Contacts posing risks of disease introduction in swine breeding herds in Quebec, Canada: Is the frequency of contacts associated with biosecurity measures?

The introduction of pathogens into swine breeding herds can occur through a variety of contacts involving people, animals, vehicle or various supplies. Appropriate biosecurity is critical to mitigate these risks. A retrospective study was conducted to describe contacts with swine breeding sites over a one-month period and to evaluate their association with biosecurity measures and site characteristics. As part of a larger project, sites which had a recent porcine reproductive and respiratory syndrome virus introduction were selected. A questionnaire, logbooks and pig traceability system were used for collecting data relative to persons or supplies entering the breeding unit, live pig transportation, service vehicles, other animal species, neighboring pig sites and manure spreading around the site. The 84 sites investigated had a median sow inventory of 675. A median of 4 farm staff and 2 visitors entered the breeding unit at least once over the one-month period. A total of 73 sites (87%) received visitor(s), mostly from maintenance and technical services. All sites received at least 3 supply deliveries (median of 8) including semen (99% of sites), small material and/or drugs (98% of sites), bags (87% of sites), and/or equipment (61% of sites). Live pig movements were observed in all sites, with a median number of 5 truck entries on the site or exits from the site. For feed mill, rendering and propane trucks, at least one entry was noted in ≥ 61% of sites. For all service vehicle categories except feed mill and manure vacuum trucks, a single service provider was involved in each site. Dogs and cats were banned from all sites, but wild birds were observed in 8% of sites. Manure spreading within a 100 m radius of pig units was noted in 10% of the sites. With a few exceptions, biosecurity measures were not associated with the frequency of contacts. A 100-sow increase in sow inventory was associated with an increase of 0.34 in the cumulated number of staff entering the breeding unit, of 0.30 in the number of visitors and of 0.19 in the number of live pig movements. Live pig movements were also positively associated with vertically integrated farrow-to-wean (vs. independent farrow-to-wean) production and time interval of 4 weeks or more between farrowing (vs. less than 4). Considering the variety and frequency of contacts observed, biosecurity should be meticulously applied in all breeding herds to prevent endemic and exotic disease introduction.

Prevalence of Coxiella burnetii seropositivity and shedding in farm, pet and feral cats and associated risk factors in farm cats in Quebec, Canada.

Cats represent a potential source of Coxiella burnetii, the aetiological agent of Q fever in humans. The prevalence and risk factors of C. burnetii infection in farm, pet and feral cats were studied in Quebec, Canada, using a cross-sectional study. Serum samples were tested using a specific enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against C. burnetii, whereas rectal swabs were assayed using real-time quantitative polymerase chain reaction (qPCR) for the molecular detection of the bacteria. Potential risk factors for farm cats were investigated using clinical examinations, questionnaires and results from a concurrent study on C. burnetii farm status. A total of 184 cats were tested: 59 from ruminant farms, 73 pets and 52 feral cats. Among farm cats, 2/59 (3.4%) were ELISA-positive, 3/59 (5.1%) were ELISA-doubtful and 1/59 (1.7%) was qPCR-positive. All pets and feral cats were negative to C. burnetii ELISA and qPCR. Farm cat positivity was associated with a positive C. burnetii status on the ruminant farm (prevalence ratio = 7.6, P = 0.03). Our results suggest that although pet and feral cats do not seem to pose a great C. burnetii risk to public health, more active care should be taken when in contact with cats from ruminant farms.

Isolation and characterization of a novel Bacillus thuringiensis strain expressing a novel crystal protein with cytocidal activity against human cancer cells.

AIMS: To characterize a novel, unusual, Bacillus thuringiensis strain, to clone its Cry gene and determine the spectrum of action of the encoded Cry protein. METHODS AND RESULTS: The B. thuringiensis strain, referred to as M15, was isolated from dead two-spotted spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae). It is an autoagglutination-positive strain and is therefore non-serotypeable. A sporulated culture produces a roughly spherical parasporal inclusion body, the crystal, tightly coupled to the spore. Although the crystal appears to be composed of at least two major polypeptides of 86 and 79 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Southern hybridization indicates that the corresponding crystal protein gene is likely present in only one copy. The crystal protein gene was cloned and, based on nucleotide sequence homology with an orthologous cry31Aa1 gene, assigned the name cry31Aa2. Although initially isolated from spider mites, B. thuringiensis M15 is non-toxic to spider mites and it does not produce the wide spectrum beta-exotoxin. Assays on mammalian cells, however, reveal that Cry31Aa2, when cleaved with trypsin, is cytocidal to some human cancer cells but not to normal human cells. No cytocidal activity was induced after protease treatment of Cry31Aa2 with either chymotrypsin or proteinase K. Trypsin, chymotrypsin and proteinase K cleavage sites were determined. CONCLUSIONS: The B. thuringiensis strain M15 exhibits specific cytocidal activities against some human cancer cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study raises questions as to the actual role of this bacterial strain and its crystal protein in the environment. It may be possible to further develop the Cry31Aa2 protein to target specific human cancer cells.

