Matricaria chamomilla CH12 decreases handling stress in Nelore calves

Matricaria chamomilla CH₁₂ is a phytotherapeutic or homeopathic product, which has been used to reduce stress. Here, we examined its effect on preventing handling stress in bovines. Sixty Nelore calves were randomly distributed into two equal groups. One group was administered Matricaria chamomilla CH₁₂ in diet and the other the ''control'' was not. Animals in both groups were maintained unstressed for 30 days to adjust to the feeding system and pasture, and were then stressed by constraint on the 31th, 38th, 45th and 60th experimental days. Blood samples were taken on these days after animals had been immobilization in a trunk contention for 5 min. Stress was followed by analyzing serum cortisol levels. These peaked on the 45th day and then decreased, but not to baseline, on the 60th day. On the 45th day cortisol levels were significantly lower in animals fed Matricaria chamomilla CH₁₂, suggesting that this product reduces stress. These effects may be a consequence of its inhibiting cortisol production and its calming and anxiolytic effects.     

Frequency of phage-infected bacterial cells in the floodwater of a Japanese paddy field

In aquatic environments, viruses play an important role in the microbial food web through microbial mortality from viral lysis. Bacteriophages (phages) compose the majority of viral communities in the floodwater of paddy fields. The present study evaluated bacterial mortality from phage lysis in the floodwater of a Japanese paddy field based on the frequency of phage-infected bacterial cells by transmission electron microscopy. Floodwater was sampled five times during the rice cultivation period from two plots of NPK plus lime (no-compost plot) and NPK plus lime and compost (compost plot) under a long-term and ongoing fertilizer trial that began in 1925. The frequency of visibly infected cells in the compost plot was larger, ranging from 2.4 to 3.6% (average 3.0%), than that in the no-compost plot, which ranged from 1.6 to 2.9% (average 2.0%). The fractions of bacterial mortality from phage lysis in the floodwater samples were estimated to range from 12.8 to 27.3% (average 17.2%) and from 21.7 to 35.0% (average 27.9%) for the samples collected from the no-compost plot and the compost plot, respectively. This is the first study to estimate bacterial mortality from phage lysis in the paddy field ecosystem, and the frequency of phage lysis in floodwater was found to be within the frequency ranges observed in other aquatic environments.     

Phylogenetic positions of Mn<Superscript>2+</Superscript>-oxidizing bacteria and fungi isolated from Mn nodules in rice field subsoils

We isolated manganous ion (Mn²⁺) oxidizing bacteria and fungi from Mn nodules collected from two Japanese rice fields. The phylogenetic position of the Mn-oxidizing bacteria and fungi was determined based on their 16S rDNA and 18S rDNA sequences, respectively. Among 39 bacterial and 25 fungal isolates, Burkholderia and Acremonium strains were the most common and dominant Mn²⁺-oxidizing bacteria and fungi, respectively. Majority of the Mn-oxidizing bacteria and fungi isolated from the Mn nodules belonged to the genera that had been isolated earlier from various environments. Manganese oxide depositions on Mn²⁺-containing agar media by these microorganisms proceeded after their colony developments, indicating that the energy produced from Mn²⁺ oxidation is poorly used for microbial growth.     

Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido, Japan

Raccoons (Procyon lotor), which have recently become feral in Japan, were examined for the presence of Babesia microti-like parasites. Out of 372 raccoons captured in the west-central part of Hokkaido, 24 animals with splenomegaly were selected and tested by nested PCR targeting the babesial 18S rRNA gene. B. microti-like parasites were detected in two of the 24 individuals, and their DNA sequences were identical to that of the B. microti-like parasite reported from raccoons in the United States, suggesting that the parasites were probably imported into Japan and that the life cycle of the parasite has already been established in the country. The potential risk of this B. microti-like parasite spreading among dogs and foxes in Japan will need to be carefully monitored, as parasitization by phylogenetically very close parasites has been reported from such animals.     

