Prevalence, molecular typing, and antimicrobial resistance of bacterial pathogens isolated from ducks

Aim: This study aimed to investigate the prevalence of different bacterial species affecting ducks as well as demonstrating the antimicrobial susceptibility and molecular typing of the isolated strains. Materials and Methods: A total of 500 samples were randomly collected from different duck farms at Ismailia Governorate, Egypt. The collected samples were subjected to the bacteriological examination. Polymerase chain reaction (PCR) was applied for amplification of Kmt1 gene of Pasteurella multocida and X region of protein-A (spA) gene of the isolated Staphylococcus aureus strains to ensure their virulence. The antibiotic sensitivity test was carried out. Results: The most common pathogens isolated from apparently healthy and diseased ducks were P. multocida (10.4% and 25.2%), Escherichia coli (3.6% and 22.8%), Staphylococcus epidermidis (10% and 8.8%), Pseudomonas aeruginosa (2% and 10%), and Proteus vulgaris (0.8% and 10%), respectively. In addition, S. aureus and Salmonella spp. were isolated only from the diseased ducks with prevalence (12.2%) and (2.8%), respectively. Serotyping of the isolated E. coli strains revealed that 25 E. coli strains were belonged to five different serovars O1, O18, O111, O78, and O26, whereas three strains were untypable. Salmonella serotyping showed that all the isolated strains were Salmonella Typhimurium. PCR revealed that four tested P. multocida strains were positive for Kmt1 gene with specific amplicon size 460 bp, while three strains were negative. In addition, all the tested S. aureus strains were positive for spA gene with specific amplicon size 226 bp. The antibiotic sensitivity test revealed that most of the isolated strains were sensitive to enrofloxacin, norfloxacin, and ciprofloxacin. Conclusion: P. multocida is the most predominant microorganism isolated from apparently healthy and diseased ducks followed by E. coli and Staphylococci. The combination of both phenotypic and genotypic characterization is more reliable an epidemiological tool for identification of bacterial pathogens affecting ducks.

Physalis angulata: A New Natural Host of Tomato chlorosis virus in Brazil

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are the two Solanaceae-infecting Crinivirus species (family Closteroviridae) of worldwide importance. In Brazil, only ToCV has been detected under natural conditions infecting tomato (Solanum lycopersicum), sweet pepper (Capsicum annuum), and potato (S. tuberosum), causing foliar chlorosis (1, 3). However, there are no formal reports of alternative weed hosts of ToCV. During crop surveys in Capão Bonito, São Paulo State, Brazil (May 2011), a high incidence (above 20%) of plants of the weed, cut leaf ground cherry (Physalis angulata L.) growing around and within a tomato (cv. Alambra) field with a high incidence of ToCV, were found displaying interveinal chlorosis on the lower leaves, similar to those induced by magnesium deficiency. The P. angulata plants also had high populations of whiteflies (Bemisia tabaci biotype B). Ten leaf samples were taken from individual symptomatic ground cherry and tomato plants for Crinivirus testing. Total nucleic acids were extracted from 2 g of symptomatic and healthy leaf tissues of both hosts using Whatman CF-11 cellulose (Sigma) as described (4). The purified double stranded RNA samples were used as a template in reverse transcription (RT)-PCR using specific primers targeting the p22 gene region in the genome of ToCV (2). Only the 566-bp ToCV-specific amplicon was detected in all field samples. Sequences of samples from the P. angulata and tomato cDNA amplicons were identical to each other (GenBank Accession No. JX187514) and they showed 99% identity with the ToCV RNA 1 from a tomato isolate from Florida (AY903447). This confirmed the initial hypothesis of Crinivirus infection. Cuttings of symptomatic P. angulata plants were also obtained and kept in a voile cage under greenhouse conditions together with healthy seedlings of P. angulata and the begomovirus-resistant inbred tomato line ‘TX-468RG.’ Fifty aviruliferous B. tabaci (biotype B) adults were placed in the cage. Similar symptoms were observed 50 days after exposure to whiteflies in both hosts. Transmission to P. angulata and to ‘TX-468RG’ was also confirmed via sequencing of ToCV-specific amplicon, demonstrating the infectivity of the isolate to both hosts. To our knowledge, this is the first report of P. angulata as a natural host of ToCV in Brazil. This weed is often present in the commercial fields because of its natural tolerance to herbicides currently used in tomato production. The ToCV-infected P. angulata plants might serve as alternative sources of inoculum for the surrounding tomato fields. The environmental persistence of P. angulata combined with its intense whitefly colonization might allow a year-round ToCV exposure for tomato plants under field conditions in this major production area of Brazil where at least 25 million tomato plants are cultivated annually. References: (1) J. C. Barbosa et al. Trop. Plant Pathol. 36: 256, 2011. (2) M. I. Font et al. Plant Dis. 86:696, 2002. (3) D. M. S. Freitas et al. Plant Dis. 96:593, 2012. (4) R. A. Valverde et al. Plant Dis. 74:285, 1990.

Rapid differentiation of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis by amplification of insertion element IS901

The aim of this study was to examine the specificity of primers designed to detect the insertion element IS901 commonly used in differentiation of Mycobacterium avium complex strains. This study shows that one of these primers non-specifically anneals to a sequence inside insertion element IS900, specific IS of M. avium subsp. paratuberculosis and to another sequence flanking this element. The resulting non-specific amplicon can be a product of amplification from some M. avium subsp. paratuberculosis strains and can simulate the presence of insertion element IS901 in these strains. However size difference between specific and non-specific amplicons allows such false-positive results to be distinguished. In addition the single PCR allows a rapid and simple differentiation between IS901+ M. avium subsp. avium and M. avium subsp. paratuberculosis strains.

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was ≈2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.

Identification of streptococci isolated from various sources by determination ofcfbgene and other CAMP-factor genes

In the present study, the CAMP-factor (cfb) gene of streptococci of serological group B (Streptococcus agalactiae) and the CAMP-factor (cfu) gene of S. uberis could be amplified by polymerase chain reaction. A cfb specific amplicon could be observed for all 128 phenotypically CAMP-positive S. agalactiae, for the phenotypically CAMP-negative S. agalactiae strain 74-360, and for 2 S. difficile reference strains. A cfu specific amplicon could be observed for all 7 phenotypically CAMP-positive S. uberis. Four S. agalactiae strains isolated from 4 cows with mastitis appeared to be phenotypically CAMP-negative and negative in the cfb gene PCR. The CAMP-positive and CAMP-negative isolates, including both S. difficile, could be identified as S. agalactiae by amplification of a S. agalactiae specific part of the V2 region of the 16S rRNA and a species-specific part of the 16S-23S rRNA intergenic spacer region. Amplification of an internal fragment of the cfb gene with a reduced annealing temperature yielded positive reactions not only for CAMP-positive S. agalactiae, but also for phenotypically CAMP-positive S. pyogenes (n = 4), S. canis (n = 28), and S. uberis (n = 7), indicating a close relation of the CAMP genes of these 4 species. The relation could be further demonstrated by sequencing the internal fragment of the CAMP-factor (cfg) gene of S. canis and comparing the sequence with those of S. agalactiae, S. pyogenes, and S. uberis.Key words: CAMP factor, cfb, cfu, S. canis.