A single phylogenetic analysis of Bacillus thuringiensis strains and bacilli species inferred from 16S rRNA gene restriction fragment length polymorphism is congruent with two independent phylogenetic analyses.

AIMS: To assess the congruence between two earlier independent phylogenetic studies on Bacillus thuringiensis strains and on Bacillus-related species and the single, all-encompassing, phylogenetic tree presented here inferred from the combination of the two earlier datasets. METHODS AND RESULTS: A dendrogram was constructed using a combination of the data from our previous studies on 16S rRNA gene fingerprinting of 86 B. thuringiensis strains and of 77 species of Bacillus and related taxa. It revealed that all B. thuringiensis strains were clustered together in four distinct groups at a DNA similarity rate of 93%, except two serovars, bolivia and finitimus. Four bacilli species, Paenibacillus alvei, P. azotofixans, B. lentus and B. licheniformis, share a DNA similarity rate of 92% with Bt Group IV. CONCLUSIONS: Most, but not all, B. thuringiensis strains could be grouped together based on the DNA similarity rate. They were also very close to some other bacilli species. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined phylogenetic study presented here, inferred from 16S rRNA gene restriction fragment length polymorphism, is congruent with two earlier independent phylogenetic studies, one on B. thuringiensis and the other on bacilli species.

Evaluation of ribosomal RNA gene restriction patterns for the classification of Bacillus species and related genera.

AIMS: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. METHODS AND RESULTS: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. CONCLUSIONS: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.

The evolutionary origin and genomic organization of SINEs in Arabidopsis thaliana.

We have characterized the two families of SINE retroposons present in Arabidopsis thaliana. The origin, distribution, organization, and evolutionary history of RAthE1 and RAthE2 elements were studied and compared to the well-characterized SINE S1 element from Brassica. Our studies show that RAthE1, RAthE2, and S1 retroposons were generated independently from three different tRNAs. The RAthE1 and RAthE2 families are older than the S1 family and are present in all tested Cruciferae species. The evolutionary history of the RAthE1 family is unusual for SINEs. The 144 RAthE1 elements of the Arabidopsis genome cannot be classified in distinct subfamilies of different evolutionary ages as is the case for S1, RAthE2, and mammalian SINEs. Instead, most RAthE1 elements were probably derived steadily from a single source gene that was maintained intact and active for at least 12-20 Myr, a result suggesting that the RAthE1 source gene was under selection. The distribution of RAthE1 and RAthE2 elements on the Arabidopsis physical map was studied. We observed that, in contrast to other Arabidopsis transposable elements, SINEs are not concentrated in the heterochromatic regions. Instead, SINEs are grouped in the euchromatic chromosome territories several hundred kilobase pairs long. In these territories, SINE elements are closely associated with genes. A retroposition partnership between Arabidopsis SINEs and LINEs is proposed.

A phylogenetic analysis of Bacillus thuringiensis serovars by RFLP-based ribotyping.

AIMS: To determine the 23S and 5S rRNA gene fingerprints in order to reveal phylogenetic relationships among Bacillus thuringiensis strains. METHODS AND RESULTS: Eighty-six B. thuringiensis strains which include 80 serovar type strains, five intraserovar strains and a non-serotypeable strain, wuhanensis, were tested. Total DNA was digested with EcoRI and HindIII. The 23S and 5S rRNA gene restriction fragment length polymorphisms showed 82 distinctive ribopatterns. The dendrogram generated by numerical analysis showed 10 phylogenetic groups and six ungrouped serovars at the 95.5% DNA relatedness rate. A second dendrogram was constructed using a combination of the data from this study and from a previous study on 16S rRNA gene fingerprinting. It revealed eight distinct phylogenetic groups and three ungrouped serovars at the 94% DNA relatedness rate. CONCLUSION: This method permitted the classification and positioning of a wide variety of B. thuringiensis strains on a phylogenetic tree. Bacillus thuringiensis strains appear to be relatively homogeneous and to share a high degree of DNA relatedness. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes a further step to the definition of valid taxonomic sublevels for the B. thuringiensis species.

Genetic diversity of Bacillus thuringiensis serovars revealed by RFLP using random DNA probes.

