, Harare. Serum (20-25 ml) was harvested from each clotted blood specimen, heat-treated for 30 minutes at 56 0 C and then stored at C. All of the other specimens (in 2ml cryotubes) were sealed with parafilm and stored at C. For air shipment of specimens to WRL the following steps had to be taken: (i) arrangements were made with an airline accepting "infectious substances" (categorised by IATA as "dangerous goods") as specimens for delivery to WRL must be sent by air-freight direct to London Heathrow and not by courier. (ii) packaging was sufficient to allow the inclusion of 50kg of dry ice as a refrigerant and sufficiently well-insulated to keep specimens frozen for at least 48 hours. (iii) a shipper's declaration and other documentation (including labels) was completed in accordance with IATA shipping regulations and the instructions provided by WRL (to ensure UK customs clearance). (iv) a contact person at the WRL was notified of the flight details and the airway bill number for the shipment. Virus detection tests Probang fluids and nasopharyngeal swab eluates were inoculated onto primary bovine thyroid cell cultures for virus isolation. Supernatant fluids from cultures showing cytopathic effects were tested for the presence of FMDV by a serotyping ELISA (Ferris and Dawson, 1988). A part of the 5 UTR and the 109

3 complete VPI gene were sequenced for a selection of the isolated viruses using RT-PCR amplification and cycle sequencing. The derived 5 UTR sequences were used to modify the forward primer employed in the diagnostic RT-PCR used at the WRL, so as to achieve perfect complementarity with the viruses that had been detected by virus isolation. The VP1 sequences were used to compare the detected viruses to previously analysed FMDVs of equivalent serotype. Probang fluids and nasopharyngeal swab eluates that had been collected in Lysis/Binding Buffer were tested by real-time RT-PCR using the procedure of Reid et al. (2003) with both the original and the newly modified diagnostic primers and TaqMan probe. RT-PCR CT values of <=45 are considered positive, values of >45 to <50 are considered inconclusive and >=50 are considered negative. However, for data analysis in this study, inconclusive results have been treated as negative. Serology Sera were tested for neutralising antibodies by virus neutralisation test (VNT) and for antibodies reactive in solid phase competition and liquid phase blocking ELISAs (SPCE and LPBE) as well as for non-structural protein (NSP) antibodies by ELISA (NSPE). The VNT, SPCE and LPBE were performed using SAT 1 and SAT 2 viruses and the procedure described in the OIE Manual. For the VNT and LPBE, the FMDV isolates used had been obtained from the present study. The SPCE employed antigens prepared from viruses isolated in the present study (SAT 1) or previously (SAT 2 Cameroon) and was also carried out using antigens and polyclonal antibody reagents prepared from serotypes O, A, Asia 1 and C (Paiba et al., 2004; Anderson et al., 2003). The specificity of the SAT 1 and SAT 2 SPCE was assessed by testing approximately 100 UK sera from each of cattle, sheep and pigs. Specificity was 100% at the 60% inhibition cut-off and approximately 99% at the 50% cut-off. The NSPEs used were the Ceditest FMDV-NS (Cedi-Diagnostics), the FMDV NSP ELISA (UBI) and the CHEKIT-FMD-3ABC (Bommeli). They were carried out according to the manufacturer s instructions. Results: Summary - collection of specimens from FMD-convalescent cattle in Zimbabwe Clinical specimens (serum, saliva, nasopharyngeal brush swabbings and oesophago-pharyngeal fluid from probang-sampling) were collected between 27 April and 7 May 2004, from 344 cattle at five outbreak locations in two different regions of Zimbabwe. Virus isolation and typing had not been attempted on specimens collected from clinically-affected cattle during the outbreaks at these locations. Therefore, at the time of sampling, it was not known which virus strain or serotype was responsible for each outbreak. At some of these outbreak locations, cattle had been vaccinated with a trivalent vaccine (comprising SAT- 1, SAT-2 and SAT-3 components) before and/or during the outbreak. As repeated vaccination might induce either serum antibody to NSPs of FMD virus and/or FMD-specific IgA in saliva, this would be a complicating factor in the interpretation of test