Species richness of the understory woody vegetation in Japanese cedar plantations declines with increasing number of rotations

The possibility of restoring natural broadleaf forests may be decreased by the effects of plantation management, particularly in sites that undergo repeated rotation. We investigated the following two working hypotheses about the effects of repeated plantation of conifers on the natural regeneration of woody saplings in cool-temperate Japanese cedar plantations: (1) that repeated plantation of conifers decreases sapling species richness, and (2) that repeated plantation of conifers changes sapling species compositions. Our result supported the first hypothesis, because species richness was significantly lower in second-rotation plantations than in first-rotation plantations. The second hypothesis was not supported, because no significant or substantial differences in species composition were observed between plantations with different numbers of rotations. However, the abundance of tree (nonshrub) and gravity-dispersed species decreased after the second rotation of large saplings, albeit not those of small saplings, suggesting that response to repeated rotation depended on sapling size. Our results suggest that it is important to consider factors affecting the maintenance of a species in the plantations, such as distance from natural forests and seed sources, to minimize the effects of repeated plantation.     

Anti-babesial activity of a potent peptide fragment derived from longicin of Haemaphysalis longicornis

Babesiosis is one of the most important tick-borne diseases affecting livestock that can cause major economic losses worldwide particularly in the tropics. Control relies on controlling both the protozoan parasite and the tick vector. Antiprotozoal drugs are most commonly used for treatment, but problems on emergence of resistant strains and food residues are encountered. Longicin, a defensin-like peptide identified from the hard tick, Haemapysalis longicornis, as well as one of its synthetic partial analogs (P4), were previously reported to exert antimicrobial, fungicidal, and parasiticidal activity. Both longicin and P4 showed babesiacidal activity, in vitro and in vivo. Here, peptide fragments of P4 were studied for in vitro activity against bovine Babesia parasites. One of the peptide fragments, antimicrobial peptide 1 (AMP1), reduced the parasitemia of Babesia bigemina. No peptide had significant effect on Babesia bovis. The sequence of AMP1 corresponded to the longicin sequence which is associated with antiparasitic activity. Although AMP1 caused reduction in parasitemia of B. bigemina, the difference in morphology of the parasite compared with the control group was not statistically significant. However, the percentage occurrence of piroplasms decreased, whereas the abnormal pycnotic form increased. The results demonstrated that this shorter peptide retained the anti-babesial activity of the parent peptide, exerting an antiparasitic effect against a bovine Babesia species. Therefore, this short peptide can be considered for chemical synthesis as an alternative therapeutic agent for babesiosis.     

Changes in peripheral leukocyte populations of weak calf syndrome of Japanese black calves

To clarify the cellular immune condition in Japanese Black (JB) calves with a weak syndrome, peripheral leukocyte populations were analyzed by flow cytometry. Twenty JB calves were divided into two groups based on clinical observations; one group of calves was weak, because they had experienced an onset of diarrhea within 3 days after birth and needed treatment (Group 1 ;n=10), and the other group of calves was healthy (Group 2; n=10). With regard to leukocyte populations, CD8(+) cells and gamma delta T cells in Group 1 were markedly lower than those in Group 2 during the experimental periods. It is possible that immune-insufficiency might be based on T lymphocyte function in weak syndrome JB calves during the growth process.     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen

A study was conducted to evaluate the therapeutic efficacy of doramectin administered intramuscularly at a dose rate of 200 microg/kg to sheep harbouring naturally acquired infections of gastrointestinal nematodes and Oestrus ovis in the southwestern region of France. On day 0, 24 sheep were selected on the basis of positive faecal egg counts (>100 EPG) and positive assessment of O. ovis infection (including positive O. ovis antibody level and positive clinical score). The sheep were randomly allocated to a non-medicated control group (T1) or a doramectin-treated group (T2) of 12 animals each. On day 0, sheep in group T2 received a single intramuscular injection of doramectin (200 microg/kg), whereas those in group T1 received an intramuscular injection of saline solution (sodium chloride, 0.02ml/kg). Individual faecal egg counts were performed on days 0, 1, 2, 3, 4, 5, 6, 7, and 14. Between days 14 and 16, all sheep were slaughtered, and worm and O. ovis burdens were determined. In doramectin-treated sheep, faecal egg counts had decreased to zero by day 4 for all recovered types of nematode eggs: strongyles, Nematodirus sp., Trichuris sp., and Rhabditidae sp. For strongyles, Nematodirus sp., and Rhabditidae, the percentage reductions in faecal egg counts (geometric means) of doramectin-treated sheep, compared to the non-medicated control sheep were 100% from days 4-7. For Trichuris sp., they were 100, 99.7, 99.9, and 100% on days 4, 5, 6, and 7, respectively. On day 14, percentage reductions were 100% for Nematodirus sp. and Rhabditidae, and 99.8 and 99.1% for strongyles and Trichuris sp., respectively. At necropsy, only adult nematodes and mainly first-stage O. ovis larvae were recovered. Doramectin was highly efficacious against the adult stages of Teladorsagia circumcincta (100%), Nematodirus battus (100%), Nematodirus filicollis (99.9%), Oesophagostomum venulosum (99.8%), and Trichuris sp. (99.3%). It was also 100% efficacious against first-stage larvae of O. ovis. No abnormal clinical signs or adverse reactions in any of the sheep treated with doramectin were observed.     

