MAL73, a novel regulator of maltose fermentation, is functionally impaired by single nucleotide polymorphism in sake brewing yeast.

For maltose fermentation, budding yeast Saccharomyces cerevisiae operates a mechanism that involves transporters (MALT), maltases (MALS) and regulators (MALR) collectively known as MAL genes. However, functional relevance of MAL genes during sake brewing process remains largely elusive, since sake yeast is cultured under glucose-rich condition achieved by the co-culture partner Aspergillus spp.. Here we isolated an ethyl methane sulfonate (EMS)-mutagenized sake yeast strain exhibiting enhanced maltose fermentation compared to the parental strain. The mutant carried a single nucleotide insertion that leads to the extension of the C-terminal region of a previously uncharacterized MALR gene YPR196W-2, which was renamed as MAL73. Introduction of the mutant allele MAL73L with extended C-terminal region into the parental or other sake yeast strains enhanced the growth rate when fed with maltose as the sole carbon source. In contrast, disruption of endogenous MAL73 in the sake yeasts decreased the maltose fermentation ability of sake yeast, confirming that the original MAL73 functions as a MALR. Importantly, the MAL73L-expressing strain fermented more maltose in practical condition compared to the parental strain during sake brewing process. Our data show that MAL73(L) is a novel MALR gene that regulates maltose fermentation, and has been functionally attenuated in sake yeast by single nucleotide deletion during breeding history. Since the MAL73L-expressing strain showed enhanced ability of maltose fermentation, MAL73L might also be a valuable tool for enhancing maltose fermentation in yeast in general.

Costs and benefits of larval jumping behaviour of Bathyplectes anurus.

Bathyplectes anurus, a parasitoid of the alfalfa weevils, forms a cocoon in the late larval stage and exhibits jumping behaviour. Adaptive significance and costs of the cocoon jumping have not been thoroughly studied. We hypothesised that jumping has the fitness benefits of enabling habitat selection by avoiding unfavourable environments. We conducted laboratory experiments, which demonstrated that jumping frequencies increased in the presence of light, with greater magnitudes of temperature increase and at lower relative humidity. In addition, when B. anurus individuals were allowed to freely jump in an arena with a light gradient, more cocoons were found in the shady area, suggesting microhabitat selection. In a field experiment, mortality of cocoons placed in the sun was significantly higher than for cocoons placed in the shade. B. anurus cocoons respond to environmental stress by jumping, resulting in habitat selection. In the presence of potential predators (ants), jumping frequencies were higher than in the control (no ant) arenas, though jumping frequencies decreased after direct contact with the predators. Body mass of B. anurus cocoons induced to jump significantly decreased over time than cocoons that did not jump, suggesting a cost to jumping. We discuss the benefits and costs of jumping behaviour and potential evolutionary advantages of this peculiar trait, which is present in a limited number of species.

Investigation of whether CLSI broth microdilution method is applicable for MICs Determination of Enterococcus species.

The broth microdilution (BMD) method is an antimicrobial susceptibility testing method defined as a guideline by the Clinical and Laboratory Standards Institute (CLSI). To date, the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM), has adopted the agar dilution (AD) method for monitoring antimicrobial resistances targeting isolates of Enterococcus spp., found in the fecal flora of food-producing animals, as indicator bacteria. However, the AD method is tedious, and time-consuming. In order to examine whether it could be replaced with the BMD method, the two methods were compared in terms of the correlation of MICs. In this study, the BMD results agreed with the AD results within ±1 log(2) dilutions in 72.3% of cases, except for the antimicrobial feed additive, Nosiheptide (NHT). Similarly, except for NHT, the MIC(50) and MIC(90) values obtained by the two methods were well correlated. In conclusion, our results indicate that the BMD method might be suitable for antimicrobial susceptibility testing targeting Enterococcus spp..

Evaluation of Salmonella enterica serovar Typhimurium and Choleraesuis slyA mutant strains for use in live attenuated oral vaccines.

SlyA protein plays a key role in virulence in Salmonella enterica. In this study, we evaluated the ability of the slyA mutant strain of S. enterica serovar Choleraesuis (S. choleraesuis) to protect against swine salmonellosis. Using a murine model infected with S. enterica serovar Typhimurium (S. typhimurium), we showed that the Salmonella strain with a deletion of slyA could be used as a highly immunogenic, effective and safe vaccine in mice. Based on these data, a slyA mutant of S. enterica serovar Choleraesuis strain RF-1 was constructed, and the ability of this mutant to protect immunized pigs from S. choleraesuis infection was examined. As with the S. typhimurium slyA mutant, immunization of pigs with the S. choleraesuis slyA mutant strain provided significant protection against subsequent challenge by the wild-type RF-1. These results demonstrate that SlyA is a potential target in the development of a novel live attenuated vaccine against S. enterica.

PCR-RFLP identification of prohibited animal derived DNA in animal feed.

