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Vaccination against foot-and-mouth disease virus confers complete clinical protection in 7 days and partial protection in 4 days: Use in emergency outbreak response
a Plum Island Animal Disease Center, Foot-and-Mouth Disease Unit PIADC, ARS, USDA P.O. Box 848 Greenport, NY 11944, USA b Department of Pathobiology and Veterinary Sciences, University of Connecticut, Storrs, CT, USA c Merial Animal Health Ltd., Pirbright, Surrey, UK
Received 7 March 2005; received in revised form 13 May 2005; accepted 22 July 2005 Available online 8 August 2005
Recent outbreaks of foot-and-mouth disease virus (FMDV) demonstrate that this highly contagious viral infection of cloven hoofed animals continues to be a signiﬁcant economic problem worldwide. Debate about the most effective way to respond to outbreaks of FMDV in disease free countries continues to center on the use of vaccines. In this report, we present data showing that a commercially available, standard dose vaccine formulation can fully protect cattle against direct challenge with the virus in as little as 7 days with no carrier transmission to na¨ıve animals. Cattle challenged 4 days after vaccination have reduced disease severity, no detectable virus in blood and little virus shedding from nasal secretions. These signiﬁcant effects at 4 days post vaccination, conﬁrmed in two separate trials, support the value of using currently available vaccines as a ﬁrst line of defense against foot-and-mouth disease (FMD) outbreaks. Published by Elsevier Ltd.
Keywords: Foot-and-mouth disease virous; Rapid acting vaccines; Emergency outbreak response
Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that continues to be a major threat to trade and commodity markets worldwide. Devastating outbreaks such as that suffered by the United Kingdom in 2001 , have cost economies in ways not limited to the livestock industry but also in tourism and travel, for example. Even in quickly controlled outbreaks like those in South Korea and Japan in 2000, countries invariably suffer signiﬁcant economic problems (reviewed by Grubman and Baxt ). Thus Japan banned imports of pork products from Korea and this ban remained in effect into 2002 [3,4]. The key to control ling this virus and thereby minimizing economic impact is to
Corresponding author. Tel.: +631 323 3249; fax: +631 323 3006. E-mail address: firstname.lastname@example.org (W.T. Golde).
0264-410X/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.vaccine.2005.07.043
have rapid and effective outbreak response programs. Besides effective and rapid diagnostics and conﬁrmatory testing, outbreak response requires rapid acting vaccine formulations that could be applied in ring vaccination. Presently approved vaccines have not been extensively tested in an outbreak response format.
The concept of banks of inactivated, concentrated FMD antigens which can be used to rapidly formulate vaccine for an emergency is not new. Such banks have existed, in one form or another, for about 30 years and many countries or groups of countries own or are joint owners of a bank [5,6]. How-ever, the prevailing philosophy on the use of emergency bank vaccines has been, for many years, one of last resort when all other approaches to eradication have been exhausted. One reason for this was the prolonged loss of export markets for livestock and livestock products (effectively a minimum of 12 months) as a consequence of the use of vaccine and in
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accordance with the previous version of the International Animal Health Code of the Ofﬁce International des Epizooties (OIE). Additionally, there has been, perhaps, a lack of conviction in the minds of many veterinary authorities on the efﬁcacy of vaccines used under emergency conditions.
Nevertheless, there are a substantial number of national and international banks in existence, several of which have been used for emergencies such as the FMD outbreak in the Balkans in 1996. Further, there is an increasing likelihood that a major outbreak in a disease free country would be controlled, in part, by the use of an appropriate vaccine.
There are several reasons for the change of emphasis from ’last resort’ to early deployment in an outbreak. The ﬁrst relates to the experience in the United Kingdom which suffered a major outbreak of the O serotype in 2001. While vaccine was not used, the UK Government has clearly indicated that it would consider vaccination in the control and eradication of a future outbreak. This was supported by several expert enquiries including that of the Royal Society (2002) in which the report recommended ‘Emergency vaccination should be seen as a major tool of ﬁrst resort along with culling of infected premises and dangerous contacts’. During this period, article 220.127.116.11 of the OIE Animal Health Code was also amended such that a country could regain its FMD-free status either 3 or 6 months after vaccination depending on whether the vaccinated livestock was slaughtered or allowed to live.