results. Therefore serum and saliva were collected from a further 60 cattle in a herd in which there had been no evidence of clinical FMD but in which all cattle had been vaccinated on four separate occasions. All specimens were labeled, catalogued and stored as they were collected. When sampling was completed at all locations a single shipment was sent to the WRL. Information on sampled herds and animals The location of the sampled herds is shown in Figure 1 whilst summary information on sampled animals at each location, including age, breed, vaccination status and estimated time since infection, is given in Table 1. Herd A Location: Harare South district, Mashonaland East; S ; E Enterprise type: intensive dairy farm Census: 1300 cattle; 550 milking cows plus calves, followers and replacement heifers Breeds: mostly Red Danish breed Vaccination status: as this farm was within 10km of a previous FMD outbreak in July 2003 all cattle had been vaccinated twice in July/August 2003 with trivalent vaccine (SAT1, SAT2 and SAT3). In addition, in an attempt to control FMD, the entire herd (including animals that were clinically-affected with FMD) was vaccinated twice with the same vaccine in February/March

4 History of most recent FMD outbreak: FMD was reported on 23 February 2004; 30 animals in a group of replacement heifers were very severely lame. The following day the disease was noticed in milking cows; 20 were clinically affected on that occasion and a further 20 cows were affected the following day. The daily milk yield of the herd (460 lactating cows) was more than 9000 litres just before the outbreak and decreased within days to less than 3000 litres. Forty calves, all of which were less than one week of age, died within the first two days after the outbreak was reported and approximately 100 lactating cows died or were culled due to severe secondary mastitis. The most likely source of infection was a kudu which had entered the paddock in which the first clinically-affected group of cattle (replacement heifers) was grazing; this occurred about 2-3 weeks before clinical FMD was observed in cattle and the kudu is thought to have been present in the paddock with the cattle for about one week. However it was also reported that a cow from a nearby premises had escaped from a truck near the entrance to this premises and had contact with replacement heifers in a paddock close to the entrance before being recaptured. Sampling and examination of animals: A total of 130 cattle were sampled, all of which had been clinically-affected during the outbreak; the sampled animals included 20 cows (each of which, as a sequel to clinical FMD, had lost a quarter due to severe mastitis) and two groups of replacement heifers (10-18 months-old). Linear breaks in the continuity of the horn forming the wall of the hoof were observed in one or more feet of 11/20 cows and 16/30 heifers. The distance of these lesions from the coronary band was estimated as approximately 20 mm in most cases. Herd B Location: Zvimba district, Mashonaland West; S E Enterprise type: communal cattle-rearing on a resettled land holding Census: 75 cattle Breeds: mostly indigenous breed (Mashona) with some European and Brahman crossbreeds Vaccination status: uncertain; it was not clear as to whether or not animals on this premises were vaccinated at any time, either before, during and/or after the outbreak. History of most recent FMD outbreak: 11 cattle were reported to have clinical FMD on 31 March There was no indication as to the likely source of infection but uncontrolled movement of livestock from other communal farming areas was most probably a factor. Sampling and examination of animals: A total of 42 cattle were sampled; most had full permanent dentition (but the sample also included two juveniles with no permanent teeth and nine adult cattle with 2-6 permanent incisors). Indistinct linear breaks were observed in one or more hooves in at least 10 cattle but handling facilities did not favour photography or measurement of these lesions. Herd C Location: Chiredzi district, Masvingo; S ; E Enterprise type: commercial beef fattening Census: 307 cattle Breeds: mostly Beefmaster with some Mashona crossbreeds Vaccination status: this herd was last vaccinated on 26 October Vaccination was not used during or after the outbreak in this herd. History of