A method for confirming identification of prohibited species tissue in animal feed has been developed on the basis of PCR-RFLP analysis. In Japan, to prevent the spread of BSE through animal feed, the use of animal protein in feed has been regulated. Species-specific PCR detection of prohibited species materials in feed has been used as one of a series of laboratory tests to ensure the proper implementation of the feed regulations. However, since the result of this PCR method is determined only by amplicon length, it is sometimes necessary to confirm whether or not the positive result is due to the effect of a non-specific reaction. For this purpose, DNA sequencing is the best way to confirm the test result but it is not suitable for routine analysis because of the required time and cost. In this study, we developed an easy and rapid method to confirm the species identification (mammals, ruminants and cattle) by using 4 restriction enzymes: SmlI, MboI, BlnI and Hpy188III. This PCR-RFLP method, which ensures identification of prohibited animal species in feed, is useful for enhancing the reliability of feed inspection for BSE prevention. This method will be added to the Official Methods of Feed Analysis.

Guidelines used in Japan to prevent the contamination of feed products with undesirable substances.

As Japan depends on imports for most ingredients used to manufacture feed products, close co-operation is indispensable between importers and manufacturers of feed and feed ingredients to effectively mitigate the risk associated with feed safety. Guidelines were issued by the Ministry of Agriculture, Forestry and Fisheries (MAFF) in March 2008 to prevent feed products from being contaminated with undesirable substances. These guidelines identify the responsibilities of feed ingredient importers, feed manufacturers and distributors, as well as the roles of the MAFF and the Food and Agricultural Materials Inspection Centre.

Conjugative transposition of Tn916 and detection of Tn916-Like transposon in Erysipelothrix rhusiopathiae.

In order to investigate the origin of tetracycline resistance in Erysipelothrix rhusiopathiae, conjugative transpositions of Tn916 were tested. The frequency of transfer between strains of E. rhusiopathiae was about 10-fold higher than that between Enterococcus faecalis and E. rhusiopathiae. In addition, detection of a Tn916-like transposon was performed by PCR assay and DNA sequencing in E. rhusiopathiae field isolates. Of 49 tetracycline-resistant isolates, 38 (77.6 %) carried a Tn916-like transposon, while 11 (22.4 %) carried tet(M) only. These results suggested that Tn916-like transposon may be widely present in the E. rhsuiopathiae field isolates resistant to tetracycline.

Development of primers for detection of heat-treated cetacean materials in porcine meat and bone meal.

The feed ban introduced after the detection of the first case of bovine spongiform encephalopathy in 2001 in Japan has been modified to allow some of the previously prohibited animal materials to be used in animal feed. Recently, porcine materials were allowed to be used in feed for pigs, poultry, and fish. Materials from other mammals, including whales, remain prohibited. In the absence of a method to detect the prohibited whale materials in porcine materials, there is a possibility that the whale materials are being used for feed for pigs, poultry, and fish. To detect illegal use of whale materials mixed with porcine materials, we have developed PCR primers specific to a group of most cetacean species, using a computer-based method we developed previously. The primer sets were capable of detecting whale meat meal that had been autoclaved at 133 degrees C for up to 20 min. The detection limit of whale material in porcine meat and bone meal was 0.1%.

Etiological and biological characteristics of Erysipelothrix rhusiopathiae isolated between 1994 and 2001 from pigs with swine erysipelas in Japan.

We investigated 66 Erysipelothrix rhusiopathiae strains isolated from pigs affected with swine erysipelas in Japan from 1994 to 2001 for serotype, pathogenicity towards mice, protection in vaccinated mice and antimicrobial susceptibility. Most of the isolates (84.8%) were serotype 1 or 2. For the first time, strains belonging to serotype 21 were isolated from cases of septicemia. Fifty isolates (75.8%) were highly virulent, 12 isolates (18.2%) were weakly virulent and 4 isolates were avirulent strains. All the mice vaccinated with the Koganei 65-0.15 vaccine strain survived challenge exposure with 50 highly virulent isolates. Six isolates (9.1%) grew on TPB-T80 agar containing 0.02% of acriflavine, and this was identical to the growth of the vaccine strain. Forty-seven isolates (71.2%) were resistant to oxytetracycline. The number of strains resistant to oxytetracycline among field isolates increased rapidly each year. Tylosin-resistant strains were also isolated (6.1%). These results suggest that certain characteristics, particularly antimicrobial susceptibility of E. rhusiopathiae isolates, change yearly in the field. Therefore, further investigation of the characteristics of E. rhusiopathiae field isolates is necessary.

Control and monitoring of antimicrobial resistance in bacteria in food-producing animals in Japan.

Increased antimicrobial resistance in bacteria that cause infections in humans is a threat to public health. The use of antimicrobials in food-producing animals in the form of veterinary medicine and feed additives may lead to the emergence or spread of antimicrobial resistance in bacteria of animal origin. In Japan, the use of antimicrobials in food-producing animals is regulated by the Pharmaceutical Affairs Law and Feed Safety Law to minimise the risk of emergence and spread of antimicrobial resistance in bacteria. Since December 2003, all antimicrobials used in food-producing animals have been subjected to risk assessment by the Food Safety Commission. In addition, an antimicrobial resistance monitoring programme has been in place since 2000 to monitor the evolution of resistance to different antimicrobials in bacteria in food-producing animals.

