Water requirements for livestock production: a global perspective.

Water is a vital but poorly studied component of livestock production. It is estimated that livestock industries consume 8% of the global water supply, with most of that water being used for intensive, feed-based production. This study takes a broad perspective of livestock production as a component of the human food chain, and considers the efficiency of its water use. Global models are in the early stages of development and do not distinguish between developing and developed countries, or the production systems within them. However, preliminary indications are that, when protein production is adjusted for biological value in the human diet, no plant protein is significantly more efficient at using water than protein produced from eggs, and only soybean is more water efficient than milk and goat and chicken meat. In some regions, especially developing countries, animals are not used solely for food production but also provide draught power, fibre and fertiliser for crops. In addition, animals make use of crop by-products that would otherwise go to waste. The livestock sector is the fastest-growing agricultural sector, which has led to increasing industrialisation and, in some cases, reduced environmental constraints. In emerging economies, increasing involvement in livestock is related to improving rural wealth and increasing consumption of animal protein. Water usage for livestock production should be considered an integral part of agricultural water resource management, taking into account the type of production system (e.g. grain-fed or mixed crop-livestock) and scale (intensive or extensive), the species and breeds of livestock, and the social and cultural aspects of livestock farming in various countries.

New developments in the diagnosis of avian influenza.

Avian influenza has become a serious concern from both veterinary and public health points of view. National and international organisations, veterinary health authorities, research institutions, diagnostic laboratories and field services make enormous efforts worldwide to detect, combat and prevent this important disease. Accordingly, the standard diagnostic protocols are being supported by a wide variety of molecular detection techniques, including improved polymerase chain reaction assays, microarray-based detection and characterisation methods, very rapid sequencing, simple pen-side tests and other on-site approaches. These recently developed 'closer to the field' methods allow rapid detection of influenza viruses and the identification of pathogenicity variants. However, in order to harmonise the diagnosis worldwide, attention has to be paid to the validation and standardisation of these technologies, to avoid erroneous interpretation of assay results, and, consequently, inappropriate epidemiological measures. This review gives an overview of the current and potential future developments related to avian influenza diagnostics.

Protective immune responses induced by different recombinant vaccine regimes to Rift Valley fever.

The glycoprotein (GP) and nucleocapsid (NC) genes of Rift Valley fever virus (RVFV) were expressed in different expression systems and were evaluated for their ability to protect mice from virulent challenge using a prime-boost regime. Mice vaccinated with a lumpy skin disease virus-vectored recombinant vaccine (rLSDV-RVFV) expressing the two RVFV glycoproteins (G1 and G2) developed neutralising antibodies and were fully protected when challenged, as were those vaccinated with a crude extract of truncated G2 glycoprotein (tG2). By contrast mice vaccinated with a DNA vaccine expressing G1 and G2 did not sero-convert with only 20% of them surviving challenge. Mice vaccinated with the DNA vaccine and boosted with rLSDV-RVFV also failed to sero-convert but 40% survived challenge. Surprisingly, although none of the mice immunised with the purified NC protein sero-converted, 60% of them survived virulent challenge. The rLSDV-RVFV construct was then further evaluated in sheep for its dual protective abilities against RVFV and sheeppox virus (SPV). Vaccinated sheep sero-converted for both viruses and were protected against RVFV challenge, however, neither the immunised or negative control animals showed any significant reactions to the virulent SPV challenge.

Genome of crocodilepox virus.

