Molecular cloning of two caspase-like genes from the hard tick Haemaphysalis longicornis

We identified two caspase-like genes from the midgut cDNA library of the hard tick, Haemaphysalis longicornis. Sequence analysis showed that these cDNAs encoded homologues of caspase-2 and caspase-8 that were categorized as apoptosis initiators. The H. longicornis caspase-2 (Hlcaspase-2) cDNA encodes 340 amino acid residues with a predicted molecular weight (Mw) of 38.5 kDa. Another cDNA identified as the H. longicornis caspase-8 (Hlcaspase-8) encodes 306 amino acid residues with an estimated Mw of 35.3 kDa. A catalytic active site was highly conserved in Hlcaspase-8 but not in Hlcaspase-2. RT-PCR analysis showed that both Hlcaspase-2 and Hlcaspase-8 were expressed in tick midgut and salivary glands. This is the first report of the molecular cloning of apoptosis-related genes in the tick.     

Genetic characterization of GRA6 genes from Toxoplasma gondii from pigs in Okinawa, Japan

Toxoplasma gondii from pigs in Okinawa Prefecture was characterized by nested PCR-restriction fragment length polymorphism (RFLP) and DNA sequence analysis of the dense granule antigen GRA6 gene. By nested PCR, parasite DNA was detected in 33 out of 91 lymph node samples with lesions similar to those found in toxoplasmosis samples that had been collected from pigs at an abattoir. RFLP analysis with MseI was successfully conducted in 29 of 33 PCR-positive samples to group the isolates into one of the three genotypes of T. gondii. Genotyping of the 29 studied samples rendered the following results: 13 of type I (44.8%), 14 of type II (48.3%), and 2 of type III (6.9%). The GRA6 genes of 12 Okinawa isolates were cloned and sequenced. Nine new nucleotide sequences were found, and nucleotide substitutions specific for the Okinawa isolates were found at 13 positions. Phylogenetic analysis indicated that all GRA6 sequences were divided into one of the 3 main groups, and Okinawa isolates of GRA6 genotypes II and III seemed to be closely related to the Beverley strain and the NED strain, respectively. The results from this study may provide basic and useful information for the analysis of the molecular epidemiology of T. gondii infection within Japan.     

Modifications of azoxymethane-induced carcinogenesis and 90-day oral toxicities of 2-tetradecylcyclobutanone as a radiolytic product of stearic acid in F344 rats

A 90-day oral toxicity test in rats was performed to evaluate the toxicity of 2-tetradecylcyclobutanone (2-tDCB), a unique radiolytic product of stearic acid. Six-week-old male and female F344 rats (n=15/group) were given 2-tDCB at concentrations of 0, 12, 60 and 300 ppm in a powder diet for 13 weeks. Slight dose-dependent increases in serum total protein and albumin in male rats were found, but these changes were not considered to be a toxic effect. The fasting, but not non-fasting, blood glucose levels of the male rats in the 300 ppm group and female rats in the 60 and 300 ppm groups were lower than those of the controls. Gas chromatography-mass spectrometry analysis showed dose-dependent accumulation of 2-tDCB in adipose tissue, notably in males. Next, we performed an azoxymethane (AOM)-induced two-stage carcinogenesis study. After injection of 6-week-old male F344 rats (n=30/group) once a week for 3 weeks, the animals received 2-tDCB at concentrations of 0, 10, 50 and 250 ppm in a powder diet for 25 weeks. The incidences of colon tumors for the 2-tDCB dosages were 34%, 45%, 40% and 37%, respectively, and were not statistically significant. These data suggest that 2-tDCB shows no toxic or tumor-modifying effects under the present conditions, and that the no-observed-adverse-effect level for 2-tDCB is 300 ppm in both sexes, equivalent to 15.5 mg/kg b.w./day in males and 16.5 mg/kg b.w./day in females.     

Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism

The polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea‐specific primers, Ar1000F and Ar1500R, or methanogen‐specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without α‐cyclodextrin‐horseradish oil complex (CD‐HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea‐specific and methanogen‐specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD‐HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR‐SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD‐HR to the rumen.     

Vaccination against Fowlpox virus via drinking water

The oral vaccination against Fowlpox was investigated via drinking water containing the F132-c strain of Fowlpox virus to be effective even though the vaccine virus-titer was 10(4) TCID (50)/dose each time. When the virus-titer of the F132-c strain was 10(4-5 )TCID(50)/dose per single drinking water vaccination, 90% or more of chickens were not protected, however, they were protected when vaccinated twice via drinking water. A weak immune response occurred by a slight infection after the first vaccination, and due to memory cells, a booster could work well after the second vaccination. These results suggest the possibility of reducing both the amount of virus required for a vaccine via drinking water and the labor cost in the field.     

Seroepidemiologic studies on Babesia equi and Babesia caballi infections in horses in Jilin province of China

The prevalence of equine piroplasmosis caused by Babesia equi and Babesia caballi in northeast China has remained unknown, although the People's Republic of China is recognized as an endemic country for the diseases. In the present study, we investigated the prevalence of equine piroplasmosis in Jilin province, a part of northeast China. A total of 111 serum samples were taken from horses in eastern Jilin, and examined for diagnosis of B. equi and B. caballi infections by the enzyme-linked immunosorbent assays with recombinant antigens, equi merozoite antigen-1 and P48, respectively. Of the 111 samples, 38 (34%) and 36 (32%) samples were sero-positive for B. equi infection and B. caballi infection, respectively. In addition, 14 (12%) samples were sero-positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in northeast China.     

Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks.     

Gene silencing of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis by RNA interference

RNA interference (RNAi) has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned Haemaphysalis longicornis serine proteinase (HlSP) gene to test the importance of the function of the HlSP gene product during blood-feeding. Growth of unfed ticks treated with HlSP dsRNA was significantly inhibited compared to that of PBS-treated ticks. This inhibition was supported by the level of HlSP mRNA. HlSP may play a crucial role for blood-feeding in these ticks. This is the first report on gene silencing of a functional serine proteinase in hard ticks.