most recent FMD outbreak: outbreak reported on 7 th April 2004; 112 clinical cases of FMD were observed; most of the affected animals had healing lesions at that time. The most likely source of infection was a feedlot owned by the same private company, from which cattle were moved to this premises (on 25 February) as FMD had been reported in the feedlot on 9 January Sampling and examination of animals: A total of 65 cattle were sampled; all had exhibited clinical signs at the time of the outbreak. All except two of these cattle had 4-6 permanent incisors, the other two animals having full permanent dentition. Linear breaks were observed in one or more hooves in 43/65 cattle (Figure 2). The distance measured between these breaks and the coronary band varied between 11 and 18 mm. Herd D Location: Mwenezi district, Masvingo; S ; E Enterprise type: communal cattle-rearing Census: 85 cattle Breeds: mostly Brahman crossbreeds Vaccination status: there are no records of this herd ever having been vaccinated for FMD (before, during or after the most recent outbreak). 111

5 History of most recent FMD outbreak: 40 animals were observed with clinical FMD on 8 January The most likely source of infection was a nearby ranch in which an outbreak had occurred; animals from both premises shared common grazing because of disruption of fences between them. Sampling and examination of animals: A total of 42 animals were sampled; most of which had full permanent dentition (but the sample also included six juveniles with no permanent teeth and ten adult cattle with 2-6 permanent incisors). Linear breaks were observed in one or more hooves in 22/42 cattle. The distance between the coronary band and these breaks varied from mm. Herd E Location: Mwenezi district, Masvingo; S ; E Enterprise type: commercial cattle-rearing (a Government-owned farm) Census: 767 cattle Breeds: mostly Brahman crossbreeds Vaccination status: Before the outbreak, this herd had last been vaccinated in However, all cattle were vaccinated twice after the outbreak (January/February 2004). History of most recent FMD outbreak: FMD was reported on 8 th December 2003; 37 clinical cases were observed when the disease was first reported but 90-95% morbidity was evident within a further 24 hours. The most likely source of infection was cattle which had been moved from a different section of the same ranch (and in which FMD had previously been reported). Sampling and examination of animals: A total of 42 cattle were examined; all were adult animals with full permanent dentition. Distinct hoof lesions were not apparent in any of these animals at the time of sampling (but the underfoot conditions in the handling facilities may have obscured some lesions). Herd F Location: Harare South district, Mashonaland East; S ; E Enterprise type: commercial beef fattening at grass; same ownership as Herd A Breeds: mostly European crossbred Vaccination status: as for Herd A; both farms were within 10km of a previous FMD outbreak and were therefore vaccinated twice (July/August 2003). In addition this herd was re-vaccinated at the time of the outbreak in Herd A (twice; February/March 2004). There has been no clinical evidence of FMD in this herd. In addition to re-vaccination at the time of the outbreak in Herd A, strict biosecurity measures were applied to prevent transmission of infection which included a complete ban on movement of personnel and vehicles between the two premises. Sampling and examination of animals: 60 heifers, all of which were months-old, were sampled; only serum and saliva were collected from these animals. Virus detection tests SAT 2 virus was recovered from herds A and B in northern Zimbabwe, whilst SAT 1 virus was recovered from herds C, D and E in southern Zimbabwe. VP1 gene sequences were obtained from isolates SAT2/ZIM/P1/2004[A2761], SAT2/ZIM/P1/2004[B7], SAT1/ZIM/P1/2004[C10], SAT1/ZIM/P1/2004[D19], SAT1/ZIM/P1/2004[E45] from herds A, B, C, D, E, respectively and their relationship to other SAT 1 and 2 viruses from southern Africa are described by Valarcher et al., elsewhere in this proceedings. Sequencing in the 5 UTR revealed 2 mismatches with the forward primer used in the WRL RT-PCR test. Therefore, a new primer was designed which gave 100% complementarity. The details of virus detection are shown in Table 2. Virus isolation combined with