Porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of Quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome.

Cytolytic and noncytolytic strains of the porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in primary cultures of porcine alveolar macrophages (PAM) from lung homogenates of stillborn fetuses or blood samples of dyspneic piglets collected from Quebec pig farms having experienced acute or chronic outbreaks of PRRS. Serological identification of the virus was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using reference antiserum prepared from experimentally-infected specific pathogen free (SPF) piglets and monoclonal antibodies (MoAbs) directed against the p15 nucleocapsid (N) protein of the reference ATCC-VR2332 isolate. Intracytoplasmic enveloped viral particles that tended to accumulate into cytoplasmic vesicles were observed in the infected PAM; no budding was demonstrated at the level of the cytoplasmic membrane. The extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter (averaged diameter of 50 particles was 58.3 nm), with an isometric core about 25-30 nm. Buoyant density of the virus in CsCL density gradients was estimated to 1.18-1.20 g/mL. No hemagglutinating activity was demonstrated. Analysis of semipurified virions of isolate IAF-exp91 by radioimmunoprecipitation (RIPA) and Western immunoblotting experiments, using reference rabbit and porcine hyperimmune sera, revealed four major viral proteins, a predominant 15 kD N protein and three other proteins with predicted M(r_ of 19, 26 and 42 kD. Progeny viral particles produced in PRRSV-infected PAM in the presence of tunicamycin lacked the 42 kD protein, thus confirming its N-glycosylated nature. Immunoprecipitation experiments using the anti-ATCC-VR2332 MoAbs confirmed the close antigenic relationships between Quebec and American reference isolates of PRRSV.     

Genomic variations and antigenic relationships among cytopathic rotavirus strains isolated in Quebec dairy herds.

Twelve isolates of bovine rotavirus, originating from eight dairy herds in Quebec known to have frequent epizootics of diarrhea in young calves in the last five years, were successfully propagated in cell cultures. The 12 isolates produced clear-cut plaques in BSC-1 cells and, except for one isolate, agglutinated human group "O" erythrocytes to an higher titer than bovine erythrocytes. Antisera to each isolate were produced in rabbits and used to study their antigenic relationships. All the isolates shared the group-specific immunofluorescent antigen and were antigenically related as demonstrated by the seroneutralization and hemagglutination-inhibition tests. However, the relationships to the Nebraska rotavirus was quite weak in cases of two Quebec isolates. When the genomes of the various isolates were compared by polyacrylamide gel electrophoresis, at least three different reproducible fractionation patterns could be identified.     

Parvovirus-like particles associated with diarrhea in unweaned piglets.

Numerous parvovirus-like particles, 18 to 26 nm in diameter, were detected by electron microscopy in the intestinal contents of two to three week old piglets with mild to severe diarrhea, in six Quebec pig herds. Hemagglutination of guinea pig and African green monkey red blood cells was obtained with clarified intestinal contents. Two isolates were found to be antigenically related to porcine and canine parvoviruses, while another differed from the porcine parvovirus using the hemagglutination-inhibition test. Three isolates could be cultivated in cell cultures as demonstrated by the development of a cytopathic effect, hemagglutination activity, immunofluorescence and identification of the virions in the cell culture fluids by electron microscopy. The possibility of a primary etiological role for these parvoviruses in diarrhea of unweaned piglets is discussed.     

Detection of bovine coronavirus and type A rotavirus in neonatal calf diarrhea and winter dysentery of cattle in Quebec: evaluation of three diagnostic methods.

