Evaluation of an ovine testis cell line (OA3.Ts) for propagation of capripoxvirus isolates and development of an immunostaining technique for viral plaque visualization.

An ovine testis cell line (OA3.Ts) was evaluated and compared with primary lamb kidney (LK) cells for its utility in capripoxvirus propagation and titration. A comparison of OA3.Ts cell growth kinetics and morphology at low (<33) and high (34-36) passage levels indicated a difference in both characteristics. However, viral titers determined in low and high passage OA3.Ts cells were comparable with those obtained using LK cells. Capripoxvirus infection of OA3.Ts and LK cells resulted in a similar cytopathic effect, which allowed for the detection of discrete viral plaques following immunostaining with capripoxvirus-specific antiserum.

Do ants enhance diversification in lycaenid butterflies? Phylogeographic evidence from a model myrmecophile, Jalmenus evagoras.

The ant-tended Australian butterfly, Jalmenus evagoras, has been a model system for studying the ecology and evolution of mutualism. A phylogeographic analysis of mitochondrial DNA cytochrome oxidase I sequences from 242 butterflies (615 bp) and 66 attendant ants (585 bp) from 22 populations was carried out to explore the relationship between ant association and butterfly population structure. This analysis revealed 12 closely related butterfly haplotypes in three distinct clades roughly corresponding to three allopatric subpopulations of the butterflies. Minimal genetic diversity and widespread haplotypes within biogeographical regions suggest high levels of matrilineal gene flow. Attendant ants are significantly more diverse than was previously thought, with at least seven well-defined clades corresponding to independent morphological determinations, distributed throughout the range of the butterflies. Nested analysis of molecular variance showed that biogeography, host plant, and ant associate all contribute significantly in explaining variation in butterfly genetic diversity, but these variables are not independent of one another. Major influences appear to come from fragmentation due to large-scale biogeographical barriers, and diversification following a shift in habitat preference. A consequence of such a shift could be codiversification of the butterfly with habitat-adapted ants, resulting in apparent phylogenetic concordance between butterflies and ants. The implications of these results are discussed in terms of possible effects of ant attendance on the diversification of Lycaenidae as a whole.

Predictions for the timing and use of culling or vaccination during a foot-and-mouth disease epidemic.

First-fortnight incidence (FFI) is a modelling parameter that can be used to predict both the prevalence and duration of a foot-and-mouth disease (FMD) epidemic at regional and national levels. With an indication of how long an epidemic may last by the end of week two, it becomes possible to estimate whether vaccination would be economically viable from the start of an epidemic. Where FFI indicates that an epidemic is unlikely to last for as long as an export ban on agricultural produce, it may be inappropriate to implement a policy of 'vaccination to live'. Alternatively where FFI indicates that an epidemic will equal or exceed the ban length, then the benefits of vaccination should be considered at an early stage, during or after the first fortnight. Since blanket vaccination of the national or regional herds and flocks would be both costly and heighten the risk of producing carrier animals, targetting vaccination through risk assessment becomes useful.

Use and abuse of mathematical models: an illustration from the 2001 foot and mouth disease epidemic in the United Kingdom.

Foot and mouth disease (FMD) is a major threat, not only to countries whose economies rely on agricultural exports, but also to industrialised countries that maintain a healthy domestic livestock industry by eliminating major infectious diseases from their livestock populations. Traditional methods of controlling diseases such as FMD require the rapid detection and slaughter of infected animals, and any susceptible animals with which they may have been in contact, either directly or indirectly. During the 2001 epidemic of FMD in the United Kingdom (UK), this approach was supplemented by a culling policy driven by unvalidated predictive models. The epidemic and its control resulted in the death of approximately ten million animals, public disgust with the magnitude of the slaughter, and political resolve to adopt alternative options, notably including vaccination, to control any future epidemics. The UK experience provides a salutary warning of how models can be abused in the interests of scientific opportunism.