antigen detection ELISA yielded only one positive result from 330 nasopharyngeal swab and only one positive result was obtained by RT-PCR out of 140 nasopharyngeal swab that had been collected in Lysis/Binding Buffer (Roche). The two positive came from different animals within herd D. A total of 314 probang were examined by virus isolation and 300 that had been collected in Lysis/Binding Buffer were examined by RT-PCR. Thirty-seven were virus isolation and antigen detection ELISA positive (12%) and the proportion of positive animals ranged from 6-26% in the different herds. Using our original diagnostic RT-PCR reagents, thirty-two (11%) were positive, whereas with the improved primer, this figure rose to 67 (22%), with a range of 8-31% between herds. Only about half of individual positive by virus isolation were also positive by RT-PCR, whilst around a third that were positive by the more sensitive of the RT-PCR methods were also positive by virus isolation. Serology VNT results for each of the six herds are summarised in Fig 3, which shows frequency distributions for SAT 1 and 2 VNT titres by herd. Cattle in herd F had apparently been vaccinated four times during the 112

6 previous nine months using a trivalent SAT vaccine but had apparently not been infected. However, only 9 of 60 cattle had SAT 1 neutralising antibody titres of greater than or equal to 1 in 45 (8 being at 1 in 45 or 1 in 64), which suggests a rather low level of vaccine induced immunity to the challenge strain from nearby herd A. The SAT 2 profile was different and suggested that some of the animals had been more effectively vaccinated, vaccinated with a strain more closely matching that used in the VNT or else infected with a SAT 2 virus. NSP testing demonstrated that between 2 and 20% of cattle were seropositive, depending on the test used, and it is therefore likely that some cattle in this herd had been previously infected with a SAT 2 virus. This would be consistent with the geographical location of the farm. In herds A, B, D and E there was a clear difference in the neutralising antibody profiles to SAT 1 and 2 viruses; in each case the highest level of antibodies being found when tested against the virus serotype isolated in the probang. Herd D, which had not been vaccinated, but had been infected with SAT 1, was largely seronegative to SAT 2. Samples from herd C neutralised both serotypes to a similar extent. SPCE reactivity profiles were similar to those of the VNTs with either a SAT 1 or SAT 2 response predominating except in herd C. A summary of the reactivity of the sera in all of the different serological tests is given in Table 3. VNT with the homologous virus detected the most seropositive animals (82%), followed by SPCE (70%), Cedi (67%), UBI (53%) and Bommeli (48%). The frequency distribution curves for the Cedi and the homologous SPCE were rather similar in all herds (data not shown). In herds D and E, sera from a significant proportion of animals had percentage inhibitions in the 50-60% range which were scored negative in SPCE, but positive in Cedi, giving the latter a higher sensitivity for these sample sets (Table 3). Table 4 shows the sensitivity of the various serological tests to detect presumed carrier animals identified by viral recovery from probang collected 1-5 months after outbreaks of disease and tested by virus isolation or RT-PCR. Results were similar, regardless of which method was used for virus detection. The VNT using homologous virus, detected 100% of carriers, although in some cases, the titres of antibody were as low as 1 in 45 (Fig 4). SPCE and Cedi tests detected approximately 90% of carriers, whereas for the Bommeli and UBI tests, the figure was around 75%. 113

