Jae-Myung Kim

Screening lactic acid bacteria from swine origins for multistrain probiotics based on in vitro functional properties

Lactic acid bacteria originated from swine feces and intestines were selected for potential probiotics based on their bile-salt resistance, low pH tolerance, potential adhesion to epithelial cells and especially functional properties, including production of antimicrobial substances, bile-salt hydrolase (BSH) and amylolytic activity. Results showed 7 isolates with antimicrobial activity, 5 with BSH activity and 3 with amylolytic activity were preliminarily selected from 485 lactic acid bacteria based on their highest potential with functional properties in vitro. The 15 isolates were further assayed on the essential characteristics as potential probiotics. All isolates were fully tolerant to 0.3% bile salts and 11 of them were able to resist pH 3 for 3 h without loss of viable cells. The eleven isolates were then evaluated on their adhesion capability. Wide variation in the hydrophobic character and specific adhesion efficiency was observed and three isolates G1-1, G22-2 and G8-5, with respective antimicrobial, BSH and amylolytic activities were finally selected. In addition, the three isolates were compatible in the coexistence assay. Isolate G1-1 was identified as Lactobacillus salivarius by API system and a 16S rRNA gene sequence analysis. Both G8-5 and G22-2 showed the closest homology to Lactobacillus reuteri according to their 16S rRNA gene sequences (99%). From the study, the three Lactobacilli strains were shown to share the functional properties necessary for probiotics use in animal additives. Their compatibility with respective in vitro activities was expected to show enhanced in vivo efficacy after combination for multistrain probiotics use.

Mycobacterium anyangense sp. nov., a rapidly growing species isolated from blood of Korean native cattle, Hanwoo (Bos taurus coreanae).

From the whole blood of Korean native cattle, Hanwoo (Bos taurus coreanae), a previously undescribed, rapidly growing, scotochromogenic isolate of the genus Mycobacterium is reported. Its 16S rRNA gene sequence, and the sequences of three other genes (hsp65, recA and rpoB) were unique and phylogenetic analysis based on 16S rRNA gene sequence (1420 bp) placed the organism into the rapidly growing Mycobacterium group close to Mycobacterium smegmatis (98.5% sequence similarity). However, phylogenetic analyses based on three different gene sequences (hsp65, recA and rpoB) revealed its location to be distinct from the branch of rapidly growing species. Culture and biochemical characteristics were generally similar to those of Mycobacterium fortuitum. Unique matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS profiles of lipids, unique fatty acid profile, unique mycolic acids profiles and a low DNA-DNA relatedness to M. fortuitum (23.6%) and M. smegmatis (39.7%) strongly supported the taxonomic status of this strain as a representative of a novel species of rapidly growing mycobacteria named Mycobacterium anyangense. The type strain is strain QIA-38(T) ( = JCM 30275(T) = KCTC 29443(T)).

Mycobacterium avium paratuberculosis in Wild Boars in Korea

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic infectious enteritis in various domestic and wild mammals and is widely distributed globally. Interspecies transmission has been frequently reported. We investigated the presence of MAP from December 2010 to March 2011 in blood and feces collected from 222 hunter-killed wild boars. We collected 197 serum and 180 fecal samples and examined them by culture, PCR, and enzyme-linked immunosorbent assay. We investigated the status of MAP infection and the MAP genotypes in the wild boar population of Korea by using IS900 PCR and IS1311-restriction endonuclease analysis typing. Of the 180 fecal samples cultured, MAP colonies were recovered from two. By PCR, 18 animals were positive for MAP and one serum sample had a strong humoral response to MAP. The PCR-positive DNA samples from the colonies and the feces samples were genotyped as “cattle type” and “bison type,” which are major MAP genotypes infecting domestic species in Korea. Our study provides new information on mycobacterial infection among wild boars, and suggests that a more effective program should be developed to monitor mycobacterial infections in wild animal populations in Korea.

QUANTITATIVE ROSE BENGAL TEST FOR DIAGNOSIS OF BOVINE BRUCELLOSIS

The Rose Bengal Test (RBT) is the most widely used screening test for brucellosis in both humans and animals. Owing to its apparent simplicity of reading, however, interpretations of the RBT results can be affected by personal experience. This study describes a simple way to improve the accuracy and uniformity of reading the RBT reaction by counting the number of agglutinated particles using transparent OHP film with Quantity One®, which was originally designed to count the bacterial colony numbers on agar plates. Using this system, the reactivities of three Rose Bengal antigens from different sources against international standard serum (1,000 units, VLA, UK) could be numerically measured: the intensity scale ranged from zero to around 1,600. This system enabled us to compare the antigenicity of Rose Bengal antigens from three different sources by using statistical analyses such as regression and mean intensity. Collectively, mathematical measuring of the reaction intensity used in this study may help interpret subtle test results by providing more reliable data and additional statistical information on the herd. In addition, the method would also be applicable to other agglutination test for other diseases.

Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.

An evaluation of the use of immunoglobulin A antibody response against mycobacterial antigens for the diagnosis of Mycobacterium bovis infection in cattle

Antibody responses are useful indicators of Mycobacterium bovis infection in cattle. Many studies have evaluated the ability of immunoglobulin G (IgG) to serodiagnose bovine tuberculosis (TB). In the current study, immunoglobulin A (IgA) and IgG responses against the MPB70 and MPB83 antigens of M. bovis, the 38 kDa phosphate-binding lipoprotein (PstS1) that is a well-known serodiagnostic M. tuberculosis antigen, and a newly identified protein, termed Rv1483c, were compared in M. bovis–infected and noninfected cattle as well as in field samples. The diagnostic utility of the IgA antibody to MPB70 and MPB83 for bovine TB was superior or comparable to that of the IgG antibody, and the sensitivity of serodiagnosis increased when the results of antigen binding by IgA and IgG were combined. The sensitivities of the IgG and IgA antibodies to the Rv1483c and PstS1 proteins were significantly lower than those to MPB70 and MPB83, and no diagnostic utility for Rv1483c was observed in field samples. Importantly, the IgA antibody reacted strongly to the MPB70 and MPB83 antigens and differentiated cattle with TB from healthy cattle in a multiantigen printed immunoassay. The results of this study support the feasibility of using IgA antibody against the MPB70 and MPB83 antigens to detect bovine TB. In addition, approaches using assays for both IgA and IgG antibodies may increase detection accuracy.

Bovine tuberculosis in an Asian small-clawed otter (Aonyx cinerea) in the Republic of Korea.

Bovine tuberculosis caused by Mycobacterium bovis has a wide range of hosts including cattle and humans, but its incidence in otters is very rare. Our report describes a case of bovine tuberculosis in an Asian small-clawed otter (Aonyx cinerea). A deceased female otter ~2–3 years of age that was raised in an aquarium was submitted to the Animal and Plant Quarantine Agency (Anyang, Republic of Korea) for autopsy in June 2013. Following gross pathological examination, many white nodules were observed in the lungs and mesentery. The nodules showed central necrosis infiltrated with lymphocytes and macrophages and surrounded by fibrous tissue. Acid-fast bacteria were detected in the necrotic foci, but no fungi were observed. Molecular analysis led to the detection of M. bovis, which is identified in otters in some European countries such as Spain and France.