Geographical and age-stratified distributions of foot-and-mouth disease virus-seropositive and probang-positive cattle herds in the Adamawa province of Cameroon.

Six of the seven known serotypes of foot-and-mouth disease (FMD) virus occur in Africa. This paper describes the results of a population-based cross-sectional study of the seroprevalence of FMD and the persistence of the virus in cattle herds and associated sheep flocks in the Adamawa province of Cameroon. Antibody titres measured by the virus neutralising test indicated that serotypes O, A and SAT2 viruses had been circulating in the province. The estimates of apparent seroprevalence in cattle herds, based on five juvenile animals (eight to 24 months old) per herd, were 74.8 per cent for serotype SAT2, 30.8 per cent for serotype A and 11.2 per cent for serotype O, indicating recent exposure; the estimates based on animals more than 24 months of age were 91.1 per cent for SAT2, 83.6 per cent for A and 34.2 per cent for serotype O. Epithelial and oropharyngeal samples were collected from cattle and small ruminants, cultured and typed by ELISA; serotypes A and SAT2 were isolated from both types of sample. The herd-level estimate of apparent prevalence of probang-positive herds was 19.5 per cent and the animal-level estimate of apparent prevalence was 3.4 per cent. The geographical distribution of the seropositive herds based on juveniles suggested that recent SAT2 exposure was widespread and particularly high in the more northern and western parts of the province, whereas recent exposure to serotype A was patchy and more concentrated in the south and east. This distribution corresponded very closely with the distribution of herds from which virus was recovered by probang, indicating recent exposure or infection. No serotype O viruses were recovered from cattle, and the distribution of seropositive herds suggested very localised recent exposure. The apparent prevalence of probang-positive animals declined with the age of the animal and the period since the last recorded outbreak in the herd.

Elevational sensitivity in an Asian 'hotspot': moth diversity across elevational gradients in tropical, sub-tropical and sub-alpine China.

South-western China is widely acknowledged as a biodiversity 'hotspot': there are high levels of diversity and endemism, and many environments are under significant anthropogenic threats not least climate warming. Here, we explore diversity and compare response patterns of moth assemblages among three elevational gradients established within different climatic bioregions - tropical rain forest, sub-tropical evergreen broad-leaved forest and sub-alpine coniferous forest in Yunnan Province, China. We hypothesised that tropical assemblages would be more elevationally stratified than temperate assemblages, and tropical species would be more elevationally restricted than those in the temperate zone. Contrary to our hypothesis, the moth fauna was more sensitive to elevational differences within the temperate transect, followed by sub-tropical and tropical transects. Moths in the cooler and more seasonal temperate sub-alpine gradient showed stronger elevation-decay beta diversity patterns, and more species were restricted to particular elevational ranges. Our study suggests that moth assemblages are under threat from future climate change and sub-alpine rather than tropical faunas may be the most sensitive to climate change. These results improve our understanding of China's biodiversity and can be used to monitor future changes to herbivore assemblages in a 'hotspot' of biodiversity.

A review of foot-and-mouth disease with special consideration for the clinical and epidemiological factors relevant to predictive modelling of the disease.

Modelling the epidemiology of foot-and-mouth disease (FMD) has been undertaken since the early 1970s. We review here clinical factors and modelling procedures that have been used in the past, differentiating between those that have proved to be more relevant in controlling FMD epidemics, and those that have showed less significance. During the 2001 UK FMD epidemic, many previously developed FMD models were available for consideration and use. Accurate epidemiological models can become useful tools for determining relevant control policies for different scenarios and, conversely, inaccurate models may become an abuse for disease control. Inaccuracy presents two opposing difficulties. Firstly, too much control (in terms of animal slaughter for 2001) would negatively impact the farming community for many subsequent years, whilst too little control would permit an epidemic to persist. Accuracy however, presents the optimal permutation of control measures that could be implemented for a given set of conditions, and is a prerequisite to boosting public confidence in the use of epidemiological models for future epidemics.