7 Discussion: The main aim of this study was to evaluate the sensitivity of various laboratory methods to detect infection of cattle with SAT serotypes. Efforts were made to obtain the fullest possible history of the herds and cattle investigated and a wide range of were collected so that both the virological and serological status of the animals could be comprehensively assessed. In order to be as sure as possible that came from infected animals, herds A-E were selected on the basis that they had all experienced outbreaks of clinical FMD in the preceding 1-5 months and sampling was targeted towards animals that had either been clinically affected or had been in close contact with affected animals. Examination of feet for hoof growth arrest lines was used to help target sampling to clinically affected groups. Information was not available on the potency of the vaccines that had been used in some herds, or on the relationship between the FMD virus vaccine strains and the virus isolates recovered from the herds. Although herds A and E had a history of prior vaccination, this had occurred more than 3 months before the outbreaks and was unable to prevent clinical disease. Herds B, C and D had no history of recent vaccination, whilst Farm F, in which clinical disease was not observed, had been vaccinated at the same time as farm A, i.e. in mid 2003 and early Hoof lesions like those observed in the present study have previously been described in cattle in Southern Africa which have recovered from clinical FMD (Thomson, 1994). Although not specific for FMD, the presence of linear breaks in one or more hooves of cattle at outbreak locations in this study was taken as an indicator of convalescence. It was presumed that acute infection with FMD virus at the time of the outbreak and viral replication in the hornproducing cells of the coronary band caused cessation of growth in the hoof wall such that when horn growth resumed after recovery from disease, a break appeared in the continuity of the hoof wall. If the rate of growth of the hoof wall is known, the distance between such lesions and the coronary band can be used to roughly estimate the time which has elapsed since acute infection occurred (Dekker et al., 2004). However when using this method it is important to realise that hoof horn growth rates vary considerably in cattle of different ages and even between the fore feet and hind feet of the same animal (Prentice, 1973). In the present field study, the distance from the coronary band of linear breaks in hooves of cattle at different sampling locations did correlate with the clinical history of when FMD outbreaks occurred at those locations. Two different serotypes of FMDV were isolated from the clinically affected herds; SAT 2 from the two northern herds and SAT 1 from the three southern herds. These viruses were genetically very similar to other SAT viruses obtained from Zimbabwe in 2003 (Valarcher et al., these proceedings). The infectivity status of individual cattle was assessed by probang sampling and testing at a single time point and although this was successful in identifying many carrier animals, it is likely to have underestimated the prevalence of such animals, since it has been estimated that without multiple sampling, up to 50% of carriers can be missed. Virus was isolated from 6-22% of probang (12% overall). A wide range of prevalence of carriers has been described in the literature at different times after outbreaks occurred (Salt, 2004). The prevalence of carrier cattle detected by VI and RT-PCR combined in herds A- E was between 14 and 38%; the lowest prevalence being found in herds D and E (17% and 14%) where the longest interval had elapsed between infection and sampling. The relative sensitivity of virus isolation and RT-PCR were compared and the results show that the conventional RT-PCR method in use at WRL approaches the sensitivity of virus isolation using highly sensitive primary bovine thyroid cell cultures (and in fact matches it, if inconclusive positive are considered as positive). Furthermore, using a RT-PCR that had been optimized for detection of SAT 1 and 2 viruses, a significantly greater proportion of cattle was scored as carriers than by virus isolation. The relatively poor concordance between virus isolation and RT-PCR may reflect the fact that the levels of virus present in probang are close to the lower limit of detection for both tests. The probang sampling cup collects mucus and cells from the back of the pharynx and the upper oesophagus where FMDV is known to persist. Because the collection method requires the use of a specialist sampling rod, considerable expertise and a means of cleaning the rod between animals, it was considered worthwhile to investigate an alternative sampling method. 114

