Application of an economic calculator to determine the cost of porcine reproductive and respiratory syndrome at farm-level in 21 pig herds in Germany.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) continues to be a major economic issue for the swine industry worldwide, not only due to acute outbreaks but also endemic infections. PRRS disease severity and consequently financial losses can vary greatly between endemically infected farms and estimation of damage is challenging. This study aimed to assess the economic effect of PRRS in a systematic way at individual farm-level for endemically infected herds, using a PRRS cost simulation tool. In total 21 German sow herds with endemic PRRSV infection were investigated. Data on health and production performance, farm management and environment to be fed into the calculator was collected on each farm, and blood samples taken to confirm the PRRSV status. RESULTS: All study farms experienced a significant loss attributable to PRRS. The median farm budget across all farms was - 31 € per sow and year, compared to a median simulated farm budget of 248 € if these farms had been PRRSV negative. The median total loss attributable to PRRS was 74,181 € per farm per year, corresponding to a median total loss per sow and year of 255 €. The impact of PRRS on farm profits was - 19.1% on average and - 41% in the worst case. CONCLUSIONS: The calculated losses give a good hint of the economic damage due to PRRS for the pig industry. Even in endemically infected farms, farmers face a non-negligible damage and profit from a concerted PRRS control. The calculator has proven itself in the field to render a valid estimation of losses due to PRRS in endemically infected farms.

Modelling the economic efficiency of using different strategies to control Porcine Reproductive & Respiratory Syndrome at herd level.

PRRS is among the diseases with the highest economic impact in pig production worldwide. Different strategies have been developed and applied to combat PRRS at farm level. The broad variety of available intervention strategies makes it difficult to decide on the most cost-efficient strategy for a given farm situation, as it depends on many farm-individual factors like disease severity, prices or farm structure. Aim of this study was to create a simulation tool to estimate the cost-efficiency of different control strategies at individual farm level. Baseline is a model that estimates the costs of PRRS, based on changes in health and productivity, in a specific farm setting (e.g. farm type, herd size, type of batch farrowing). The model evaluates different intervention scenarios: depopulation/repopulation (D/R), close & roll-over (C&R), mass vaccination of sows (MS), mass vaccination of sows and vaccination of piglets (MS + piglets), improvements in internal biosecurity (BSM), and combinations of vaccinations with BSM. Data on improvement in health and productivity parameters for each intervention were obtained through literature review and from expert opinions. The economic efficiency of the different strategies was assessed over 5 years through investment appraisals: the resulting expected value (EV) indicated the most cost-effective strategy. Calculations were performed for 5 example scenarios with varying farm type (farrow-to-finish - breeding herd), disease severity (slightly - moderately - severely affected) and PRRSV detection (yes - no). The assumed herd size was 1000 sows with farm and price structure as commonly found in Germany. In a moderately affected (moderate deviations in health and productivity parameters from what could be expected in an average negative herd), unstable farrow-to-finish herd, the most cost-efficient strategies according to their median EV were C&R (€1'126'807) and MS + piglets (€ 1'114'649). In a slightly affected farrow-to-finish herd, no virus detected, the highest median EV was for MS + piglets (€ 721'745) and MS (€ 664'111). Results indicate that the expected benefits of interventions and the most efficient strategy depend on the individual farm situation, e.g. disease severity. The model provides new insights regarding the cost-efficiency of various PRRSV intervention strategies at farm level. It is a valuable tool for farmers and veterinarians to estimate expected economic consequences of an intervention for a specific farm setting and thus enables a better informed decision.

Cost of porcine reproductive and respiratory syndrome virus at individual farm level - An economic disease model.

