Influence of exposure intensity on the efficiency and speed of transmission of Foot-and-mouth disease.

Foot-and-mouth disease virus (FMDV) can be spread by direct animal-to-animal contact, indirect contact facilitated by contaminated materials or by airborne spread. The rate of spread and the incubation period, as well as the severity of disease, depends on many variables including the dose received, the route of introduction, the virus strain, the animal species and the conditions under which the animals are kept. Quantitative data related to these variables are needed if model predictions are to be used in practical disease control. This experimental study quantifies the risk of transmission of FMDV in pigs exposed by contact, sheep exposed by indirect contact with pigs and sheep exposed to airborne FMDV. Groups of pigs were inoculated with the FMDV O UKG 34/2001 strain and susceptible pigs were then exposed to the inoculated animals at different stages of the infection cycle. The mean incubation period in the susceptible pigs ranged from 1 to 10 days. The length of the incubation period, severity of clinical disease and efficiency of spread were related to dose (i.e. infectiousness of source and intensity of contact). Low intensity transmission increased the proportion of subclinical or abortive infections. Local conditions are important in the efficiency and speed of transmission of FMDV. The results of the experiments described above suggest that transmission is frequency dependent rather than density dependent. The sheep experiments provided further evidence that development of infection and clinical disease is dependent upon local conditions. Dose, infectiousness, intensity of contact and local factors are thus important determinants for the outcome of an initial outbreak and must be truthfully accounted for in mathematical models of epidemiological spread.

Reduction of foot-and-mouth disease (FMD) virus load in nasal excretions, saliva and exhaled air of vaccinated pigs following direct contact challenge.

In future, a policy of "vaccinate-to-live" may be included in the repertoire of foot-and-mouth disease (FMD) control measures and in support of this approach, we have investigated the hypothesis that vaccine-induced reduction in virus replication and excretion from pigs can be correlated to the severity of clinical signs of FMD by measuring excretion of virus in natural secretions and aerosols. The other aims of this study were to verify the existence of sub-clinical infection in vaccinated pigs, to evaluate the correlation between this and seroconversion to foot-and-mouth disease virus (FMDV) non-structural protein antibodies and to re-examine the occurrence of FMDV persistence in the oro-pharynx of pigs. Therefore, pigs were vaccinated (O1 Manisa) and challenged (O1 UKG) in a manner calculated to produce a broad range of clinical outcomes and were monitored for a minimum of another 33 days post-challenge. Eighty-one percent of the early (10 days vaccinated) challenged pigs and 25% of the late (29 days vaccinated) challenged pigs were clinically infected and all other vaccinated pigs were sub-clinically infected. Although vaccination could not provide complete clinical or virological protection, it reduced the severity of the disease, virus excretion and production of non-structural FMDV antibodies in vaccinated and subsequently infected pigs. As hypothesised, vaccine-induced reduction of virus replication and excretion was found to be correlated to the severity of clinical disease. RNA copies, but no live virus was detected from the pharyngeal and soft palate tissues of a minority of vaccinated and infected pigs beyond the acute stage of the infection.

Cytokine and Toll-like receptor mRNAs in the nasal-associated lymphoid tissues of cattle during foot-and-mouth disease virus infection.

The pharyngeal region is known to play an important role in foot-and-mouth disease virus (FMDV) infection in relation to acute disease and viral persistence. In this study, the local mucosal immune response in nasal-associated lymphoid tissue (NALT) of cattle infected with FMDV (strain O UKG 34/2001) was examined. Quantitative "real-time" RT-PCR assays were used to measure mRNA expression of cytokines (IFN-alpha, beta and gamma, IL-2, IL-1alpha and TNF-alpha) and Toll-like receptors (TLR)-3 and -4. NALTs from dorsal soft palate were collected from cattle at 7 days post-infection (dpi) and from carriers and non-carriers at 64 dpi. Expression of IFN-alpha mRNA was significantly greater in NALT during acute disease than in uninfected animals. Increased expression of IFN-gamma and IL-1alpha mRNA was also observed but was much lower than IFN-alpha expression. There was a slight increase in mRNA expression of TNF-alpha and IL-2. During persistence, TNF-alpha mRNA expression in carrier cattle was much higher than in non-carrier cattle. Expression of TLR-4 in NALT during the acute stage of infection was greater than in uninfected animals. Carrier and non-carrier cattle did not differ in respect of expression of TLR-3 and -4 mRNA in NALT.

Liver biopsy in the diagnosis of malignancy.