Over the last decade or so, it has become quite clear that high potency, emergency vaccines are capable of providing substantial levels of protection in livestock and there has been a particularly active interest in the rate of development of immunity in relation to the need to provide protection in the face of an approaching disease front [7–10]. Using high potency vaccines prepared from antigens held by the International Vaccine Bank, Doel and colleagues demonstrated that cattle could be protected against virulent challenge 4 days after vaccination . Furthermore, there was evidence that the extent to which virus could be recovered from animals that developed a carrier state was related to the interval between vaccination and challenge . The general obser vations have been extended by other workers to sheep and pigs [8,10] and there is growing evidence that high potency vaccines can reduce the potential level of virus shedding following challenge .
A critical question prompted by these studies is whether or not the results observed are due uniquely to the use of high potency bank vaccines or whether conventional potency vaccines are capable of conferring a similar quality of immunity in relation to early onset of protection and inﬂuence on the carrier state. If indeed, the latter is the case, such results will provide added conﬁdence for veterinary authorities contemplating the use of vaccine to prevent the spread of an outbreak as well as point to a common mechanism(s) for prevention of viral replication in and virus recovery from the oropharynx of challenged cattle.
In the work reported here, the responses of cattle vaccinated with a conventional potency vaccine and challenged with a virulent strain of FMDV by the intradermolingual route have been analyzed. These vaccine tests were specifically designed to use the only vaccination and challenge methods accepted by OIE  i norder to provide results that have met these strict standards. Commercially available vaccines would not require reformulation and would provide more doses for outbreak response compared to high potency vaccines. In two independent trials, the standard, commercial vaccine, inoculated at the standard dose and route as indicated by the manufacturer, was able to protect all cattle from clinical disease 7 days after vaccination. Further, animals challenged on day 4 showed delayed and less severe disease as well as reduced virus in plasma and nasopharyngeal secretions. We also tested for the transmission of FMDV to na¨ıve animals by this group of vaccinated and protected cattle. In a 2-week period, when the vaccinated/challenged animals tested positive for FMDV, no virus or response to virus was detected in sentinel animals kept in direct contact with carrier animals.
2. Materials and methods
Holstein steers, between 400 and 500 lbs, were obtained from Thomas Morris Inc., Reiserstown, MD, and allowed to acclimatize from shipping for 1 week before testing was initiated. Baseline vital signs and serum and plasma samples were taken before the ﬁrst vaccinations.
2.2. Vaccine and vaccine trials
A commercial, double oil emulsion vaccine was prepared by Merial Animal Health Limited, Pirbright, UK using inactivated puriﬁed O Manisa strain of FMDV. The potency was consistent with a 3 PD50 vaccine as determined from companion lots of vaccine from the same FMDV strain and the vaccine was administered at the manufacturers recommended dose of 2.0 ml/animal, intramuscularly in the neck. Animals were vaccinated in two different experiments. In the ﬁrst one, groups of three animals were vaccinated at 4, 7, 14 and 21 days before challenge (named as group −4, −7, −14 and −21 below). In the second experiment, groups of 5 animals were vaccinated at 4 and 7 days before challenge (named as group −4 and −7). Each experiment included two na¨ıve animals used as controls. In each experiment vaccinated and na¨ıve animals were mingled in direct contact until the end of each experiment, day 19 for ﬁrst trial and day 22 for second trial.
The challenge virus consisted of tongue epithelium macerate harvested from two cattle deliberately infected with
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FMDV strain O Manisa. After preparation, virus aliquots were maintained at −70 ◦C until use. The challenge virus was previously titrated in the tongue of a cow to determine 50% bovine tongue infectious doses (BTID50). Following the OIE guidelines , all experimental animals were challenged with 104 BTID50 of FMDV, strain O Manisa, intradermolingually in 0.1 ml of PBS.
