THE DESIGN AND ANALYSIS OF EXPERIMENTS. By Oscar Kempthorne. New York, John Wiley and Sons, Inc., 1952. 631 pp. $8.50. This book by a teacher of statistics (as well as a consultant for \"experimenters\") is a comprehensive study of the philosophical background for the statistical design of experiment. It is necessary to have some facility with algebraic notation and manipulation to be able to use the volume intelligently. The problems are presented from the theoretical point of view, without such practical examples as would be helpful for those not acquainted with mathematics. The mathematical justification for the techniques is given. As a somewhat advanced treatment of the design and analysis of experiments, this volume will be interesting and helpful for many who approach statistics theoretically as well as practically. With emphasis on the \"why,\" and with description given broadly, the author relates the subject matter to the general theory of statistics and to the general problem of experimental inference. MARGARET J. ROBERTSON

The Design and Analysis of Experiments

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

Manual for the Identification of Medical Bacteria.

Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide each year. The causative agent of the disease, PRRS virus (PRRSV), is a member of the family Arteriviridae. Here we report our investigation of the unparalleled large-scale outbreaks of an originally unknown, but so-called “high fever” disease in China in 2006 with the essence of PRRS, which spread to more than 10 provinces (autonomous cities or regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. Different from the typical PRRS, numerous adult sows were also infected by the “high fever” disease. This atypical PRRS pandemic was initially identified as a hog cholera-like disease manifesting neurological symptoms (e.g., shivering), high fever (40–42°C), erythematous blanching rash, etc. Autopsies combined with immunological analyses clearly showed that multiple organs were infected by highly pathogenic PRRSVs with severe pathological changes observed. Whole-genome analysis of the isolated viruses revealed that these PRRSV isolates are grouped into Type II and are highly homologous to HB-1, a Chinese strain of PRRSV (96.5% nucleotide identity). More importantly, we observed a unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2). Taken together, this is the first comprehensive report documenting the 2006 epidemic of atypical PRRS outbreak in China and identifying the 30 amino-acid deletion in NSP2, a novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, and will stimulate further study by using the infectious cDNA clone technique.

/pdf/emergence-of-fatal-prrsv-variants-unparalleled-outbreaks-of-53lggebfud.pdf

Emergence of Fatal PRRSV Variants: Unparalleled Outbreaks of Atypical PRRS in China and Molecular Dissection of the Unique Hallmark

In 1987, porcine reproductive and respiratory syndrome (PRRS) was recognized in the USA as a new disease of swine causing late-term reproductive failure and severe pneumonia in neonatal pigs. The syndrome is caused by an RNA virus referred to as PRRS virus (PRRSV), which is classified in the family Arteriviridae. Swine macrophages are the only indigenous cell type known to support PRRSV replication. Direct contact between infected and naive pigs is the predominant route of PRRSV transmission. Exposure of a mucosal surface to PRRSV leads to virus replication in regional macrophages, a prolonged viremia and systemic distribution of virus to other macrophage populations. Reproductive failure induced by PRRSV infection in late-gestation sows is characterized by premature farrowing of stillborn, partially autolyzed, and mummified fetuses. Pneumonia caused by PRRSV infection is more severe in young pigs compared to adults and may be complicated by concurrent bacterial infections. Gross lung lesions associated w...

https://journals.sagepub.com/doi/pdf/10.1177/030098589803500101

Porcine reproductive and respiratory syndrome

Secondary specific pathogen-free (sSPF) piglets were inoculated intranasally with Streptococcus suis serotype 2 alone, porcine reproductive and respiratory syndrome virus (PRRSV) alone, or with PRRSV followed by S suis. Uninfected piglets were used as controls. Pigs inoculated with PRRSV (ATCC VR-2332) followed by challenge with a virulent strain (87555) of S suis serotype 2 developed clinical signs, suppurative meningitis and large numbers of S suis in their tissues, including the brain and meninges. Pigs inoculated with PRRSV alone, S suis (87555) alone, or with PRRSV and the DH5 strain of S suis serotype 2 (lacking a protein associated with virulence) and the uninfected piglets did not develop clinical signs or lesions or have large numbers of bacteria in their tissues. The results suggest that PRRSV predisposes sSPF pigs to infection and disease caused by virulent S suis serotype 2. Co-infection of piglets with PRRSV and a virulent strain of S suis may provide a useful model for the study of S suis septicaemia and meningitis.

Interaction between Streptococcus suis serotype 2 and porcine reproductive and respiratory syndrome virus in specific pathogen-free piglets.

The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.

https://journals.sagepub.com/doi/pdf/10.1177/104063879901100307

Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs.