8 Guarded nasopharyngeal brush swabs have been used in cattle for many years as a means of detecting respiratory viruses. Provided that the swab is sufficiently long, it can be passed to the back of the nasopharynx and used to collect mucus and cells from the bovine tonsillar region (Nuttall et al., 1980). However, this study did not find the method very successful for detecting FMDV carriers and the nasopharyngeal sampling procedure was also less well tolerated by the cattle than was insertion of the probang sampling cup. A representative of each FMDV SAT serotype was grown in cell culture and used for virus neutralization tests and to prepare antigens for SPCE. However, the SAT 2 virus selected did not generate a suitable antigen for use in SPCE and therefore was substituted with another antigen prepared from SAT 2 Cameroon The prevalence of seroreactors varied substantially between tests with the highest level being detected by homologous VNT used at a cut-off of 1 in 45 (Table 3). At this cut-off, the specificity of the VNT probably approaches 100% (Paiba et al., 2004). The test does not distinguish antibodies due to infection from those attributable to vaccination. However, the level of vaccine-induced neutralizing antibodies appeared to be low; for example there was little difference between the low levels of SAT 2 neutralising antibody found in herds D and E (Fig 2), although animals in the former herd had never been vaccinated, whilst those in herd E had been vaccinated twice approximately three months prior to the recent outbreak of FMD. The occurrence of SAT 1 neutralising antibody seroreactors in herds A, B and F, which had higher titres of SAT 2 antibodies may be attributed to either vaccination, undisclosed SAT 1 infection or cross-reactivity of SAT 2 antibodies. In herd C, where there had been no recent vaccination, the presence of high levels of SAT 1 and SAT 2 neutralising antibodies is suggestive of previous infection by SAT 2 as well as with the SAT 1 viruses that were recovered from probang. The finding of significant numbers of SAT 2 neutralising antibody seroreactors in herd F, is suggestive of previous SAT 2 infection rather than vaccination and this could be explained by the occurrence of subclinical infection around the time that SAT 2 was infecting neighbouring herds A and B. It would therefore be useful to examine sera from herd F for IgM antibodies to see if there was evidence that infection was recent or not. The SAT 1 and SAT 2 SPCEs showed similar specificity profiles when tested with naïve sera from the UK, and these were also similar to the profiles seen with SPCEs for other serotypes (Paiba et al., 2004; Anderson et al., 2003; Paton et al., this proceedings). The use of a 60% cut-off is likely to result in a test with specificity close to 100% and therefore, a lower cut-off of around 50% could probably have been used to boost sensitivity. Even at the 60% cut-off, the test was quite sensitive, scoring 70% of sera as positive, compared to 82% for VNT. All of the NSPE tests evaluated were able to detect SAT 1 and 2 antibodies. However, there were considerable differences in the seroprevalence recorded with each test; overall, the Cedi test scored 67% of as positive, compared to 48% and 53% with the NSPE from Bommeli and UBI. Similarly, more carriers were scored positive by the Cedi test (89-91%) compared to the Bommeli (74-76%) and UBI (70-75%) tests (Table 4). Figures of 92% and 47% were reported for detection of carriers (mixture of serotypes SAT 2 and A) in unvaccinated cattle from the Cameroon using the precursor to the Cedi test and the Bommeli test respectively (Bronsvoort et al., 2004). There is considerable interest in estimating the prevalence of FMDV carrier animals that can be identified by NSPE tests in vaccinated herds that have been infected with FMDV. This investigation should complement studies with similar aims that have been carried out using experimentally vaccinated and/or infected animals, since the experimental models yield fewer and are unlikely to fully mimic natural transmission conditions. However, not all of the herds had been vaccinated and even in those that had, their levels of protective immunity as assessed by neutralizing antibody titre and protection against disease were low. Authors conclusions: 1. Linear breaks in the hooves of cattle at known outbreak locations may be used as both an indicator of convalescence and to roughly estimate the time that has elapsed since infection occurred. 115