Porcine reproductive and respiratory syndrome (PRRS) is reported to be among the diseases with the highest economic impact in modern pig production worldwide. Yet, the economic impact of the disease at farm level is not well understood as, especially in endemically infected pig herds, losses are often not obvious. It is therefore difficult for farmers and veterinarians to appraise whether control measures such as virus elimination or vaccination will be economically beneficial for their farm. Thus, aim of this study was to develop an epidemiological and economic model to determine the costs of PRRS for an individual pig farm. In a production model that simulates farm outputs, depending on farm type, farrowing rhythm or length of suckling period, an epidemiological model was integrated. In this, the impact of PRRS infection on health and productivity was estimated. Financial losses were calculated in a gross margin analysis and a partial budget analysis based on the changes in health and production parameters assumed for different PRRS disease severities. Data on the effects of endemic infection on reproductive performance, morbidity and mortality, daily weight gain, feed efficiency and treatment costs were obtained from literature and expert opinion. Nine different disease scenarios were calculated, in which a farrow-to-finish farm (1000 sows) was slightly, moderately or severely affected by PRRS, based on changes in health and production parameters, and either in breeding, in nursery and fattening or in all three stages together. Annual losses ranged from a median of € 75'724 (90% confidence interval (C.I.): € 78'885-€ 122'946), if the farm was slightly affected in nursery and fattening, to a median of € 650'090 (90% C.I. € 603'585-€ 698'379), if the farm was severely affected in all stages. Overall losses were slightly higher if breeding was affected than if nursery and fattening were affected. In a herd moderately affected in all stages, median losses in breeding were € 46'021 and € 422'387 in fattening, whereas costs were € 25'435 lower in nursery, compared with a PRRSV-negative farm. The model is a valuable decision-support tool for farmers and veterinarians if a farm is proven to be affected by PRRS (confirmed by laboratory diagnosis). The output can help to understand the need for interventions in case of significant impact on the profitability of their enterprise. The model can support veterinarians in their communication to farmers in cases where costly disease control measures are justified.

Effect of natural exposure to vaccine-derived North American genotype PRRS virus on the serological response in naïve pigs.

Diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) is often performed by serological testing, but ELISA does not differentiate between infections with wild-type or vaccine virus. Two attenuated live vaccines [European (EU) or North American (NA) genotype] are used. In addition to wild-type isolates, vaccine or vaccine-derived viruses occur frequently. This is often not considered when the ELISA results are used to differentiate between epizootic and enzootic infections. In this study, an infection with the NA genotype vaccine-derived virus was detected in two herds previously PRRSV negative and ELISA results [sample to positive (s/p) ratios] were analysed. The virus was identified by RT-PCR and nucleotide sequences of ORF5 had 97% (herd A) and 99% (herd B) identity with the genome of a ML PRRSV vaccine belonging to the NA genotype. Pigs of different age became positive with an average s/p ratio of 2.24 (A) and 1.18 (B). The data clearly demonstrate that spontaneous infection with a vaccine-derived virus of the NA genotype induces ELISA s/p ratios similar to those induced by vaccination or by infection with wild-type virus. We conclude that for a correct interpretation of serological results the circulation of vaccine or vaccine-derived virus isolates has to be excluded by RT-PCR, even if vaccination is not ongoing.

Folding intermediates of SNARE complex assembly.

SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions. Sso1 switches to an 'open' state when its Hcore domain binds Sec9. Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.

Structural and dynamical properties of a denatured protein. Heteronuclear 3D NMR experiments and theoretical simulations of lysozyme in 8 M urea.

