Appendix 21

Appendix 21

Foot-and-Mouth disease virus transmission between individually housed calves

A. Dekker, A. Bouma and M.C.M. de Jong

Introduction

Foot-and-Mouth disease (FMD) virus was most likely introduced in the Netherlands by calves imported from Ireland via an FMDV-contaminated resting point in Mayenne, France. The calves were sent to a mixed veal-calf/dairy-goat farm and housed individually (Figure 1). The first clinical signs of FMD were reported in the goats, three weeks after arrival of the calves (index case 2001/03). At culling of the farm, serum samples were taken from all calves (74) and 100 goats on that farm. From the 74 calves 4 were positive for antibodies against FMDV, whereas 87 of the 100 goats sampled were serological positive. The serological positive calves were not found in an isolated part of the stable (Figure 1). Based on the information given in Figure 1, it seemed that these calves were able to have contact with other calves.

Since only a small percentage of the calves was serologically positive, it was questioned whether the hypothesised route via Mayenne was likely or even possible. After all, FMD is known to be very contagious and the spread of FMD virus among calves appeared to be only limited. The calves on the index farm (NET/2001/03) were initially housed individually. As for every contagious disease, the contact structure between infectious and susceptible animals is important and in part determines whether an infection will spread. In theory, infection of animals housed in a row will stop spreading, if transmission relies on direct contact between animals, because the chance of transmission will always be smaller than 1. However, we had no information on the reduction of transmission of FMD virus if such a housing system is used. In a previous challenge experiment using tethered calves no transmission from infected calves (10 vaccinated and 2 controls) to 5 non-immune contact calves was observed when the

25 calves 2 + 23 calves

10 calves 15 calves 2 +

Storage room

Milking parlour (goats) Milk tank

Figure 1: schematic drawing of the stables where the calves where housed on outbreak NET/2001/03

calves were culled 7 days after infection (personal observation). This indicated that transmission between calves that are individually fed does not happen very easily. In the current experiments we try to estimate the transmission rate of FMD virus can in individually housed calves without direct and with direct contact.

Materials and methods

Animal experiments

Conventionally reared calves of 4 to 9 weeks of age were randomly allocated to the experimental groups. In experiments 1-4, one inoculated calf was housed at 1 metre distance from the contact calf. In experiments 5 and 6, the inoculated calf was housed between two contact calves (Photo 1). The animals arrived at "High Containment" facilities 5 - 7 days before the start of the experiment. At day 0, the experiment started with inoculation of one of the calves in each group was intranasally inoculated with 1.5 ml of FMD virus in each nostril. The other animal(s) in each group were in-contact calves. The body temperature off all calves was recorded daily. After inoculation heparinised blood samples were taken from all calves daily until seven days, buccal swabs using cotton gauze and a forceps until 14 days after inoculation, and serum samples weekly. Animal caretakers first took care of the contact calves, and changed shoes and clothing before taking care of the infected calves. Calves had their own feeding and drinking bucket. In experiment 1and 2 all calves were culled 30 days after inoculation, in experiment 2 and 3 the calves were culled 43 days after inoculation and in the last two experiment 71 days after inoculation.

Virus

Samples from outbreak NET/2001/01 and NET/2001/03 were grown in the tongue of FMD serological negative calf. After approximately 25 hours all vesicles were collected and used to prepare the challenge virus, which was stored at -70 °C. The challenge virus was titrated on cattle tongue before use, and a titre of 105.9 cattle ID50 was found. In the experiments the virus was diluted 1500 times and 1.5 ml was inoculated in each nostril, resulting in approximately 1500 cattle ID50 per calf. After inoculation samples of the freshly prepared inoculum as well as the inoculum used in the stable were titrated on secondary porcine kidney cells as well as on secondary lamb kidney cells.

Photo 1: Housing of the calves in the experiments with direct contact.

Virus neutralisation test

The virus neutralisation test was performed as described previously (Dekker and Terpstra, 1996) using O1Manisa virus and secondary porcine kidney cells.

Results

Titration of the inoculum on secondary pig and lamb kidney cells resulted in a dose of 6800 PFU and 9800 PFU per calf, respectively. In 5 out of the 6 inoculated animals clinical signs of foot-and-mouth disease were observed, with a marked rise in body temperature for one or two days between 5 and 7 days after inoculation. Vesicles were observed for the first time 6 to 9 days after infection, in some animals reddening of the dental pad was seen one or two days earlier. Lameness was not observed, but due to the housing system (Photo 1) also unlikely to be observed easily. In all inoculated animals a four-fold increase in neutralisation titre was observed between 7 and 11 days after inoculation. In one of the inoculated calves no vesicles or lesions were detected, but the animal had fever for one day and became serological positive after 15 days. In the inoculated calves the peak in antibody titre was detected between 10 and 27 days after inoculation. Antibody titres declined towards the end of the experiment, but stayed in all inoculated animals well above the cut-off defined by the FAO cut-off reference serum. None of the contact calves showed a rise in body temperature, or any vesicles or lesion. All contact calves remained serologically negative.

Table 1: Clinical and serological observations.