The use of direct electron microscopy, enzyme-linked immunosorbent assay, and protein A-gold immunoelectron microscopy for the identification of bovine coronavirus and type A rotavirus were examined. Two hundred and forty-nine samples from diarrheic calves and winter dysenteric cattle from seven geographic areas in Quebec were examined for the presence of viruses by direct electron microscopy of negatively stained preparations. In addition, all the samples were analyzed by enzyme-linked immunosorbent assay, and a random selection of 47 samples were also analyzed by protein A-gold immunoelectron microscopy. Thirty-nine percent of samples examined by direct electron microscopy contained viral particles; bovine coronavirus and type A rotavirus were the most common viruses involved. Overall agreement between any two of the methods used compared favorably with results obtained by others using similar methods. The presence of coronavirus and rotavirus in fecal samples obtained from neonatal calves and the presence of coronavirus in samples from winter dysenteric adult cattle suggested their etiological roles in the respective diseases. Furthermore, results from protein A-gold immunoelectron microscopy of coronavirus-like particles implied that a different coronavirus or some other viruses might be involved in these diseases. Finally, the efficiency of direct electron microscopy, enzyme-linked immunosorbent assay and protein A-gold immunoelectron microscopy as diagnostic tools is discussed.     

Environmental conditions encountered during typical consumer retail display affect fruit and vegetable quality and waste

Temperature and relative humidity (RH) are the most important environmental factors affecting the sensory quality of fresh produce, and therefore, the consumer acceptability for fruits and vegetables displayed in a produce department. Poor temperature management inevitably occurs in commercial handling and reduces the quality and maximum potential shelf life of fruits and vegetables. Since there is a lack of information on the actual temperatures registered inside local distribution trucks or in consumer retail displays, and the effects on the produce quality, the current study was designed to evaluate the segment of the distribution chain that comprises the time the produce arrives from the distribution center to the store, is displayed at the store, and then stored under household conditions. Temperature and RH were recorded in three different produce departments, at reception and during a 6-week retail display period. Truck and produce temperatures were collected immediately upon opening of the doors, and display temperatures were monitored inside 27 refrigerated and non-refrigerated retail displays. Visual quality of 37 different produce items was evaluated, and surface or pulp temperatures were measured immediately upon unloading from the delivery truck. Reasons for produce waste were recorded on a daily basis during a 6-week period. Shelf life studies were conducted under simulated household conditions using samples collected at the stores. Results from this study showed that chilling-sensitive commodities were transported under too cold conditions, whereas heat-sensitive commodities were transported under too warm conditions. Visual quality of the produce received at the store ranged from good to poor depending on the fruit or vegetable evaluated, with raspberry receiving the lowest visual quality score in 6.7% of store deliveries, and banana, fresh-cut vegetables, peach and pineapple receiving the best visual quality score in 100% of store deliveries. Temperatures measured inside retail displays showed a wide variation, depending on the store and location inside the display, ranging from ?1.2 °C to 19.2 °C in refrigerated displays and from 7.6 °C to 27.7 °C in non-refrigerated displays. RH ranged from 55.9% to 92.9% in refrigerated displays and from 29.7% to 86.6% in non-refrigerated displays. Poor temperature management was the major cause of produce waste (55%) whereas expired date and mechanical damage accounted for 45% of the produce wasted. Results from this study show that fruits and vegetables are often handled under adverse environmental conditions, resulting in produce with poor quality and shorter shelf life, and increased waste at the retail and consumer levels.     

Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes.

Three distinct antigenic profiles were identified by comparing the reactivities of 15 Canadian field isolates, the attenuated U.S. vaccine (Ingelvac MLV) strain and 2 European reference strains (Lelystad and Weybridge) of the porcine reproductive and respiratory syndrome virus (PRRSV) by indirect immunofluorescence with a set of 4 monoclonal antibodies to the nucleocapsid (N) protein and 2 other to the matrix (M) protein. In the present study, 9 Canadian isolates for which the sequences were determined appeared closely related to 2 U.S. reference strains (ATCC VR-2332 and ATCC VR-2385) with amino acid identities varying between 90 to 98% for the M and N proteins; substitutions in the nucleotide sequences were distributed randomly throughout the ORFs 6 and 7 genes, and most were 3rd base silent mutations. In comparison, more than 30% divergence was demonstrated with the Lelystad virus. Furthermore, differentiation between North American and European isolates, and between field isolates and the MLV strain could be achieved by cutting PCR-amplified products encompassing both ORFs 6 and 7 genes with 4 restriction endonucleases. When taken individually, BsaJI and AluI were the more appropriate restriction enzymes for distinguishing the vaccine strain from field isolates. The results obtained suggest that the restriction fragment length polymorphism of the genomic region covering the ORFs 6 and 7 genes may be a valuable tool to differentiate among PRRSV isolates.     