High GC contents of primer 5'-end increases reaction efficiency in polymerase chain reaction.

Many studies have suggested that regulation of the polymerase chain reaction (PCR) is influenced by several factors. However, the understanding of reaction efficiency factors is not sufficient. Here we propose that high GC contents of primer 5'-end increases reaction efficiency in PCR. Using 71 primers (45 pairs), we analyzed factors that affect reaction efficiency, and statistically tested the correlation between the amplification signals and several factors. As a result, there were significant correlations between the amplification signals and the GC contents in the first 1 approximately 3 bps of primer 5'-end.

A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells.

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.

Pathogenicity for mice and swine of Erysipelothrix isolates from the tonsils of healthy cattle.

The pathogenicity of 79 Erysipelothrix isolates from bovine tonsils for mice and swine was determined. Five (6.3%) isolates were lethal for mice. These isolates belonged to serovars 1b (one isolate), 2 (2), 19 (1) and 21 (1). The 50% lethal dose values of the isolates ranged from 0.33 to 5x10(2) CFUs in mice. Twenty Erysipelothrix isolates (25.3%) were weakly virulent inducing only emaciation while 12 (15.2%) inducing emaciation and ruffled hair. In swine, clinical signs of varying severity were observed. Four isolates were virulent, capable of inducing localized or generalized urticarial lesions accompanied with a rise in body temperature after intradermal inoculation. One isolate each of serovars 1b, 2 and 19 was highly virulent, capable of inducing generalized urticarial lesions while another Erysipelothrix isolate of serovar 2 induced only a localized urticarial lesion at the site of inoculation. Another isolate of serovar 1b induced itching and irritation without obvious urticarial lesion at the site of inoculation. On the other hand, one isolate of serovar 21 and two other isolates of serovar 2 could not induce experimentally any clinical sign of erysipelas other than rise in body temperature. There was a rise in growth agglutination (GA) titer of serum in all the inoculated swine. These observations suggest that Erysipelothrix isolates from cattle are pathogenic for mouse and swine, and may also be pathogenic for other animals and humans.

Molecular analysis of a haemagglutinin of Haemophilus paragallinarum.

The gene encoding a haemagglutinin of H. paragallinarum, hagA, has been identified and the full-length nucleotide sequence determined. A approximately 39 kDa protein, recognized by an anti-haemagglutinin monoclonal antibody, mAb4D, was purified from H. paragallinarum strain 0083 and the N-terminal sequence obtained. The full-length nucleotide sequence was obtained by inverse PCR and the deduced amino acid sequence of the protein encoded was shown to be similar to other outer-membrane proteins of closely related organisms in the HAP group (Haemophilus, Actinobacillus, Pasteurella), especially the P5 protein of Haemophilus influenzae. The hagA gene was cloned into a His-tag expression vector and overexpressed in Escherichia coli strain M15(pREP4). The identity of the purified recombinant protein as a H. paragallinarum haemagglutinin was confirmed by haemagglutination of chicken red blood cells and reactivity, in a Western blot, with the monoclonal antibody specific for the serovar A haemagglutinin.

The reproductive strategy of the gregarious parasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae) : 2. Host size discrimination and regulation of the number and sex ratio of progeny in a single host.

Host size of Pteromalus puparum, a gregarious pupal parasitoid, shows a wide inter- and intraspecific variation. Experiments were made to study the regulation of the number and sex ratio of progeny per host by the parasitoid. The parasitoid could discriminate inter- and intraspecific size differences of the host and regulate the number of eggs according to the host size when a single female attacked the host. The sex ratio of progeny (proportion males) was about 0.1. The number of progeny laid by the female agreed with the energetically most efficient number og eggs in order to maximize total weight of progeny per host but not with the reproductively most efficient number of eggs to maximize the total fecundity of the progeny. The parasitoid laid smaller number of eggs in a half buried host, but the number was much larger than a half of those in a fully exposed host. When more than one female attacked a single host, the number and sex ratio of progeny per host increased with the number of females attacking the host, but the number of progeny per female decreased. The change of the sex ratio agreed with the prediction of the local mate competition model.

The reproductive strategy of the gregarious parasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae) : 1. Optimal number of eggs in a single host.

Pteromalus puparum is a gregarious parasitoid of many butterfly pupae. Adult size, mortality, and sex ratio of P. puparum, as a parasitoid of Papilio xuthus, were unit weight of the host. Effects of female size on fecundity, wing load, and longevity were also examined.The highest total weight of progeny from the host was attained when the number of eggs per gram of the host was approximately 150. Positive correlations were observed between the size of the females and their fecundity and wing load. The maximum longevity of the female kept with honey but without hosts was attained when the initial number of parasitoids per g of the host was 150.Considering the total fecundity of all female progeny, the reproductively most efficient number of eggs to be deposited per g of the host was estimated to be approximately 300. However, as shortage of food for the adult females strongly affects their fecundity, the reproductively most efficient number of eggs to be deposited per g of the host was about 70 when the adult female progeny was not provided with food.The optimal number of eggs to be deposited when the emale oviposits in the host under field conditions is discussed.