Experiment Inoculated / contact Vesicles or lesions Maximum rectal temp (°C) Day first four-fold rise antibodies

Maximum VNT titre (10log) 1 I Foot and mouth lesions 40.4 8 2.4 1 C No lesions <0.3 2 I No lesions 40.1 15 1.05 2 C No lesions <0.3 3 I mouth lesions 40.5 7 >2.4 3 C No lesions 39.1 <0.3 4 I Foot and mouth lesions 40.4 7 >2.4 4 C Suspect mouth lesion 39.1 <0.3 5 C No lesions 39 <0.3 5 I Foot lesions 40.3 7 >2.4 5 C No lesions 39.2 <0.3 6 C No lesions 39.2 <0.3 6 I Foot and mouth lesions 40.4 11 >2.4 6 C No lesions 39.6 <0.3

Discussion

The purpose of this study was to determine the transmission rate of FMD virus among calves that had only limited contact. In neither of the experiments virus transmission was observed, therefore, the transmission rate could not be estimated. These results indicate that the occurrence of minor outbreaks, as observed on two veal-calf herds during the Dutch FMD epidemic in 2001, were possible.

Although it is known that FMD virus can spread by air it has recently been established that the virus dose required to infect pigs with a recent European isolate is much higher than previously was determined for other strains (Alexandersen and Donaldson, 2002). New data on the minimal infectious dose of these isolates for cattle and sheep are not available. In the 2001 FMD outbreak in the Netherlands two outbreak farms were found in which infected calves were found in the same stable as non-infected calves. On these outbreak-farms a housing system similar to the one in this study was used. These field data suggested that airborne transmission, although well described previously, did not occur in these situations. As mentioned before, when an infection spreads only via direct contact, theoretically an infection chain ends when animals are housed in a row and have only direct contact to their neighbours. Only large outbreaks are seen in calves on a row when a calf can also infect calves further away via indirect contact, for example by air. We, therefore, started the experiment with a distance of 1 metre between animals. However, none of the 4 contact calves at 1 metre distance did contract the infection. Subsequently we tried to quantify the transmission when direct contact was possible (Photo 1). Also in this situation transmission did not occur, despite the clinical signs in both inoculated animals. Because none of the contact calves got infected it is not possible to make a quantitative estimation of the transmission rate. The results of these experiments do question the role of airborne transmission in FMD infection of cattle. In the housing system applied in these experiments calves have their own feeding and drinking bucket, which is cleaned daily. Whereas in traditional housing systems calves used to be fed by drinking out of the same through or by sharing the drinking buckets. Also in housing systems for dairy cattle sharing drinking and feeding places is common. In the outbreaks in the Netherlands there was no indication that the virus was not able to spread in dairy farms or goat farms. Additionally it is also very unlikely that young cattle are less susceptible for FMD virus than older cattle. Therefore the most likely conclusion is that the contact structure of the housing system applied in these experiments is the major cause of the reduction in transmission. This would indicate that airborne transmission between cattle is a very unlikely route. Sharing feeding, drinking, milking machine and e.g. injection needles probably causes transmission in other housing systems. Because ingestion of FMD virus is not known to be a very efficient route of infection, small wounds in the mouth or on teats are more likely the port of entry. In these experiments each inoculated calf received 1500 times the dose that in 50% of the cases produces vesicles on cattle tongue. In cell culture, however, the calculated dose was even 4.5 to 6.5 times higher, which is common when comparing sensitivity of cell culture to the sensitivity of cattle. One of the inoculated calves did not show clinical signs. Based on the four-fold increase of antibodies the virus dose used did result in infection of all inoculated calves. Antibodies in the subclinical infected animal appeared later and the maximum titre was lower (table 1). The titre, however, stayed above the titre (0.55 10log) defined by the FAO cut-off serum (Moonen et al., 2000). Extrapolation and generalisation of these results from these experiments to the field situation, indicating a very low risk of infection if young cattle are involved, is dangerous. In our containment facilities the direction of the air was from head to tail, and the complete air content of the stable is refreshed approximately 7 times per hour. Moreover, there was no contact between the animals via animal caretakers or drinking buckets. In conclusion, the experimental data are in agreement with the field observations and suggest that the occurrence of the minor outbreaks as observed on the two veal-calf herds during the Dutch FMD epidemic in 2001 were possible.

References

Alexandersens, S., Donaldson, A.I. 2002. Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs. Epidemiology and infection 128 (2); 313-323. Dekker, A., Terpstra, C. 1996. Prevalence of foot-and-mouth disease antibodies in dairy herds in the Netherlands, four years after vaccination. Research in Veterinary science 61; 89-91. Donaldson, A. I., C. F. Gibson, R. Oliver, C. Hamblin, and R. P. Kitching. 1987. Infection of cattle by airborne foot-and-mouth disease virus: minimal doses with O1 and SAT 2 strains. Research in veterinary science 43; 339-346. Moonen, P., Miedema, G.K.W., van Hemert-Kluitenberg, F., Chenard, G., Dekker. A. 2000.

Comparison of FMDV neutralisation tests using three different cell lines: validation of the new FAO reference sera. Session of the research group of the European commission for the control of foot-and-mouth disease, Borovets, Bulgaria, 5 to 8 September 2000.