In a National Audit of 1500 liver biopsies, 38% were for suspected malignancy. To measure their contribution to clinical decisions, the initial diagnoses, biopsy diagnoses, final diagnoses, and outcomes were coded by computer and compared. Most patients (92%) were investigated for advanced malignancy. The accuracy of clinical diagnosis was 78% against final diagnosis. Liver biopsy was seen as 'confirming' clinical diagnosis overall. This was achieved in 67% (75% with ultrasound guidance), and specificity was almost 100%. However, hepatocellular cancer was confirmed by biopsy in only 32% and haematological malignancy in 13% of suspected cases. Within 3 months, 44% of patients with histological malignancy had died. Histological tumour type was not used in 36% of final diagnoses. Of patients with a malignancy-negative liver biopsy--showing reactive hepatitis, normality, or cholangitis/cholestasis--25%, 47% and 60%, respectively, had final malignant diagnoses. In 6% of patients, biopsy showed chronic liver disease. Only 12% of deaths were autopsied. Liver biopsy contributes very high specificity to the diagnosis of malignancy, and detects non-malignant disease. Failure to use tumour type may result in sub-optimal therapy. Improving diagnostic practice requires more information on outcomes, including autopsies.

Use of the polymerase chain reaction in differentiating rinderpest field virus and vaccine virus in the same animals.

In 1991, a disease with clinical signs indicative of rinderpest was reported in yaks in the former Soviet Union, near the border with Mongolia. At the peak of the epizootic, mortality among affected yaks was 32-42% in adults and 65% in animals less than one year old. Pathological samples were examined independently at two institutes in Russia. Both institutes confirmed the presence of rinderpest using complement fixation, agar gel diffusion and immunoassays. Since vaccination had been initiated to control an outbreak of a similar disease several months earlier, the later cases were possibly due to the vaccine and field rinderpest may not have been present. However, the disease had occurred in non-vaccinated animals and these were then vaccinated against the disease. Tissue samples obtained from these animals, which were examined at the Pirbright Laboratory using gel diffusion assays and specific nucleic acid probes, were found to be positive for rinderpest antigen and nucleic acid. Ribonucleic acid derived from the post-mortem tissue samples was amplified using the polymerase chain reaction and rinderpest-specific primers. Sequence analysis of the amplified deoxyribonculeic acid from the samples revealed the presence of two distinct virus strains, one identical to the Plowright rinderpest tissue culture vaccine and the other related to field strains of rinderpest virus circulating in Asia and the Middle East.

Detection of foot-and-mouth disease viral sequences in clinical specimens and ethyleneimine-inactivated preparations by the polymerase chain reaction.

The polymerase chain reaction method (PCR) has been applied to the diagnosis of foot-and-mouth disease viral RNA in tissues and, particularly, oesophageal-pharyngeal fluid (probang) samples from cattle. Using primer sets which corresponded to conserved regions of the VP1 sequence of the viral genome, it was possible to amplify sequences regardless of the serotype/strain of the virus. In comparison with infectivity assays, the PCR was generally more sensitive although there were a number of examples where only infectivity was detectable. In experiments with uninfected probang samples deliberately seeded with a dilution series of virus, the PCR proved to be approximately 10(4) times more sensitive than infectivity assays. This greater sensitivity was attributed, in part, to the ability of the PCR to amplify specifically from non-infectious RNA preparations. This enabled the identification, by sequencing, of viral RNA from chemically inactivated virus concentrates typical of those used for commercial vaccine production. Amplification of specific PCR products was also achieved with virus eluted from commercial vaccine, including preparations which had been stored for more than 10 years at 4 degrees C. The PCR technique is of considerable value, therefore, both as a complement to infectivity assays and as a powerful tool in vaccine-associated studies.

Cross-reactive and serotype-specific antibodies against foot-and-mouth disease virus generated by different regions of the same synthetic peptide.

Synthetic peptides based on the VP1 proteins of two serotypes of foot-and-mouth disease virus (FMDV) and having the general formula C-C-(200-213)-P-P-S-(141-158)-P-C-G induce heterologous as well as homologous protection against challenge. Substitution of the sequence consisting of residues 200 to 213 (200-213 sequence) with a second copy of the homologous 141-158 sequence (i.e., homodimers) resulted in failure of either serotype peptide to protect heterologously. The antiviral and antipeptide titers of sera from guinea pigs immunized with the homodimeric 141-158 peptides showed serotype specificity and, with the data from the heterodimeric peptide vaccines, suggested that the C-terminal 141-158 sequence was more effectively recognized by the immune system than the N-terminal sequence. Whereas heterologous antiviral titers as measured by enzyme-linked immunosorbent assay and virus neutralization tests have not been observed with sera from cross-protected animals, epitope-mapping studies established that there was heterologous recognition of an octapeptide within the 200-213 sequence. That the 200-213 sequence was required for the induction of heterologous protection was also confirmed with a number of peptides, including hybrids based on the 200-213 sequence of one virus and the 141-158 sequence of a second virus. Thus, peptides of the general formula given above induce serotype-specific and serotype-cross-reactive protective antibodies and are unique in their induction of significant levels of heterologous protection, a property which has never been reported for whole FMDV vaccines.