2.4. Carrier state and transmission to na¨ıve cattle
Four of the ﬁve animals of the group −7 in the second experiment were evaluated beyond day 28 to detect if there was a persistence of the virus in the pharyngeal area. For this, oral-pharyngeal samples (probang), plasma and nasal swabs were collected for virus isolation together with serum samples for antibody detection twice a week, beginning on day 29 postchallenge for a period of 6 weeks (71 days post challenge). To evaluate transmission from potential carrier animals to na¨ıve animals, three new cattle were placed in the same room, in direct contact, for a period of 2 weeks, starting on day 28 postchallenge. Samples (serum, plasma, nasal swabs and probangs) were also collected from these animals twice a week. On day 41 postchallenge the contact animals were moved to a new room and samples collected up to day 59 postchallenge. A last sample collection was done on day 93 postchallenge.
2.5. Clinical assessment of cattle
Cattle were monitored for clinical signs of FMD during vaccination and challenge periods. Temperatures were monitored daily through out the experiment. Animals were monitored for clinical signs daily, for 7 days after the challenge. Cattle were examined closely after sedation on days 3 or 4 (for ﬁrst or second trial, respectively) and day 7 (for both trials). Clinical scores were determined by assigning a score of 1 for each foot that developed vesicles. The maximum clinical score is 4. Since the challenge virus was inoculated in the tongue, vesicles in the tongue and mouth were not considered in the clinical scoring system.
2.6. Virus isolation from plasma, nasal swabs and probang samples
Heparanized blood was obtained daily from day 0–5 postchallenge, aliquoted and stored at −70 ◦C. Nasal swabs were collected on the same days and transported in MEM1% antibiotics, aliquoted and stored at −70 ◦C until further use. An aliquot of plasma or nasal swab was thawed for inoculation of BHK-21 cell monolayers for virus isolation and quantiﬁcation as previously described . Cell cultures were examined for cytopathic effect (CPE) for 48–72 h. If no CPE was detected, the cells were frozen and thawed and used to inoculate fresh cultures and examined for CPE for another 48 h (blind passage). Speciﬁcity of CPE was determined by identiﬁcation of virus in freeze-thawed extracts from the inoculated cell cultures using an indirect ELISA assay similar to one used for virus isolation .
Probang samples were collected and added to equal amounts of MEM with 10 mM Hepes and frozen at −70 ◦C until further use. When thawed, to remove antibodies that may be present in the samples, 5 ml of the sample were treated with 5 ml of trichlorotriﬂuoroethane (TTE) in an OmniTMmix tube and after clariﬁcation by centrifugation, supernatant was used to inoculate preformed monolayers of BHK-21, in 25 cm2 cell culture ﬂasks. Flasks were observed daily for CPE and positive samples were conﬁrmed by ELISA. For negative samples, a second and a third passage over cells were performed.
2.7. Determination of neutralizing antibody titer
Serum samples were drawn before vaccination and 4 days after vaccination followed by weekly sampling until challenge. Post challenge, serum samples were taken on days 0, 4, and 7 and then weekly through day 28. Samples, together with known positive and negative sera as controls, were heat inactivated (56 ◦C, 30 min) and used for microtiter neutralization assay on BHK-21 cells. Serial dilutions of serum were incubated with a virus dose of 100 TCID50 of O1 Manisa for 1 h at 37 ◦C and then transferred to preformed monolayers of BHK-21 cells and incubated at 37 ◦C for 48–72 h. CPE was used to determine the end-point titers that were calculated as the reciprocal of the last serum dilution to neutralize 100 TCID50 of virus in 50% of the wells.
2.8. Detection of antibody to nonstructural proteins
Sera obtained before vaccination and after challenge (days −21 and 28 of the experiment, respectively) were also tested for the presence of antibodies against viral NS proteins (3ABC) by a indirect enzyme linked immunosorbent assay (3ABC-ELISA), using an ELISA similar to that one described by Meyer et al. .