Genomic relationship between capripoxviruses.

Capripoxvirus DNAs from field isolates and vaccine samples were analysed by digestion with the restriction enzyme Hind III. The patterns of fragments generated by digestion with Hind III are sufficiently similar to show that all capripoxviruses are closely related, although patterns of different isolates can be grouped in a way which correlates with the animal of origin. The close relatedness was also demonstrated by the high level of sequence homology detected using the Southern Cross hybridization system. Despite the sequence homology, the molecular weights of the genomes of different isolates varied from 73 to 91 MDa. The presence of two rapidly reannealing restriction fragments in the Hind III digests of capripoxvirus DNA indicated the presence of terminal cross-links.

Geographic distribution and epidemiology of peste des petits ruminants virus.

Peste des petits ruminants (PPR) is an important viral disease of goats and sheep prevalent in West Africa and the Middle East. In recent years, PPR has emerged in India, first in the South India and later in North India. To study the genetic relationships between viruses of distinct geographical origin we have sequenced a 322 nucleotide cDNA fragment of the fusion protein gene generated using reverse transcription followed by polymerase chain reaction (PCR) amplification. Viruses from nineteen independent PPR outbreaks were compared; these included the prototype African strain from Senegal and viruses from disease outbreaks which have occurred at different times and locations across Africa, Arabia, the Near East and the Indian subcontinent. Four separate lineages of the virus were identified and the virus isolates from Asia over the past 2 years were all of one lineage which had not previously been identified in Africa or Asia.

Genetic heterogeneity of Indian field isolates of foot-and-mouth disease virus serotype O as revealed by partial sequencing of 1D gene.

The sequence of 165 nucleotides at the 3' end of the 1D gene, determined from RT PCR amplified cDNA fragments, of 25 type O strains isolated from different parts/regions of India during 1987 1995 and the vaccine strain (R2/75) currently in use in India were subjected to phylogenetic analysis. One isolate from the neighbouring country Nepal was also included in the study. The virus/ field strains showed high degree of genetic heterogeneity among themselves with % divergence in nucleotide sequence ranging from 1.2 to 19.4%. The Indian strains were much away (13.3 20.6%) from the exotic type O strains of O1BFS, O1K, and O1Campos. The type O strains analyzed were classified into three genotypes basing on level of divergence observed in nucleotide sequence. The type O vaccine virus (R2/75) was > 71% divergent (7.3-15.2%) from the field strains which revealed significant ( > 5%) genetic heterogeneity between the two. The phylogenetic analysis identified three distinct lineages, viz., (i) lineage 1 represented by the exotic strains, (ii) lineage 2 represented by 25 of the field strains which clustered into seven subgroups/sublines (2a-2g), and (iii) lineage 3 represented by a unique field isolate which shared the branching/origin with the vaccine strain. The lineage 2 which comprised of 25 of the 26 type O field strains analyzed, was placed almost at equidistance from the lineages 1 and 3 in the phylogenetic tree. The vaccine strain was closer to the viruses in lineage 2. Though there was no specific distribution pattern of sequences in different geographical regions of India, the viruses/ sequences in subgroup 2f appeared to be restricted to the southern states. Comparison of deduced amino acid sequence in the immunodominant regions 133-160 and 200-208 of the 1D gene product (VP1) showed that the two viruses in lineage 3 had unique amino acid residues at the positions 138 (D), 139 (G), 144 (I), and 158 (A) compared to rest of the strains including the exotic ones. Comparison of amino acid residues at critical positions 144, 148, 149, 151, 153, 154, and 208 revealed similarity between the type O strains analyzed. The virus strains showed variation (V/L/I) at position 144. One field strain showed replacement from Q149-->E and another from P208-->L. Thus, the study revealed that the type O FMD virus populations circulating in India and causing disease outbreaks are genetically much heterogeneous but related at the immunodominant region of VP1 polypeptide, and there are more than one genetically distinct virus populations in almost every region of the country which is possible due to unrestricted animal movement in the country. The involvement of vaccine virus in disease outbreaks was ruled out as the field strains (excluding the one in lineage 3) were phylogenetically distinct from it.