Swine reproductive and respiratory syndrome in Québec: Isolation of an enveloped virus serologically-related to Lelystad virus

Sera were collected from convalescent sows and sick piglets from six pig farms in southern Quebec that have experienced outbreaks of the so-called porcine reproductive and respiratory syndrome. By indirect immunoperoxidase, a few of these sera (4 of 14) (28.6%) were found to be positive for antibody to the Lelystad virus, whereas by indirect immunofluorescence 30 of 36 (83.3%) were positive for antibody to the antigenically-related American isolate ATCC-VR2332. Pregnant sows inoculated intranasally with filtered homogenates prepared from the lungs of necropsied piglets obtained from a seropositive farm developed fever, inappetence, and reproductive failure characterized by stillbirths and various stages of mummification. Lesions of interstitial pneumonia were induced in experimentally-infected specific pathogen-free piglets. A virus, having morphological and biological characteristics of viruses assigned to the family Togaviridae, was isolated from lung tissues of experimentally-infected animals; it could only be propagated in primary cultures of porcine alveolar macrophages. Identification of the virus was confirmed by indirect immunofluorescence using a monoclonal antibody directed against the nucleocapsid protein of the ATCC-VR2332 isolate and porcine sera that were found positive for antibody to both the Lelystad and ATCC-VR2332 isolates.     

Transmissible enteritis of turkeys: experimental inoculation studies with tissue-culture-adapted turkey and bovine coronaviruses.

Four Quebec isolates of turkey enteric coronaviruses (TCVs) and three isolates of bovine enteric coronaviruses (BCVs) were serially propagated in HRT-18 and compared for their pathogenicity in turkey embryos and turkey poults. By immunoelectron microscopy, hemagglutination-inhibition, and Western immunoblotting assays, tissue-culture-adapted Quebec TCV isolates were found to be closely related to the reference Minnesota strain of TCV and the Mebus strain of BCV. Genomic relationships between TCV isolates and the reference BCV strain were confirmed by hybridization assays with BCV-specific radiolabeled recombinant plasmids containing sequences of the N and M genes. Only TCV isolates could be propagated by inoculation in the amniotic cavity of chicken and turkey embryonating eggs, and induced clinical disease in turkey poults. Nevertheless, coronavirus particles or antigens were detected by electron microscopy or indirect enzyme-linked immunosorbent assay in the clarified intestinal contents of BCV-infected poults up to day 14 PI, and genomic viral RNA was detected by slot-blot hybridization using BCV cDNA probes.     

Rapid pneumococcal evolution in response to clinical interventions

Epidemiological studies of the naturally transformable bacterial pathogen Streptococcus pneumoniae have previously been confounded by high rates of recombination. Sequencing 240 isolates of the PMEN1 (Spain(23F)-1) multidrug-resistant lineage enabled base substitutions to be distinguished from polymorphisms arising through horizontal sequence transfer. More than 700 recombinations were detected, with genes encoding major antigens frequently affected. Among these were 10 capsule-switching events, one of which accompanied a population shift as vaccine-escape serotype 19A isolates emerged in the USA after the introduction of the conjugate polysaccharide vaccine. The evolution of resistance to fluoroquinolones, rifampicin, and macrolides was observed to occur on multiple occasions. This study details how genomic plasticity within lineages of recombinogenic bacteria can permit adaptation to clinical interventions over remarkably short time scales.