Here, we present the genome sequence, with analysis, of a poxvirus infecting Nile crocodiles (Crocodylus niloticus) (crocodilepox virus; CRV). The genome is 190,054 bp (62% G+C) and predicted to contain 173 genes encoding proteins of 53 to 1,941 amino acids. The central genomic region contains genes conserved and generally colinear with those of other chordopoxviruses (ChPVs). CRV is distinct, as the terminal 33-kbp (left) and 13-kbp (right) genomic regions are largely CRV specific, containing 48 unique genes which lack similarity to other poxvirus genes. Notably, CRV also contains 14 unique genes which disrupt ChPV gene colinearity within the central genomic region, including 7 genes encoding GyrB-like ATPase domains similar to those in cellular type IIA DNA topoisomerases, suggestive of novel ATP-dependent functions. The presence of 10 CRV proteins with similarity to components of cellular multisubunit E3 ubiquitin-protein ligase complexes, including 9 proteins containing F-box motifs and F-box-associated regions and a homologue of cellular anaphase-promoting complex subunit 11 (Apc11), suggests that modification of host ubiquitination pathways may be significant for CRV-host cell interaction. CRV encodes a novel complement of proteins potentially involved in DNA replication, including a NAD(+)-dependent DNA ligase and a protein with similarity to both vaccinia virus F16L and prokaryotic serine site-specific resolvase-invertases. CRV lacks genes encoding proteins for nucleotide metabolism. CRV shares notable genomic similarities with molluscum contagiosum virus, including genes found only in these two viruses. Phylogenetic analysis indicates that CRV is quite distinct from other ChPVs, representing a new genus within the subfamily Chordopoxvirinae, and it lacks recognizable homologues of most ChPV genes involved in virulence and host range, including those involving interferon response, intracellular signaling, and host immune response modulation. These data reveal the unique nature of CRV and suggest mechanisms of virus-reptile host interaction.

Aspects of kit validation for tests used for the diagnosis and surveillance of livestock diseases: producer and end-user responsibilities.

The Joint Food and Agriculture Organization/International Atomic Energy Agency (IAEA) Division of Nuclear Techniques in Food and Agriculture, based at the IAEA in Vienna, Austria, has extensive experience in helping to develop and validate assays and has provided strong support in developing World Organisation for Animal Health (OIE) norms. This paper will focus on enzyme-linked immunosorbent assay and polymerase chain reaction as the major technologies exploited in diagnosis and surveillance. Problems involving the terminology and factors in kit production, supply and validation are examined, in particular emphasising the importance of robustness and ruggedness of tests. The authors discuss the responsibilities of the various stakeholders (producers, distributors, users, and national/international organisations) in achieving quality controlled data to solve diagnostic and surveillance problems. The roles of internal quality control (internal proficiency testing) and external quality assurance (external proficiency testing) as well as aids to solving problems with kits are examined.

In ovo inhibition of fowlpoxvirus replication by a gall extract from Guiera senegalensis.

Several field isolates of fowlpoxvirus (FPV) from Burkina Faso, West Africa, were isolated and partly evaluated by molecular analysis. In addition, the in ovo antiviral activity against FPV of a gall extract from Guiera senegalensis was determined. Three viral isolates were obtained from suspected fowlpox cases after passage in embryonating chicken eggs and their poxviral identity confirmed by electron microscopy. All isolates were found to be pathogenic for chicks and all grew well in cell culture. Polymerase chain reaction and sequencing of amplicons revealed sequences identical with those of other FPV strains. The most studied isolate was then employed for use in an antiviral assay. An aqueous acetone extract from the galls of G. senegalensis was found to inhibit both virus-induced pock formation and to reduce viral titre in embryonating chicken eggs. The suggested mechanism of action is the activation of the alternative complement pathway and the inhibition of FPV-induced cholesterogenesis in ovo by constituents of the gall extract.

Discrimination between sheep-associated and wildebeest-associated malignant catarrhal fever virus by means of a single-tube duplex nested PCR.

A single-tube duplex nested polymerase chain reaction (sdn-PCR) was developed for the detection of and discrimination between ovine herpesvirus-2 (OvHV-2) and alcelaphine herpesvirus-1 (AIHV-1). These viruses respectively cause sheep- and wildebeest-associated malignant catarrhal fever (SA-MCF and WA-MCF). In the first step of the sdn-PCR, two primers with high annealing temperatures based on conserved regions of the tegument genes were used for DNA amplification. In the second step, two primer sets based on variable regions of the respective OvHV-2 and AIHV-1 genes and with annealing temperatures > 11 degrees C below the primers used in the first step, were used. Internal regions of different sizes from amplicons produced in the first step were amplified. This single-tube test obviates the need for two separate assays to detect both viral types, thereby reducing time, labour and cost.

Characterization of a pigeon paramyxovirus (PPMV-1) isolated from chickens in South Africa.

A paramyxovirus with a thermostability of 60 min (typical of velogenic viruses) and a mean death time of > 90 h (typical of lentogenic viruses) was isolated from layers near Mooi River, South Africa. Our results, based on comparative nucleotide sequence data indicated that the virus is pigeon paramyxovirus 1 (PPMV-1), a variant of Newcastle disease virus. The F0 cleavage site contains a 112RRKKRF117 motif, and the virus had 98% sequence identity with PPMV-1 strains from the Far East. PPMV-1 was last reported in South Africa during the 1980s, with this being the first report of PPMV-1 isolated from chickens in South Africa.