The role of the World Reference Laboratories for Foot-and-Mouth Disease and for Rinderpest.

The World Reference Laboratories for Foot-and-Mouth Disease and for Rinderpest provide a worldwide diagnostic and surveillance service for these disease for FAO and OIE. Both laboratories are housed within the high security facility of the Institute for Animal Health, Pirbright, UK. Foot-and-mouth disease (FMD) and rinderpest (RP) are OIE List A diseases and historically have caused huge losses to agricultural economies around the world, prompting the establishment of veterinary colleges in Europe and environmentally controversial control programs in Africa. FMD and RP have now been geographically restricted, but the large legal and illegal world trade in live animals and animal products constantly threatens to allow them to spread back into disease-free areas. The Reference Laboratories provide a center of excellence for the development of improved diagnostic techniques and a repository of isolates collected over many years. These libraries provide material for investigations of the molecular epidemiology and evolution of the viruses and a data base against which new isolates can be compared. Thus it is possible to individually characterize new outbreak strains, identify their likely origin and provide the most up-to-date support for their control.

OIE List A disease as a constraint to international trade.

World Trade Organisation agreements have swept aside many of the previous constraints to international trade in animals and animal products and have looked critically at those that still survive. The presence of disease, in particular the OIE list A diseases, still provide legitimacy for barriers to trade, and as a consequence the importance of reliable animal disease surveillance has increased. However, the economic consequences of reporting the occurrence of a List A disease have also increased, as this provides trading partners with sufficient reason to impose an embargo that could severely compromise the national agricultural industry. The dilemma for some developing economies, reliant on agricultural exports, is how to balance a transparent and efficient disease reporting service, sufficient to provide the necessary information for importing countries to make realistic risk assessments, with the perceived political damage from being honest with trading partners who might take advantage of the information to require additional safeguards and health certification.

Use of a recombinant antigen in an indirect ELISA for detecting bovine antibody to capripoxvirus.

The gene coding for the capripoxvirus structural protein P32 was cloned, expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and purified on glutathione Sepharose. An indirect enzyme linked immunosorbent assay (ELISA) using this antigen was developed to screen bovine sera for antibodies to capripoxvirus. Sequential serum samples from experimentally infected animals tested by ELISA and by virus neutralisation test (VNT) showed that the ELISA was more sensitive and detected antibodies to capripoxvirus earlier post-infection than the VNT.

A hemi-nested PCR assay for the detection and identification of vesicular stomatitis virus nucleic acid.

This paper is the first to describe the development of a hemi-nested PCR assay for the detection of vesicular stomatitis virus (VSV) nucleic acid. This assay was developed as it combines high sensitivity for virus genome detection with the identification of the external amplification product in the reamplification step, thus confirming the specificity of the reaction. The assay did not depend on the presence of infectious virus in samples, as demonstrated by its detection of VSV in blood samples which were non-infectious in tissue culture. One further advantage was that the VSV-New Jersey and VSV-Indiana serotypes could be differentiated through the selective use of the appropriate hemi-nested primer. This assay is ideal for the study of VSV pathogenesis and persistence.

Use of inactivated foot-and-mouth disease virus antigen in liquid-phase blocking ELISA.

A liquid-phase blocking ELISA is used by the World Reference Laboratory for Foot-and-Mouth Disease for the quantification of antibodies to foot-and-mouth disease virus. The potential for using inactivated FMDV antigens in the assay has been assessed by titrating bovine convalescent sera to all seven serotypes and comparing the titres obtained with live or inactivated antigens. The titres were similar indicating that either live or inactivated antigens can be used in the liquid-phase blocking ELISA. Removing the need to use live antigens in tests for FMD antibody would reduce disease security risk and widen the acceptability of kits for FMD antibody detection and assay.