Foot-and-mouth disease vaccines formulated for emergency use have been previously shown to be protective in cattle challenged through contact with infected animals as early as four days post vaccination . These data indicated that high potency vaccines are effective at early times post vaccination. These preliminary results warranted studies using vaccines containing standard potency vaccines and with direct intradermolingual challenge, following OIE guidelines for FMD vaccine efﬁcacy trials.
3.1. Protection from clinical disease
Two separate trials were performed utilizing the same vaccine and virus challenge. In the ﬁrst trial, three animals per
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group were vaccinated at 21, 14, 7, and 4 days before challenge. All animals were housed in a single room and were challenged on the same day, including two control na¨ıve cattle. Vaccinated animals that showed clear, clinical signs of FMD in the form of vesicles on the feet were removed to a separate room. The two na¨ıve animals, however, remained in the room for the duration of the experiment as directed by OIE guidelines. Animals vaccinated on days −21, −14 and −7 were completely protected from clinical disease when they were assessed for clinical signs on days 4 and 7 after challenge (Table 1).Animals vaccinated on day -4 had vesicles on all four feet by day 4 after challenge and were clinically indistinguishable from na¨ıve animals. A second trial was performed following the same protocol as trial 1, but this time groups of 5 animals were vaccinated at days −7or −4. As in trial 1, all animals vaccinated on day −7 were free of clinical disease when assessed on days 3 and 7 after challenge. Four of ﬁve animals vaccinated 4 days before challenge had reduced and delayed clinical symptoms compared to controls. In addition,
Table 2 Virus isolation results in clinical samples from vaccinated and naive cattle
one animal in this group showed no clinical signs. As in the ﬁrst trail, na¨ıve animals had vesicles on all 4 feet by day 3 post challenge ( Table 1).
3.2. Virus isolation in blood and nasal swabs
To further determine the relative level of protection in the different treatment groups, blood samples, nasal swabs and probangs taken from all of the animals were analyzed for the presence of virus. In the ﬁrst trial, only the na¨ıve animals had virus isolated from plasma after challenge ( Table 2). All animals vaccinated 7 days or longer before challenge were negative for virus isolation. Notably, the animals vaccinated 4 days before challenge were also negative for virus in plasma even though all three had clinical scores of 4 (vesicles on all 4 feet). One animal in this vaccinated group had virus isolated from nasal swabs, as was the case in the na¨ıve group.
Results from the second trial conﬁrmed these observations ( Table 2). Again, na ¨ıve animals were positive for FMD
Vaccination day Trial 1 Trial 2 Virus in plasma Virus in nasal swabs Virus in plasma Virus in nasal swabs −21 0/3 0/3 ND ND −14 0/3 0/3 ND ND −7 0/3 0/3 0/5 0/5 −4 0/3 1/3 (3) 0/5 2/5 (3,3) Na¨ıve 2/2 (2,3)a 1/2 (2) 2/2 (1,1) 2/2 (1,1)
a Indicates the ﬁrst day after challenge that virus was detected. Viremia lasted 1–3 days.
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Fig. 1. Mean serum antibody responses in cattle following vaccination and challenge with FMDV O Manisa. Groups of cattle were vaccinated at 21 (open diamond), 14 (open squares), 7 (open triangles) and 4 (closed diamond) days prior to challenge, or not vaccinated (closed triangles). Arrows mark the day of challenge. VNT: virus neutralizing titer.
virus in plasma. No virus was isolated from plasma or nasal swabs in the group −7. Even though 4 of 5 animals in the group −4 in trial 2 showed clinical disease, none of these animals had detectable virus in plasma and only 2 of 5 of these animals had virus in nasal swabs. In both trials, animals from groups −4, whether showing clinical signs or not, had no virus in blood and reduced amount of virus from nasal swabs when compared with naives animals (results not shown).