9 2. This study has provided data on the prevalence of SAT 1 and 2 virus carriers in cattle herds 1-5 months after FMDV infection and on their ease of detection by different virological and serological methods. 3. Virological tests on nasopharyngeal brush swabs scored very few cattle as infected compared to the conventional approach of testing obtained with a probang sampling cup. 4. Optimised RT-PCR was more sensitive than virus isolation for the detection of SAT 1 and SAT 2 FMDV in probang cup. 5. SPCE and NSPE tests readily detected antibodies to SAT 1 and SAT 2 FMD viruses. 6. Sensitivity estimates of NSPE for detection of FMDV carriers (75-90%) were similar to those obtained from a study of unvaccinated Cameroon cattle and accord with analyses of experimentally derived sera from a recent NSPE workshop (DeSimone et al., this proceedings). 7. However, none of the herds from which virological data were available had been optimally vaccinated and the study therefore provides limited insight into the prevalence of carriers likely following subclinical infection in such herds. Authors recommendations: 1. Some outstanding work remains to be finished, concerning antibody detection tests on saliva, use of RT-PCR internal standards and completion of data analysis. 2. It would be useful to conduct similar exercises involving herds with a more certain vaccination status and preferably following use of emergency vaccination in a previously disease-free region; also in areas where disease has occurred in vaccinated pigs and sheep. References Anderson, J., Corteyn, M., Gibson, D., Hamblin, P. & Paton, D Further validation of the solid-phase competitive ELISA for FMDV types A, C & Asia 1. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Gerzensee, Switzerland, September Rome: FAO, Appendix 24: Bronsvoort, B. M. D., Sorensen, K. J., Anderson, J., Corteyn, A., Tanya, V. N., Kitching, R. P. & Morgan, K. L Comparison of two 3ABC enzyme-linked immunosorbent assays for diagnosis of multiple-serotype foot-and-mouth disease in a cattle population in an area of endemicity. J. Clin. Microbiol. 42: Dekker, A., Moonen, A. & Pol, J.M.A. Linear hoof defects discovered in foot-and-mouth disease infected sheep; a case report. Veterinary Record. in press. DeSimone, F., DeClercq, K., Brocchi, E., Grazioli, S., Paton, D., Dekker, A., Haas, B., Yadin, H., Bulut, N., Tjornehoj, K. and Sammin, D. Preliminary report of a workshop for comparative evaluation of NSP antibody ELISAs, IZSLER, Brescia, 4-15 May Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Chania, Crete, Greece, th October Rome: FAO, Appendix 6 (This proceedings). Reid, S. M., Grierson, S. S., Ferris, N. P., Hutchings, G. H. & Alexandersen, S Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus. J. Virol. Methods 107: Ferris, N. P. & Dawson, M Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. Vet. Microbiol. 16:

10 Nuttall, P. A., Stott, E. J., & Thomas, L. H Experimental infection of calves with two strains of bovine virus diarrhoea virus: virus recovery and clinical reactions. Res. Vet. Sci. 28: Office International des Epizooties, World Organisation for Animal Health Foot and mouth disease, in: OIE Standards Commission (Ed.), Manual of standards for diagnostic tests and vaccines, 5th ed., Office International des Epizooties, Paris, France, Chapter Paiba, G. A., Anderson, J., Paton, D. J., Soldan, A. W., Alexandersen, S., Corteyn, M., Wilsden, G., Hamblin, P., Mackay, D. K. J. & Donaldson, A. I Validation of a Footand-mouth disease antibody screening Solid-phase competition ELISA (SPCE). J. Virol. Methods 115: Paton, D.J., Armstrong, R.M., Fernandez, R., Hamblin, P.A., Turner, L., Corteyn, M, Gibson, D, Parida, S., Wright, C. & Anderson, J. FAO Phase XVIII FMD serological standardisation; progress and future prospects. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and- Mouth Disease, Chania, Crete, Greece, 12-15th October Rome: FAO, Appendix 10 (This proceedings). Prentice, D. E Growth and wear rates of hoof horn in Ayrshire cattle. Res. Vet. Sci. 14: Salt, J Foot and Mouth Disease, Current Perspectives, edited by Francisico Sobrino and Estaban Domingo, Horizon Bioscience, Wymondham, England. Chapter 6, Persistence of Footand-Mouth disease. Chapter 6, pp Thomson, G.R Foot and mouth disease. In: Infectious diseases of livestock with special reference to Southern Africa. eds. J. A. W. Coetzer, G. R. Thomson & R. C. Tustin. Cape Town, Oxford University Press. pp Valarcher J. F., Knowles, N. J., Fernandez, R., Davies, P. R., Midgley, R. J. Statham, B., Hutchings, G., Newman B. J., Ferris N. P. & Paton D. J. Global foot-and-mouth disease situation Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Chania, Crete, Greece, th October Rome: FAO, Appendix 21 (This proceedings). Zhang, Z. & Alexandersen, S Detection of carrier cattle and sheep persistently infected with foot-and-mouth disease virus by a rapid real-time RT-PCR assay. J. Virol. Methods 111:

11 Table 1. Summary of the sampled animals in each herd Herds Province No. of cattle sampled and tested Breed Age Vaccination status Time elapsed since outbreak Hoof Lesions* Serotype of virus detected in probang SAT 2 A Mashonaland East 130 Red Danish 110 < 18 m. 20 adults x2 pre-outbreak x2 post-outbreak >2 months 27/50; approx. 20mm B Mashonaland 42 Mashona Adult unknown >1 month 10/42; SAT 2 West not measured C Masvingo 65 Beef-master Not since 2001 ~1 month 43/65; SAT 1 years mm D Masvingo 42 Brahmin X Adult Never ~4 months 22/42; SAT mm E Masvingo 65 Brahmin X Adult x2 post-outbreak ~5 months NOT seen SAT 1 F Mashonaland East 60 European X m. x2 Jul/Aug 03 x2 Feb/Mar 04 No history of infection NOT seen No probang collected * the number of animals in which linear breaks were apparent in one or more hooves/the number of animals whose feet were examined; distance of linear breaks in the hoof-wall from the coronary band 118

12 Table 2. Virus detection results Herds Serotype of virus detected in probang Number of nasopharyng eal swab tested by virus isolation/rt -PCR* Number of probang tested by virus isolation/rt -PCR Number and percentage of probang found positive by virus isolation Number and percentage of probang found positive by RT-PCR 1 Number and percentage of probang found positive by RT-PCR 2 Number of probang found positive by virus isolation and RT-PCR / positive by any method Prevalence of probang sample positive animals detected by one or more detection methods A SAT / / / 6% 19 / 16% 36 / 31% 4 / 40 34% B SAT 2 36 / / 37 4 / 11% 2 / 5% 5 / 14% 1 / 8 22% C SAT 1 62 / / / 26% 8 / 16% 16 / 31% 9 / 23 38% D SAT 1 40 / / 36 4 / 11% 1 / <1% 3 / 8% 1 / 6 17% E SAT 1 63 / / 59 5 / 9% 2 / 3% 7 / 12% 4 / 8 14% Overall 330 / / / 12% 32 / 11% 67 / 22% 19 / 85 28% no probang collected from herd F; * only one nasopharyngeal swab sample (from herd D) was found positive by virus isolation and one (other) sample (from herd D) was found positive by RT-PCR; RT-PCR 1 = RT-PCR using diagnostic primers of Reid et al. (2003); RT-PCR 2 = using modified forward primer with enhanced match to isolated SAT viruses 119

13 Figure 1. Map of Zimbabwe showing herd locations Figure 2. Linear breaks 11 mm from the coronary band in the hoof of a convalescent animal 120

14 Herd A Herd B <= <= Herd C Herd D <= <= Herd E Herd F <= <= Fig 3. VNT results presented as frequency (y axis) versus titre expressed as reciprocal serum dilution (x axis) plots. SAT 1 VNT = solid line; SAT 2 VNT = dotted line 121

15 Table 3. Comparative numbers of seroreactors and seroprevalence within herds Herd VNT* SPCE* Cedi Bommeli UBI A 128 (98%) 129 (98%) 120 (91%) 86 (66%) 101 (77%) B 39 (95%) 39 (95%) 30 (74%) 20 (49%) 25 (61%) C 65 (100%) 65 (100%) 63 (97%) 65 (100%) 65 (100%) D 38 (93%) 9 (22%) 16 (39%) 10 (24%) 7 (17%) E 42 (65%) 21 (32%) 30 (46%) 10 (15%) 10 (15%) F 17 (28%) 20 (33%) 12 (20%) 1 (2%) 7 (12%) Combined 329 (82%) 283 (70%) 271 (67%) 192 (48%) 215 (53%) * Using virus or antigen of the same serotype as that isolated from probang Sera were scored positive at the following cut-offs: VNT, >=1 in 45; SPCE, >60%; Cedi, >=50%; Bommeli, >=20 percent positivity; UBI, OD >= 0.23 x positive control Table 4. Comparative serological detection of virus carriers VNT* SPCE* Cedi Bomme UBI li % of 37 VI carriers detected % of 65 RT-PCR carriers detected** *Using virus or antigen of the same serotype as that isolated from probang ** No matching blood sample found for two cattle with RT-PCR positive probangs Sera were scored positive using the same criteria as in Table 3. Fig 4. Homologous VNT results by herd for carrier animals detected by virus isolation or RT-PCR, presented as frequency (y axis) versus titre (x axis) plots. Frequency <= Reciprocal serum dilution (VNT) A B C D E 122