Resolution of phenotypically distinct strains of Enterococcus spp. in a complex microbial community using cpn60 universal target sequencing.

Characterization of complex microbial communities is frequently based on the examination of polymerase chain reaction amplified sequences from a single phylogenetic marker, usually the 16S rRNA gene. However, this commonly used target often does not offer robust resolution of species or sub-species and is thus not a sufficiently informative target for understanding microbial population dynamics occurring at the strain level. We have used the cpn60 universal target sequence to characterize Enterococcus isolates from feces of growing pigs and have shown that sub-species groups, not detected using 16S rRNA sequences, can be resolved. Furthermore, groups resolved by cpn60-based phylogenetic analysis have distinct phenotypes. We report changes in the structure and function of Enterococcus communities in pig feces sampled from individual animals at three times, from suckling through to maturity. Enterococcus faecalis was largely replaced by Enterococcus hirae between suckling and 9 weeks of age, and a shift from one sub-species group of E. hirae to another was observed in all animals between 9 and 15 weeks. Conversely, E. faecalis strains remained consistent throughout the study period. Our results demonstrate that cpn60 sequences can be used to detect strain level changes in Enterococcus populations during succession in the fecal microbiota of growing pigs.

Characterization and quantification of feline fecal microbiota using cpn60 sequence-based methods and investigation of animal-to-animal variation in microbial population structure.

The complex microbial community of the intestine plays a major role in animal health and diseases. Despite its significance to feline health and the significance of intestinal and fecal populations to the public health, little is known about the actual composition of the normal microbiota of the cat. To create a sequence-based inventory of feline fecal microbiota, we applied established methods exploiting the gene encoding the universal 60kDa chaperonin (cpn60) to create libraries of cloned cpn60 sequences from pooled fecal samples from five exclusively indoor and four outdoor, known predatory cats. Sequencing of 1248 clones from each library revealed diverse populations dominated by Actinobacteria (particularly bifidobacteria) and Firmicutes (particularly lactobacilli). To investigate the degree of animal-to-animal variation in species abundance, ten targets were selected from the libraries for analysis by quantitative real-time PCR. Quantitative PCR results showed substantial animal-to-animal variation in target abundance although most targets were detected in all cats. This study lays the foundation for future work aimed at understanding the dynamics of intestinal microbial communities and their role in feline health.

Effect of metC mutation on Salmonella Gallinarum virulence and invasiveness in 1-day-old White Leghorn chickens.

Salmonella enterica serotype Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid (FT) in chickens. FT is a severe systemic disease of chickens causing heavy economic losses to the poultry industry through mortality, reduced egg production and culling of precious breeding stocks. In this study, a metC (encoding cystathionine beta lyase) mutant was produced from a virulent strain of S. Gallinarum by Mini-Tn5 insertional inactivation. The mutant was significantly attenuated in virulence for 1-day-old White Leghorn chickens. Inactivation of metC resulted in 10(4)-fold increase in the LD50 when compared with the wild type parent. The metC mutant showed an in vivo competitiveness defect in the challenged chickens and significantly lower (P < 0.01) bacterial burden in the reticuloendothelial organs when compared with the wild-type parent. These results indicate that metC gene is important for virulence of S. Gallinarum in chickens.

Molecular detection of feline hemoplasmas in feral cats in Korea.

The purpose of this study was to determine if Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' exist in Korea. Three hundreds and thirty one feral cats were evaluated by using PCR assay targeting 16S rRNA gene sequence. Fourteen cats (4.2%) were positive for M. haemofelis, 34 cats (10.3%) were positive for 'Candidatus M. haemominutum' and 18 cats (5.4%) were positive for both species. Partial 16S rRNA gene sequences were closely (>98%) related to those from other countries. This is the first molecular detection of feline hemoplasmas in Korea.

An allele-specific PCR assay for the rapid and serotype-specific detection of Salmonella pullorum.

Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.

Tick-borne rickettsial pathogens in ticks and small mammals in Korea.

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.