Temporal and spatial variation of mortality in field populations of Danaus plexippus L. and D. chrysippus L. Larvae (Lepidoptera: Nymphalidae).

Mortality estimates for the immature stages of two butterfly species, Danaus plexippus and D. chrysippus, were obtained by observing the survival of egg cohorts on different sized patches of food plants (Asclepias spp.), over a one-year period. Losses were variable (0-100%) but usually high (90% and over) throughout the year for both species. Most of the losses in both species occurred in the early stages. The mortality by the third instar accounts for 86-100% of the total losses by instar V. Accordingly both species fall into Price's (1975) type A survivorship category. The size of patches of host plants affected losses. The trend was for increasing losses with increasing patch size. A full life-budget is presented for D. plexippus and implications of the observed mortality levels for competition between the two butterfly species is discussed.

Quantifying the distribution and abundance of an animals resource using aerial photography.

Infra-red aerial photography was used on two occasions to map the abundance and dispersion of milkweeds (Asclepias spp.). The scale at which the aerial photographs could be interpreted showed that milkweeds occur in large contiguous areas or patches. These patches are abundant, the number in any size-class declining exponentially with increasing size. Analysis of the maps using various dispersion indices and spatial autocorrelation statistic showed that patches have a clumped dispersion. Large patches tend to be surrounded by smaller sized patches. The scales at which clumping occurred indicated a close association between milkweeds and the degree of disturbance of the 'natural' environment by human activities. The utility of resource mapping using aerial photography is demonstrated.

Effects of simvastatin and enalapril on serum lipoprotein concentrations and left ventricular mass in patients on dialysis. The Perfect Study Collaborative Group.

A randomised trial of simvastatin and enalapril in patients with chronic renal failure on dialysis: effects on serum lipoprotein concentrations and left ventricular mass. Left ventricular hypertrophy and abnormalities of lipoprotein metabolism are both possible contributors to the high risk of cardiovascular death in patients with chronic renal failure on dialysis. We investigated the effects of simvastatin on lipid and lipoprotein concentrations and the effects of enalapril on left ventricular mass in 107 patients receiving haemodialysis or continuous ambulatory peritoneal dialysis. Patients were randomised in a factorial design to receive simvastatin (10 mg daily) or placebo and enalapril (2.5-5 mg daily) or placebo. During follow-up, there was a significant excess of patients withdrawn from enalapril because of hypotension (2p = 0.002), and after 6 months only 55% of those assigned enalapril were still on treatment. From baseline to 6 months, there were no statistically significant differences in left ventricular mass or left ventricular dimensions between patients assigned enalapril and those assigned placebo. Among the patients assigned simvastatin, total cholesterol was reduced by 13% (2p = 0.001), LDL cholesterol was reduced by 17% (2p = 0.003) and apolipoprotein B was reduced by 12% (2p = 0.005) compared to patients assigned placebo. There were borderline significant (2p = 0.05 to 0.08) reductions in VLDL cholesterol, total triglyceride and VLDL triglycerides of 26%, 12% and 17% respectively. Large-scale trials are now required to determine whether reductions in lipid and lipoprotein concentrations confer a reduction in coronary heart disease morbidity and mortality in patients on dialysis.

The isolation of peste des petits ruminants virus from northern India.

The aetiological agent responsible for an epizootic of a rinderpest-like disease afflicting sheep and goats in three states of northern India was confirmed as peste des petits ruminants virus. To differentiate the virus from rinderpest a number of diagnostic tests were used, including immunocapture ELISA, specific oligonucleotide primers in a reverse transcriptase polymerase chain reaction, immunofluorescence with virus specific monoclonal antibodies and virus isolation. The virulence profile of one isolate in cattle sheep and goats was established. Infected animals developed specific antibody responses and excreted specific antigen in their lachrymal secretions.