Oxidized and reduced hen lysozyme denatured in 8 M urea at low pH have been studied in detail by NMR methods. 15N correlated NOESY and TOCSY experiments have provided near complete sequential assignment for both 1H and 15N resonances. Over 900 NOEs, including 130 (i, i + 2) and 23 (i, i + 3) NOEs, could be identified by analysis of the NOESY spectra of the denatured states, and 3J(HN, Halpha) coupling constants and 15N relaxation rates have been measured. The coupling constant and NOE data were analyzed by comparisons with theoretical predictions from a random coil polypeptide model based on amino acid specific phi,psi distributions extracted from the protein data bank. There is significant agreement between predicted and experimental NMR parameters suggesting that local conformations of the denatured states are largely determined by short-range interactions within the polypeptide chain. This result is supported by the observation that the chemical shift, coupling constant, and NOE data are little affected by whether or not the four disulfide bridge cross-links are formed in the denatured protein. The relaxation data, however, show significant differences between the oxidized and reduced protein. Analysis of the relaxation data in terms of simple dynamics models provides evidence for weak clustering of hydrophobic groups near tryptophan residues and increased barriers to motion in the more compact conformers formed when the polypeptide chain is cross-linked by the disulfide bridges. Using this information, a structural description of these denatured states is given in terms of an ensemble of conformers, which have a complex relationship between their local and global characteristics.

The concept of a random coil. Residual structure in peptides and denatured proteins.

Non-native states of proteins are of increasing interest because of their relevance to issues such as protein folding, translocation and stability. A framework for interpreting the wealth of experimental data for non-native states emerging from rapid advances in experimental techniques involves comparison with a "random coll' state, which possesses no structure except that inherent in the local interactions. We review here the concept of a random coil, from its global to its local properties. In particular, we focus on the description of a random coil in terms of statistical distributions in psi, phi space. We show that such a model, in combination with experimental data, provides insight into the structural properties of polypeptide chains and has significance for understanding protein folding and for molecular design.

Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro.

The membrane-associated coenzyme F420-reactive hydrogenase of the anaerobic methanogenic archaeon Methanosarcina barkeri Fusaro has been purified 95-fold to apparent homogeneity. A new purification procedure and altered storage conditions gave substantially higher yield (13.4% versus 4.3%) and specific coenzyme F420-reducing activity (82.8 mumol.min-1.mg protein-1 versus 11.5 mumol.min-1.mg protein-1) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79-88]. The predominant coenzyme F420-reactive form of the hydrogenase has an apparent molecular mass of 198 kDa and is composed of three non-identical subunits with apparent molecular masses of 48 (alpha), 33 (beta), and 30 kDa (gamma), apparently in a stoichiometry of alpha 2 beta 2 gamma 1. This minimal coenzyme F420-reducing hydrogenase formed aggregates with apparent molecular masses of approximately 845 kDa. 1 mol of the 198-kDa form of hydrogenase contained 2 mol FAD, 2 mol nickel, 28-32 mol non-heme iron, and 34 mol acid-labile sulfur; in addition, 0.2 mol selenium was detected. The isoelectric point was 5.30. The amino acid sequence PXXRXEGH, where X is any amino acid, was found to be conserved in the N-termini of the putative nickel-binding subunits of most [NiFe]- and [NiFeSe]hydrogenases of methanogenic Archaea and Bacteria. However, this motif was not detected in the protein sequences of [Fe]hydrogenases. Maximal coenzyme F420-reducing activity was obtained with reductively reactivated enzyme at 55 degrees C in the pH range 6.5-7.25. The Km values of the purified enzyme for H2 with coenzyme F420 or methylviologen as electron acceptor were extremely low, namely 3 microM and 4 microM. The catalytic efficiency coefficients (kcat/Km) for H2 with both reducible cosubstrates were high: 2.5 x 10(7) M-1.s-1 with coenzyme F420 and 6.9 x 10(7) M-1.s-1 with methylviologen.

Principles of protein folding--a perspective from simple exact models.

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse.

A test of lattice protein folding algorithms.

