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.

Detection of Bartonella species from ticks, mites and small mammals in Korea.

We investigated the prevalence of Bartonella infections in ticks, mites and small mammals (rodents, insectivores and weasels) collected during 2001 through 2004, from various military installations and training sites in Korea, using PCR and sequence analysis of 16S rRNA, 23S rRNA and groEL heat shock protein genes. The prevalence of Bartonella spp. was 5.2% (n = 1,305 sample pools) in ticks, 19.1% (n = 21) in mesostigmatid mites and 13.7% (n = 424 individuals) in small mammals. The prevalence within the family Ixodidae was, 4.4% (n = 1,173) in Haemaphysalis longicornis (scrub tick), 2.7% (n = 74) in H. flava, 5.0% (n = 20) in Ixodes nipponensis, 11.1% (n = 9) in I. turdus, 33.3% (n = 3) in I. persulcatus and 42.3% (n = 26) in Ixodes spp. ticks. In rodents, the prevalence rate was, 6.7% (n = 373) in Apodemus agrarius (striped field mouse) and 11.1% (n = 9) in Eothenomys regulus (Korean red-backed vole) and in an insectivore,Crocidura lasiura, 12.1% (n = 33). Neither of the two weasels were positive for Bartonella spp. Phylogenetic analysis based on amino acid sequence of a portion of the groEL gene amplified from one A. agrarius spleen was identical to B. elizabethae species. We demonstrated the presence of Bartonella DNA in H. longicornis, H. flava and I. nipponensis ticks, indicating that these ticks should be added to the growing list of potential tick vectors and warrants further detailed investigations to disclose their possible roles in Bartonella infection cycles.

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.

Cloning and expression of 51-kDa antigenic protein of Neorickettsia risticii NR-JA1.

Neorickettsia (Ehrlichia) risticii is a causative agent of acute diarrheal syndrome in horses, commonly known as Potomac horse fever. Korean isolate of N. risticii NR-JA1 was cultivated in mouse macrophage cell line P388D1. A complete ORF of p51 antigenic protein gene was amplified and cloned into pQE32 and pcDNA3.1 vectors and the resultant clones were named as pQE32/Nr-51 and pcDNA3.1/Nr-51, respectively. Recombinant p51 (rp51) protein antigen was expressed in E. coli (pQE32/Nr-51) and cos-7 cell line (pcDNA3.1/Nr-51). The rp51 protein showed immunoreactivity with anti- mouse p51 antibodies. BALB/c mice were inoculated with recombinant plasmid DNA (pcDNA3.1/Nr-51). The serum samples collected from these BALB/c mice showed IgG ELISA titers of 1:128. In a Western immunoblot assay, these serum samples showed a strong reactivity to rp51 expressed in cos-7 cell line transfected with pcDNA3.1/Nr-51. The results of this preliminary indicate that N. risticii p51 protein is an immmuno-dominant antigen and may be a good target for the development of serological or a molecular diagnostic test and possibly an improved recombinant DNA based vaccine against Potomac horse fever.

Allele-specific PCR method based on rfbS sequence for distinguishing Salmonella gallinarum from Salmonella pullorum: serotype-specific rfbS sequence polymorphism.

Cloning and sequence analysis of rfbS gene identified two polymorphic nucleotides, one at position 598 (Salmonella gallinarum-specific) and other at position 237 (Salmonella pullorum-specific). Based on S. gallinarum-specific nucleotide found at position 598, an allele-specific PCR method was developed for serotype-specific detection of S. gallinarum. This PCR method was able to discriminate pure cultures of S. gallinarum from S. pullorum and other Salmonella serotypes from serogroup D in less than 3 h. Serotype-specific detection of S. gallinarum was possible in less than 24 h when the PCR was applied on the presumptive Salmonella colonies obtained after overnight incubation of selective media plates streaked with the clinical material from diseased chickens. As little as 100 pg of genomic DNA could be detected with S. gallinarum-specific primers; no PCR product was detected in non-S. gallinarum serotypes of serogroup D and other closely related non-salmonella organisms. This rfbS allele-specific amplification assay is specific, reproducible and less time consuming than the standard bacteriological methods used to detect S. gallinarum and could be an effective molecular tool for rapid definitive diagnosis of fowl typhoid in the areas of endemicity where fowl typhoid infection exists.