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.     

Detection of Cryptosporidium parvum oocysts in environmental water in Hokkaido, Japan

Control of cryptosporidiosis is important in public health. Rivers that are polluted with Cryptosporidium and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptosporidiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptosporidium oocysts in environmental water, the filtration method was used for 28 samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency (U.S. EPA) filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by discontinuous sucrose gradient method. Twelve samples were collected from 10 rivers and parasites were purified by iron (III) flocculation method. Cryptosporidium parvum oocysts were identified with the immunofluorescence antibody technique using DIF kit (Cellabs Pty. Ltd., Sydney/Australia). We detected Cryptosporidium oocysts in 6 out of 10 rivers sampled. Fifty percentage (14/28) of the samples were Cryptosporidium-positive. The average number of Cryptosporidium oocysts was 16.73/100 L (max. 80/100 L).     

Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.     

Comparison of noninvasive cardiac output measurements by transthoracic bioimpedance, partial carbon dioxide rebreathing, and transesophageal echocardiography with the thermodilution technique in beagle dogs

Most methods for determining cardiac output (CO) have limited application in clinical practice due to the invasive techniques required. This study compared the thermodilution technique (TDCO) with three noninvasive methods for determining CO in anesthetized dogs: transthoracic bioimpedance (BICO), partial CO₂ rebreathing (NICO), and transesophageal echocardiography (TEECO). TDCO was compared to BICO, NICO, and TEECO in six adult sevoflurane anesthetized beagle dogs (9.1–13.0 kg). All dogs were administered midazolam [0.3 mg kg^?1, intravenously (IV)] and butorphanol (0.1 mg kg^?1 IV), followed by ketamine (5.0 mg kg^–1 IV) and sevoflurane in nitrous oxide (1 L minute^–1) and oxygen (1 L minute^–1) and mechanically ventilated. Dogs were maintained at 2.2% end‐tidal sevoflurane (ETsev) concentration for instrumentation and baseline measurements. Low (5.0% ETsev), intermediate (3.3% ETsev), and high cardiac output values were achieved by varying the end‐tidal sevoflurane concentration and the administration of dobutamine (3–10 g kg^–1 minute^–1 and 2.2% ETsev). A minimum of thirty data sets was obtained for each comparison. The correlation coefficients when compared to TDCO were 0.684 for BICO (p < 0.0001), 0.883 for NICO (p < 0.0001), and 0.991 for TEECO (p < 0.0001). Cardiac output values ranged 50–444 mL kg^–1 minute^–1 for TDCO, 100–253 mL kg^–1 minute^–1 for BICO, 64–214 mL kg^–1 minute^–1 for NICO, and 52–401 mL kg^–1 minute^–1 for TEECO. The differences when compared to TDCO ranged – 62–235 mL kg^?1minute^?1 for BICO, 18–220 mL kg^?1 minute^?1 for NICO, and – 35–32 mL kg^–1 minute^–1 for TEECO. Differences were maximum at the highest CO in BICO and NICO. In conclusion, this study demonstrated that BICO and NICO underestimate CO in sevoflurane anesthetized dogs. TEECO is a viable noninvasive method for determining CO in sevoflurane anesthetized dogs.     

Plaque assay of equine influenza virus

ESK cells, a stable cell line derived from a swine embryo kidney, were found to be a good medium for plaque formation of the Prague and Miami strains of equine influenza virus. Factors influencing the plaque formation were investigated and a plaque assay for these viruses was worked out. The method is not only simple enough for routine use, but also is as sensitive as the egg inoculation method. The method was readily adapted for a neutralization test.