We report a blind test of lattice-model-based search strategies for finding global minima of model protein chains. One of us (E.I.S.) selected 10 compact conformations of 48-mer chains on the three-dimensional cubic lattice and used their inverse folding algorithm to design HP (H, hydrophobic; P, polar) sequences that should fold to those "target" structures. The sequences, but not the structures, were sent to the UCSF group (K.Y., K.M.F., P.D.T., H.S.C., and K.A.D.), who used two methods to attempt to find the globally optimal conformations: "hydrophobic zippers" and a constraint-based hydrophobic core construction (CHCC) method. The CHCC method found global minima in all cases, and the hydrophobic zippers method found global minima in some cases, in minutes to hours on workstations. In 9 out of 10 sequences, the CHCC method found lower energy conformations than the 48-mers were designed to fold to. Thus the search strategies succeed for the HP model but the design strategy does not. For every sequence the global energy minimum was found to have multiple degeneracy with 10(3) to 10(6) conformations. We discuss the implications of these results for (i) searching conformational spaces of simple models of proteins and (ii) how these simple models relate to proteins.

Modeling compact denatured states of proteins.

We propose a model for the conformations of compact denatured states of globular proteins: that they are broad ensembles of chain backbone conformations that involve common localized hydrophobic clustering and helical contacts, depending on the amino acid sequence. We construct representative ensembles for chain lengths up to 136 monomers on three-dimensional cubic lattices using the "hydrophobic zippers" method (Fiebig & Dill, 1993). We find that model conformations with radii of gyration about 20% larger than native conformations commonly have bimodal distributions of P(r), of the pairwise interatomic distances, r, and Kratky plots in agreement with recent small-angle X-ray scattering (Sosnick & Trewhella, 1992; Flanagan et al., 1992; Kataoka et al., 1993; Flanagan et al., 1993) experiments on three different proteins. We also find that the lattice model of the Shortle 1-136 fragment of staphylococcal nuclease does not appear capable of forming a single hydrophobic core by hydrophobic zippering, consistent with experiments.

Oculocutaneous albinism, immunodeficiency, hematological disorders, and minor anomalies: a new autosomal recessive syndrome?

We report on 2 related children, a boy and a girl, from a large Turkish clan. Their parents are both first cousins and have several common ancestors. Both children have tyrosinase-positive oculocutaneous albinism, recurrent bacterial infections, granulocytopenia, intermittent thrombopenia, and microcephaly, a protruding midface, rough and projecting hair, and mild mental retardation. Chromosomes are normal. Metabolic disorders were excluded. None of 14 well-known types of albinism, including Hermansky-Pudlak syndrome and Chediak-Higashi syndrome, nor any other genetic syndrome, characterizes our patients sufficiently. Thus, this combination of symptoms is considered a new autosomal recessive syndrome.

Cooperativity in protein-folding kinetics.

How does a protein find its native state without a globally exhaustive search? We propose the "HZ" (hydrophobic zipper) hypothesis: hydrophobic contacts act as constraints that bring other contacts into spatial proximity, which then further constrain and zip up the next contacts, etc. In contrast to helix-coil cooperativity, HZ-heteropolymer collapse cooperativity is driven by nonlocal interactions, causes sheet and irregular conformations in addition to helices, leads to secondary structures concurrently with early hydrophobic core formation, is much more sequence dependent than helix-coil processes, and involves compact intermediate states that have much secondary--but little tertiary--structure. Hydrophobic contacts in the 1992 Protein Data Bank have the type of "topological localness" predicted by the hypothesis. The HZ paths for amino acid sequences that mimic crambin and bovine pancreatic trypsin inhibitor are quickly found by computer; the best configurations thus reached have single hydrophobic cores that are within about 3 kcal/mol of the global minimum. This hypothesis shows how proteins could find globally optimal states without exhaustive search.

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro.

The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.

Purification of the F420-reducing hydrogenase from Methanosarcina barkeri (strain Fusaro).