3.3. Neutralizing antibody responses
It is generally accepted that there is a good correlation between the virus neutralizing antibody titers and protection from challenge . Fig. 1 shows that in our experiments, vaccination induced measurable levels of anti-FMDV antibody detected 6–7 days after vaccination. In the groups −4, serum neutralizing antibody titers were not detected in either trial after vaccination. After challenge, there was a greater increase in neutralizing antibody titer in groups −4 than in any of the other treatment groups. This increase was more rapid than in the na¨ıve animals, indicating some response to the vaccine at this early, 4 day time point. This result was consistent across both of trials and indicated an antigenic boost early after challenge in these animals.
3.4. Serum antibody responses to nonstructural proteins of FMDV
The presence of serum antibody reactivity against FMDV non-structural proteins (NSP) is indicative of active virus infection (i.e. active viral replication). Various tests that measure antibodies to NSP are routinely utilized to distinguish vaccinated from infected animals . Table 3 s hows that in both trials, all na¨ıve, control animals seroconverted for NSP when tested 19 or 22 dpc. In addition, all the animals of the group −4 also had serum antibody reactive with NSP. Only 1 of 3 cattle seroconverted in the group −21 and1of8inthe group −7. No animals in the group −14 seroconverted.
Even with a combined cohort of n = 8 for both trials for vaccination groups day −4 and day −7, these cohorts are still too small to correlate antibody reactivity to NS proteins and disease. These data collectively indicate that by day 7 post vaccination, the immune response to vaccination, reduced the level of viral infection and consequently virus shedding by infected animals. As there were animals reactive with NSP of FMDV even in group −21, protective immunity does not block infection completely and some level of viral replication appears to occur in these cattle, as has been previously noted .
3.5. Carrier state and transmission to naive cattle
An important consequence of vaccination of cattle followed by direct exposure to virus is the potential for these animals to become subclinically infected carriers posing a risk to contact, na¨ıve animals . We analyzed four of the ﬁve animals of the group −7 in the second trial for presence of virus starting at 28 days post challenge. As described before, these animals remained clinically normal after direct challenge in the tongue and while in direct contact exposure to clinically infected animals (the na¨ıve controls), that were kept in the room up to 22 dpc. Table 4 shows that analy sis from day 28 through day 71 after challenge revealed that three of four animals had infectious virus detectable by virus isolation in their oropharyngeal secretions at least twice during this time period. These results indicated that these three animals were subclinically infected carriers.
In order to assess their potential as a source of viral infection, we housed three na¨ıve animals in direct contact with the carrier cattle for a period of 2 weeks starting on day 28 and ending on day 41 post challenge. After separation, sample collection continued twice a week for 52 more days when ﬁnal samples of plasma and probang were collected. Virus was detected intermittently in probang samples in three of the four vaccinated and challenged animals over the 2-week exposure period ( Table 4),and antibodies were detected continuously in serum but no virus was isolated from plasma or nasal swabs in this period. All plasma and nasal swabs samples from na¨ıve, contact animals tested negative for virus isolation in two blind passages in tissue culture. All probang samples tested negative after three blind passages.
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Neg: negative, Pos: positive. No animals were vaccinated at −21 and −14 days of challenge in the second trial. Table 4 Isolation of virus from oropharyngeal samples obtained four vaccinated and protected animals Bovine # 28a 29 31 36 38 43 45 50 52 57 59 64 66 71 21 N P1 N P1 P1 P3 N N P1 P3 N N N N 31 N P1 N P1 P3 P1 N N ND ND ND ND ND ND 32 P1 P1 N N N N P3 N N N N N N N 33 N N N N N N N N N N N N N N
P1: positive in the ﬁrst passage over cells. P2: positive in the second passage over cells. P3: positive in the third passage over cells. N: negative after three blind
passages. ND: not determined. a Days postchallenge.
In addition, no serum antibodies were detectable in the na¨ıve animals by either serum neutralization or ELISA for non-structural proteins of FMDV 52 days after being separated from the 4 carrier animals.