EcoRI and HindIII restriction fragment length polymorphism (RFLP) profiles using 2 random DNA probes, named 104 and 106, were generated for 85 B. thuringiensis strains. These include 80 serovars, 4 intra-serovar strains: kurstaki HD-1, dendrolimus, tenebrionis and sandiego, and a non-serotypeable strain B. thuringiensis var. wuhanensis. A total of 47 EcoRI and 65 HindIII restriction patterns were generated when hybridization results from both probes were combined. Seventy-seven B. thuringiensis strains showed distinctive hybridization profiles. The dendrogram resulting from the numerical analysis of the distance matrix revealed fourteen distinct phylogenetic groups at the 96% banding patterns similarity. The intra-serovar strains showed higher similarity with their respective type serovars. However, different serovars from a common H-serotype did not always cluster in the same phylogenetic group. Alternatively, several mosquitocidal serovars clustered in a single phylogenetic group. The correlation between serotyping and banding pattern similarity is discussed.

Cloning and nucleotide sequence of a new insertion sequence, IS231N, from a non-serotypable strain of Bacillus thuringiensis.

A new IS231 variant, IS231N, has been isolated from an autoagglutinable, non-serotypable strain of B. thuringiensis. IS231N is 1654 bp in length and is delimited by two incomplete 20-bp inverted repeats (IRL and IRR) with two mismatches. No direct repeats (DRs) were found at the right and left borders of IS231N. Surprisingly, IS231N contains three open reading frames (ORFs) that could code for polypeptides of 329 (ORF1), 118 (ORF2), and 17 (ORF3) amino acids, respectively. IS231N lacks the 5th conserved amino acid domain, called C2, owing to the addition of an adenine residue at nucleotide 1319. IS231N shows the highest nucleotide identity (99%) with IS231M, another insertion sequence previously isolated from the same bacterial strain. IS231N, however, shares only 83% amino acid identity with IS231M because of nucleotide substitutions and additions. The ORF1 of IS231N has five fewer amino acids than ORF1 of IS231M. Furthermore, the ORF2-3 putative fusion product in IS231N contains eight fewer amino acids than ORF2 in IS231M. The dendrogram showing the evolutionary relationship between members of the IS231 family and IS231N indicates that IS231N is phylogenetically more closely related to IS231M (83%), followed by IS231F(74%), and is more distant from IS231V and W(46%).

A new insertion sequence, IS231M, in an autoagglutinable isolate of Bacillus thuringiensis.

An insertion sequence was isolated from an autoagglutinable strain of Bacillus thuringiensis. Analysis of its DNA sequence revealed high homology to the IS231 family. The name IS231M is proposed for this new insertion sequence. IS231M is 1652 bp long and is delimited by two imperfect 20-bp inverted repeat sequences with two mismatches, which are flanked by two perfect 11-bp direct repeats (DRs). The region upstream of the open reading frame, presumed to be able to form a stable hairpin structure, is particularly well conserved in IS231M. Based on primary nucleotide sequences, IS231M is most homologous to IS231F and IS231G and most distant from IS231V and IS231W. However, as opposed to the single transposase A ORF found in IS231A, -B, -C, -D, -F, and -G, IS231M has two overlapping open reading frames, ORF1 and ORF2, that could code for polypeptides of 334 and 143 amino acids, respectively. Whether IS231M is a functional transposable element remains to be determined.

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms.

AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.

Nucleotide sequence of the rice cytochrome f gene and the presence of sequence variation near this gene.

The rice (Oryza sativa L. var. Labelle) chloroplast (cp) gene encoding cytochrome f has been isolated and sequenced. The coding region of this rice gene displays 95.1%, 85.3% and 85.2% nucleotide sequence homology with that of wheat, pea and spinach, respectively. To examine the cpDNA sequence variation in rice, cpDNA from Labelle and its parents, Belle Patna and Dawn was compared. Using the cytochrome f gene as the probe for hybridization, we found several differences in the size and number of restriction fragments in the cp genome of three rice varieties. An additional restriction fragment found in the Belle Patna cp suggests that this cp genome is either heterogeneous or contains two copies of cytochrome f gene per cpDNA.

The beta chorionic gonadotropin-beta luteinizing gene cluster maps to human chromosome 19.

We used a cloned human cDNA probe homologous to the placenta chorionic gonadotropin beta subunit (CGB) and to the pituitary luteinizing hormone beta subunit (LHB) and Southern blotting techniques to analyse DNA from a series of rodent X human somatic cell hybrids for the presence of specific gonadotropin beta subunit related sequences. Our results provide evidence for the assignment and linkage of the eight genes (or pseudogenes) coding for the beta subunit of these glycoprotein hormones to chromosome 19. Moreover, we observed a strict concordance between the permissivity of mouse X man hybrid cells to enteroviruses (which is linked to the presence of specific cell receptors encoded by human chromosome 19) and the presence of CGB and LHB related sequences, thus confirming the localization of the structural genes for the beta subunits on chromosome 19.