The 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing and methyl-viologen-reducing hydrogenase of the anaerobic methanogenic archaebacterium Methanosarcina barkeri strain Fusaro has been purified 64-fold to apparent electrophoretic homogeneity. The purified enzyme had a final specific activity of 11.5 mumol coenzyme F420 reduced.min-1.mg protein-1 and the yield was 4.8% of the initial deazaflavin-reducing activity. The hydrogenase exists in two forms with molecular masses of approximately 845 kDa and 198 kDa. Both forms reduce coenzyme F420 and methyl viologen and are apparently composed of the same three subunits with molecular masses of 48 kDa (alpha), 33 kDa (beta) and 30 kDa (gamma). The aerobically purified enzyme was catalytically inactive. Conditions for anaerobic reductive activation in the presence of hydrogen, 2-mercaptoethanol and KCl or methyl viologen were found to yield maximal hydrogenase activity. Determination of the apparent Km of coenzyme F420 and methyl viologen gave values of 25 microM and 3.3 mM, respectively. The respective turnover numbers of the high molecular mass form of the hydrogenase are 353 s-1 and 9226 s-1.

The lactose carrier of Klebsiella pneumoniae M5a1; the physiology of transport and the nucleotide sequence of the lacY gene.

A comparison has been made between the physiology and amino acid sequence of the lactose carriers of Klebsiella pneumoniae M5a1 and Escherichia coli K-12. The membrane transport of lactose was much weaker in Klebsiella than in E. coli. On the other hand o-nitrophenylgalactoside uptake by Klebsiella was distinctly greater than with E. coli. In spite of the differences in sugar transport between the two organisms, the amino acid sequences of the respective lactose carriers were remarkably similar (60% of the amino acids are identical).

Characterization of lactose carrier mutants which transport maltose.

Brooker and Wilson (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963) previously isolated lactose carrier mutants which were able to transport maltose. All of the mutants were found to be single amino acid substitutions for alanine 177 or for tyrosine 236. In the present study, we have examined the ability of these mutants to transport maltose, lactose, o-nitrophenyl-beta-D-galactopyranoside, methyl-beta-D-thiogalactopyranoside, and H+. Both the position 177 and 236 mutants have enhanced rates of maltose transport and exhibit apparent Km values for maltose which are substantially less than that of the wild-type strain. The position 177 mutants transport lactose and other galactosides at a normal rate and with normal affinity during downhill transport and show counterflow transport rates which are faster than the wild-type strain. Interestingly, these mutants are markedly defective in accumulating substrates against a concentration gradient, yet retain a normal H+:galactoside stoichiometry. The position 236 mutants appear to be defective in the downhill, uphill, and counterflow transport of galactosides but exhibit a normal H+:galactoside stoichiometry.

Sodium ions and an energized membrane required by Methanosarcina barkeri for the oxidation of methanol to the level of formaldehyde.

Methanogenesis from methanol by cell suspensions of Methanosarcina barkeri was inhibited by the uncoupler tetrachlorosalicylanilide. This inhibition was reversed by the addition of formaldehyde. 14C labeling experiments revealed that methanol served exclusively as the electron acceptor, whereas formaldehyde was mainly oxidized to CO2 under these conditions. These data support the hypothesis (M. Blaut and G. Gottschalk, Eur. J. Biochem. 141: 217-222, 1984) that the first step in methanol oxidation depends on the proton motive force or a product thereof. Cell extracts of M. barkeri converted methanol and formaldehyde to methane under an H2 atmosphere. Under an N2 atmosphere, however, formaldehyde was disproportionated to CH4 and CO2, whereas methanol was metabolized to a very small extent only, irrespective of the presence of ATP. It was concluded that cell extracts of M. barkeri are not able to oxidize methanol. In further experiments, the sodium dependence of methanogenesis and ATP formation by whole cells was investigated. Methane formation from methanol alone and the corresponding increase in the intracellular ATP content were strictly dependent on Na+. If, in contrast, methanol was utilized together with H2, methane and ATP were synthesized in the absence of Na+. The same is true for the disproportionation of formaldehyde to methane and carbon dioxide. From these experiments, it is concluded that in M. barkeri, Na+ is involved not in the process of ATP synthesis but in the first step of methanol oxidation.