A phylogenetic study of South African Newcastle disease virus strains isolated between 1990 and 2002 suggests epidemiological origins in the Far East.

Genetic comparisons were made of the fusion protein sequences of 155 Newcastle disease virus isolates collected in South Africa between 1990 and 2002. Their evolutionary relationships and origins are described. All of the lentogenic field isolates were shown to be derived from commercial vaccines. No true South African lentogenic wild type strain was identified. Furthermore, it was shown that almost all mesogenic isolates had avirulent F(0) cleavage site sequences. Three major epizootics occurred in South Africa during the period of this study. The first outbreak (1990/1991) was caused by viruses endemic to South Africa since the 1960's (genotype VIII) but were occasionally also isolated in 2000. Genotype VIIb viruses, implicated in the severe outbreaks during 1993/1994, persisted until 1999. Genotype VIId viruses, responsible for the most recent outbreak in 1999/2000, had their origins in the Far East like those of the two previous outbreaks.

PCR-based detection of the transovarial transmission of Uruguayan Babesia bovis and Babesia bigemina vaccine strains.

Bovine babesiosis is responsible for serious economic losses in Uruguay. Haemovaccines play an important role in disease prevention, but concern has been raised about their use. It is feared that the attenuated Babesia bovis and Babesia bigemina vaccine strains may be transmitted by the local tick vector Boophilus microplus, and that reversion to virulence could occur. We therefore investigated the possibility that these strains could be transmitted via the transovarial route in ticks using a Babesia species-specific polymerase chain reaction (PCR) assay. DNA was extracted from the developmental stages of the tick vector that had fed on calves immunized with the haemovaccine. It was possible to detect Babesia DNA not only in adult ticks, but also in their eggs and larvae. In addition, it was shown that calves infested with larvae derived from eggs laid by ticks fed on acutely infected calves, were positive for Babesia using PCR. Caution should therefore be shown with the distribution of the haemovaccine in marginal areas. It is still advisable that suitable tick control measures be used to prevent transovarial transmission and the potential risk of attenuated Babesia reverting to virulence.

Comparative sequence analysis of the South African vaccine strain and two virulent field isolates of Lumpy skin disease virus.

The genomic sequences of 3 strains of Lumpy skin disease virus (LSDV) (Neethling type) were compared to determine molecular differences, viz. the South African vaccine strain (LW), a virulent field-strain from a recent outbreak in South Africa (LD), and the virulent Kenyan 2490 strain (LK). A comparison between the virulent field isolates indicates that in 29 of the 156 putative genes, only 38 encoded amino acid differences were found, mostly in the variable terminal regions. When the attenuated vaccine strain (LW) was compared with field isolate LD, a total of 438 amino acid substitutions were observed. These were also mainly in the terminal regions, but with notably more frameshifts leading to truncated ORFs as well as deletions and insertions. These modified ORFs encode proteins involved in the regulation of host immune responses, gene expression, DNA repair, host-range specificity and proteins with unassigned functions. We suggest that these differences could lead to restricted immuno-evasive mechanisms and virulence factors present in attenuated LSDV strains. Further studies to determine the functions of the relevant encoded gene products will hopefully confirm this assumption. The molecular design of an improved LSDV vaccine is likely to be based on the strategic manipulation of such genes.

Development of a diagnostic one-tube RT-PCR for the detection of Rift Valley fever virus.

Diagnosis of Rift Valley fever (RVF) is based on serology and virus isolation. The disadvantages of the former include poor sensitivity, high cost, risks associated with using infectious virus as antigen, the lengthy duration of ELISA as well as cross-reactivity with other Phleboviruses. We developed, optimised and evaluated a one-tube reverse-transcription-polymerase chain reaction (RT-PCR) for the detection of Rift Valley fever virus (RVFV) in ruminants. The PCR primers for this assay were designed to anneal to a region within the M segment of the virus genome, encoding glycoproteins G1 and G2. A PCR amplicon of 363 bp was obtained. The sensitivity of the assay was determined to be 0.25 TCID50. This test should allow for the early and rapid detection of RVFV in both serum and whole blood. In addition, it could facilitate the quantification of antigen for the manufacture of current vaccines.

Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation.

An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.

Importance of thymidine kinase activity for normal growth of lumpy skin disease virus (SA-Neethling).

In order to study the importance of an intact thymidine kinase (TK) gene for the vaccine strain of a southern African capripoxvirus, namely, lumpy skin disease virus (LSDV) (type SA-Neethling), a TK disruption recombinant was generated expressing the Escherichia coli beta-galactosidase (lacZ) reporter gene. A comparative growth study of the recombinant and wild-type (wt) LSDV in TK-positive primary and secondary cells and TK-negative secondary cells was performed. It was found that although recombinant and wt virus both grew in TK-positive cells without selection, the recombinant was unable to grow in TK-negative cells (with or without selection), indicating that TK activity is important, if not essential, for normal growth of LSDV.

Rapid detection and differentiation of Newcastle disease virus isolates by a triple one-step RT-PCR.

A triple one-step RT-PCR was developed to screen and differentiate virulent from avirulent Newcastle disease virus (NDV) isolates. Three sets of oligonucleotides were designed, each specific for amplifying NDV fusion protein gene-specific RNA from virulent, avirulent or all isolates respectively. The sensitivity of one-step RT-PCR was determined using viral RNA extracted from serially diluted NDV-infected allantoic fluid and found to be 10(-5) HA units. Application of one-step RT-PCR to various NDV samples, including wild-type virulent isolates and avirulent vaccine strains, demonstrated the potential for rapid identification (3-4 h) of NDV isolates as well as the differentiation of virulent from avirulent strains.

Eliciting antigen-specific egg-yolk IgY with naked DNA.

Immunization with naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol precipitation. Specific antibodies were present in the yolks of eggs from hens immunized with each of the three different plasmids. This approach to raising polyclonal antibodies obviates the need to produce and purify large quantities of proteins for immunization and can potentially yield large amounts of diagnostically or therapeutically useful reagents.

Immune responses in a horse inoculated with the VP2 gene of African horsesickness virus.

The ability of a DNA vaccine to elicit an immune response in a horse was evaluated. The outer capsid protein VP2 of African horsesickness virus is known to elicit protective immunity in horses. Reverse transcribed DNA of the gene encoding VP2 was placed under the transcriptional control of the cytomegalovirus immediate-early enhancer/promoter and was injected on several occasions intramuscularly into a horse. Low antibody levels could be detected by ELISA. Antibodies directed against VP2 alone were shown by Western blot while low levels of neutralizing antibodies were detected by a 50% plaque reduction assay. In contrast to a relatively poor humoral response, a significant lymphoproliferative response in the presence of whole virus proteins, as well as a cytotoxic cellular reaction against virus-infected syngeneic target cells was shown.

Identification and characterisation of an early/late bi-directional promoter of the capripoxvirus, lumpy skin disease virus.

Identification and characterisation of an early/late bi-directional promoter element of lumpy skin disease virus (LSDV) is described. The 56 bp element shows substantial structural similarities with other poxvirus promoters, providing further evidence that transcriptional elements are conserved within the Poxviridae. The relative strengths of the LSDV early and late promoters were compared to the vaccinia virus (VV) P7.5K and P11K promoters in transient expression assays. These transient assays also verified the temporally regulated nature of the promoter. Favourable expression levels suggest that the identified LSDV promoters could be used for expressing foreign genes in a recombinant LSDV system.

Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E. coli.

Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprotein. Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3Cpro) sequence together with flanking sequences from a region of the viral genome 3'-distal to the putative helicase-encoding region. Expression from these vectors in E. coli resulted in discrete protein products with smaller than expected molecular sizes. This confirmed that an active viral protease was produced in E. coli and that the protease was capable of cleaving the expressed protein at defined sites. Expressed cleavage products surrounding the protease region of the viral polyprotein were separated by SDS-PAGE, transferred to PVDF membranes and subjected to N-terminal sequence analysis. Cleavage occurred at an EG dipeptide at the N terminus of the putative VPg (961E/GKNKG) and also at the protease/polymerase boundary (1280E/GGDKG). The N terminus of the protease was released from the VPg C terminus at an EA dipeptide in the sequence 1099E/APPTL. These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products.