Sub-clinical infection as an effective protocol for obtaining anti-Leishmania chagasi amastigote antibodies of different animal species

This work aims at identifying an effective protocol to raise anti-Leishmania chagasi amastigote antibodies in different animal species. Protocols of immunization by subcutaneous injections of L. chagasi promastigote and amastigote lysates or by either intravenous or subcutaneous inoculation of live metacyclic promastigotes were assessed in mice, rabbits, and dogs. The immunization with live promastigotes produced a strong humoral immune response against L. chagasi amastigotes in all three animal species. The sera from animals immunized with the promastigote lysate did not react with amastigotes and, conversely, the sera from mice immunized with the amastigote lysate did not react with promastigotes. Taken all data together, the immunization through infection with metacyclic promastigotes was considered the most satisfactory way to immunize animals for obtaining anti-amastigote and anti-promastigote antibodies, since it did not only allowed the obtention of antibody against the two forms of the parasite, but it is also cheap, less laborious than carrying out the purification of amastigotes from infected tissues and avoid the use of a large number of hamsters for obtention the amastigotes, necessary to produce the immunogenic lysates. Furthermore, this immunization protocol was comparable to the amastigote lysate immunization protocol for the obtaining of mouse monoclonal antibodies (mAbs).     

U.S.-type Babesia microti isolated from small wild mammals in Eastern Hokkaido, Japan

Our previous report demonstrated that small wild rodents in Japan harbored two types of novel Babesia microti-like parasites (Kobe and Hobetsu types), but not the type widely distributed throughout the temperate zones of North American and Eurasian Continents (U.S. type). In this study, we surveyed small wild mammals collected at various places in the northern part of Japan, seeking for U.S.-type B. microti. A total of 197 small mammals comprising 10 species, Apodemus speciosus, A. argenteus, Clethrionomys rufocanus, C. rutilus, Eothenomys andersoni, Microtus montebelli, Tamias sibiricus, Sorex unguiculatus, S. caecutiens, and Urotrichus talpoides, were examined. Babesia parasites were detected in A. speciosus, C. rufocanus, C. rutilus, M. montebelli, S. unguiculatus, and S. caecutiens by microscopy of blood smears and by PCR targeting babesial nuclear small-subunit rRNA (rDNA) and beta-tubulin genes. Inoculation of their bloods into experimental animals gave rise to 23 parasite isolates, which included 16 from A. speciosus, 4 from C. rufocanus, and 1 each from C. rutilus, M. montebelli and S. unguiculatus. Sequencing analyses of their rDNA and beta-tubulin genes revealed that, of the 23 isolates, 20 and 3 were of Hobetsu and U.S. types, respectively. The U.S.-type B. microti strains isolated in Japan, however, were distinguishable from the isolates in the United States when their beta-tubulin gene sequences and antigen profiles in Western blots were compared. We conclude that U.S.-type B. microti exists in Japan although it has been genetically and antigenically diversified from that distributed in the United States. The results also suggest that not only rodents, but also some insectivores may serve as reservoirs for the agent of human babesiosis.     

Green fluorescent protein gene insertion of Sendai Virus infection in nude mice: possibility as an infection tracer

The green fluorescent protein (GFP) marker from jellyfish Aequorea victoria is considered to have potential use in the study of host-pathogen relationships, by tracing infections in living cells, organs and animals. We compared the pathogenicity of Sendai virus with an inserted GFP gene (GFP-SeV) with that of its wild-type (Wt-SeV) to determine the usefulness of the recombinant virus in long-term infection of BALB/c nude (nu/nu) mice. The results indicated that the presence of GFP in infected cells could be analyzed easily and sensitively. GFP helped in identifying and in understanding the cellular sites of viral replication in vitro and in vivo. However, the GFP insertion into the Wt-SeV genome, led to decreased pathogenicity, altering the in vivo viral kinetics.