Methods: Ninety-six 4-week-old pigs from a PRRSV-naive source were organized into six groups individually housed in isolation rooms (four replicate trials, 24 pigs per trial). Group 1 pigs were inoculated intranasally with PRRSV strain VR-2332 (2 mL, 105 median tissue culture infective doses per mL.) On days 5, 6, and 7 post inoculation, investigators exposed to Group 1 pigs attempted to transmit PRRSV to sentinel pigs (Groups 2 to 5) by contact. After exposure to the infected pigs, an investigator entered the Group 2 room (Direct Contact group) wearing contaminated boots and coveralls and without washing hands. In contrast, investigators who entered the rooms housing Groups 3 to 5 were required to complete specific sanitation protocols, which included changing boots and coveralls and washing hands (Danish System, Group 3); changing boots and coveralls, showering, and 12 hours down time (Standard Protocol, Group 4); and changing boots and coveralls and showering, with no down time (Alternative Protocol, Group 5).

/pdf/transmission-of-porcine-reproductive-and-respiratory-22ci02ewrp.pdf

Transmission of porcine reproductive and respiratory syndrome virus by fomites (boots and coveralls)

Objective To document airborne transmission of Actinobacillus pleuropneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV) infection in nursery pigs. Animals 32 two-week-old pigs obtained from 3 farms, but with similar Landrace X Yorkshire genetics for trial 1 of each experiment; 16 pigs for trial 2 of the A pleuropneumoniae experiment; and 14 pigs for trial 2 of the PRRSV experiment. Procedure In experiment 1, pigs were inoculated with A pleuropneumoniae serotype 1 (6/8) or were left as contacts (2/8). At the beginning of trial 1, pigs were seronegative to A pleuropneumoniae serotypes 1 and 5 on the basis of results of an ELISA, but had positive results on the A pleuropneumoniae hemolysin I (Apx1)-neutralization test. Pigs in trial 2 had negative results on both tests. Pigs of trial 1 of experiment 2 were inoculated with a PRRSV virulent field isolate (MN-1b); pigs of trial 2 were inoculated with the virus reference strain VR-2332. Aerosol-exposed pigs were placed on the other side of the air duct and kept there for 2 to 7 weeks depending on evidence of airborne transmission. Results In trial 1 of experiment 1, evidence of airborne transmission was not found. In trial 2, most airborne-exposed pigs died as a result of A pleuropneumoniae infection 12 days after initiation of the experiment. In trial 1 of experiment 2, all inoculated pigs (8/8) seroconverted, but only 2 of 8 contact-exposed pigs seroconverted. Aerosol-exposed pigs did not seroconvert nor was virus isolated. In trial 2, all inoculated and contact-exposed pigs seroconverted. All aerosol-exposed pigs seroconverted after 21 days, and virus was isolated at 16 days. Conclusions A pleuropneumoniae was transmitted by air at a distance of 1 m when pigs were fully susceptible to the organism. Transmission of PRRSV appeared to be strain dependent; when reference strain VR-2332 was used, airborne transmission of PRRSV was documented.

Airborne transmission of Actinobacillus pleuropneumoniae and porcine reproductive and respiratory syndrome virus in nursery pigs

Using a field-based model, mechanical transmission of porcine reproductive and respiratory syndrome virus (PRRSV) was assessed throughout a coordinated sequence of events that replicated common farm worker behavior during cold weather (< 0°C). The model involved fomites (boots and containers), vehicle sanitation, transport, and the movement of personnel. A field strain of PRRSV was inoculated into carriers consisting of snow and water, and carriers were adhered to the undercarriage of a vehicle. The vehicle was driven approximately 50 km to a commercial truck washing facility where the driver's boots contacted the carriers during washing, introducing the virus to the vehicle interior. The vehicle was then driven 50 km to a simulated farm site, and the driver's boots mechanically spread virus into the farm anteroom. Types of containers frequently employed in swine farms (styrofoam semen cooler, metal toolbox, plastic lunch pail, and cardboard animal health product shipping parcel) contacted drippings from footwear on the anteroom floor. The truck wash floor, vehicle cab floor mats, boot soles, anteroom floor, and the ventral surface of containers were sampled to track the virus throughout the model. Ten replicates were conducted, along with sham-inoculated controls. At multiple sampling points PRRSV nucleic acid was detected in 8 of 10 replicates. In each of the 8 PCR-positive replicates, infectious PRRSV was detected on the surfaces of containers by virus isolation or swine bioassay. All sham-inoculated controls were negative. These results indicate that mechanical transmission of PRRSV can occur during coordinated sequence of events in cold weather.

https://www.gitep.com.ar/novedades/11.pdf

Carlos Pijoan

Papers

Interaction between Streptococcus suis serotype 2 and porcine reproductive and respiratory syndrome virus in specific pathogen-free piglets.

Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs.

Transmission of porcine reproductive and respiratory syndrome virus by fomites (boots and coveralls)

Airborne transmission of Actinobacillus pleuropneumoniae and porcine reproductive and respiratory syndrome virus in nursery pigs

Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during cold weather