In this report, we have demonstrated that a commercial FMD vaccine with a standard antigen payload used according to the manufacturer’s instructions for inoculation, completely protects cattle from clinical disease in as short as 7 days after vaccination. Further, analysis of clinical samples for the presence of virus showed that animals vaccinated 4 days before challenge not only had milder and delayed development of clinical disease, but also had signiﬁcantly lower viral loads in plasma and in nasal secretions compared to na¨ıve animals, based on quantiﬁcation of virus in those samples (results not shown). These results support the approach of controlling outbreaks of FMD through the use of standard potency FMD vaccines. These data also support the prediction that vaccination would have a real effect on slowing the spread of FMDV even if complete protection of all animals is not achieved. With recent development of rapid, nucleic acid based diagnostic tests [16,17] itis possible to detect out breaks and identify the causative strain early in the outbreak. Intervention with vaccination and implementation of movement restrictions will therefore greatly reduce virus shedding and the availability susceptible animals in as early as 4 days post vaccination.
An important consequence of vaccinated animals being exposed to the virus is the potential of these animals becoming FMDV carriers. Our work clearly documented this, as a number of protected animals became carriers. However, the ability of carriers to pass the virus to na¨ıve individuals seemed to be very low or perhaps even nonexistent, at least under the conditions of this study. We evaluated the potential of carrier animals to infect na¨ıve animals in direct contact for a period of 2 weeks. We clearly demonstrated that three of these four animals had virus present albeit at low levels, in their oropharyngeal ﬂuid. Virus detection always required at least one and sometimes two blind passages in
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cells. None of three naive animals kept in direct contact for 2 weeks with these carrier animals became infected despite the fact that three of four animals of the latter had virus present in their oropharynx several times during this period. Under the OIE guidelines governing our method of challenge of vaccinated cattle, the carrier effect was documented, however, these carrier animals did not transmit viral infection. In determining whether to use vaccination as a response to FMDV outbreak in cattle, these data indicate that the carrier effect may be less of a risk making the beneﬁts of vaccination more substantial. This is of particular note considering previous reports showing virus isolated from probang samples required the emulsiﬁcation of 3–15 ml of oropharingeal (OP) ﬂuid specimen with triﬂuorotrichloroethane (TTE) to liberate the virus from antibodies or other inhibitors . Itis described by Sutmoller et al. () that early after infection treatment with TTE did not increase the FMD virus titers of the OP ﬂuid, but, from 14 days post-infection onwards titers of the treated OP ﬂuid increase 10–100-fold over the untreated sample.
Although the carrier state has been documented and studied in cattle [20,21] t ransmissionof FMD has never been convincingly demonstrated under controlled conditions . Again, the animal of interest is one vaccinated and known to be exposed to virus, becoming infected without showing clinical signs. In this study, we are able to demonstrate active infection by induction of antibodies to nonstructural proteins in addition to the isolation of the virus. These animals have had exposure to inactivated antigen and live virus and have a broad speciﬁcity antibody response to viral antigens. Much of the risk of carrier transmission could be mitigated by speciﬁc restrictions on animal movement, repeated testing of animals with highly sensitive techniques capable of detecting virus even when coupled with antibodies (e.g. real-time RT-PCR) and a ban on the introduction of na¨ıve animals until carrier animals have been removed and decontamination procedures of the premises are carried out.
In conclusion our study supports the use of current standard vaccine as a ﬁrst-line of defense in the control of FMD outbreaks. We demonstrated protection from clinical disease in 7 days and dramatic decrease in virus shedding at 4 days despite the fact that protection against clinical disease was not always achieved at this early time. The issue of carrier animals as a potential source of virus to naive animals appears not to be relevant at least in the short term and testing with very sensitive tests available today allows their removal. Vaccination achieves two important goals in FMD control: rapidly protecting animals from clinical disease and dramatically reducing virus shedding even in animals that are not completely protected from clinical disease.
The authors would like to thank Drs. Min-Tsung Yeh and He Wang for their assistance with the ELISA assay measuring antibody responses to nonstructural proteins of FMDV. We are very grateful for the efforts of Sam Trivedi, a summer intern from the Veterinary Medical College at UC Davis, for assistance during the ﬁrst cattle trial and to the animal care staff at PIADC for excellent assistance throughout these studies.
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