Quantitative capillary reversed passive latex agglutination test for C-reactive protein (CRP) in the dog

A capillary reversed passive latex agglutination test (capillary RPLA) was developed which allows quantification of serum C-reactive protein (CRP) within approximately 15 min. The logarithmic regression line (calibration curve) obtained after measuring each CRP concentration three times in twofold dilutions of a standard canine serum containing 222 g/ml of CRP was y=6.394+0.030x (r=0.995). Capillary RPLA permitted quantification of CRP in the range 6.9–222 g/ml. The coefficients of variation ranged from 10.28% to 12.40%. The recovery rates (percentage recovery) of CRP by capillary RPLA were within the range 87% to 106%. On measuring the CRP concentrations in sera from 78 dogs by capillary RPLA, single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA), close correlations were demonstrated between SRID and capillary RPLA (y=7.250+1.109x, r=0.978), between SRID and ELISA (y=3.042+1.059x, r=0.967), and between capillary RPLA and ELISA (y=1.778+0.929x, r=0.962). Capillary RPLA may be considered useful as a routine biochemical technique for measurement of serum CRP concentration in the dog.Abbreviations CRP C-reactive protein - ELISA enzyme-linked immunosorbent assay - RPLA reversed passive latex agglutination test - SRID single radial immunodiffusion     

Ultrastructural Localization of Hydrogen Peroxide in Host Leaves Treated with AK-toxin I Produced by Alternaria alternata Japanese Pear Pathotype

The rapid generation of reactive oxygen species (ROS), called the oxidative burst, is one of the earliest host responses to pathogen infection or elicitor treatments. Therefore, we looked for the induction of ROS generation in Japanese pear leaves by the host-specific toxin, AK-toxin I using a cytochemical method for detecting H₂O₂. A small amount of non-specific generation of H₂O₂ was found in the cell walls in toxin- and water-treated susceptible and resistant leaves. Thus, the generation of H₂O₂ at cell walls appears to be caused by wounding stress during sampling. Specific generation of ROS, however, was found only in the membrane fragments and extended desmotubules characteristic of modified sites of the plasma membrane in the toxin-treated susceptible leaves. In addition, generation of H₂O₂ at plasma membranes was observed with higher frequency in toxin-treated susceptible leaves. This result indicates that the H₂O₂ generation was associated with damaged sites in the plasmalemma after toxin treatment and perhaps with the formation of membrane fragments from altered portions of the invaginated plasma membrane. Received 21 September 2001/ Accepted in revised form 25 October 2001     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002     

Evaluation of truncated LipL32 expressed by Escherichia coli and Pichia pastoris for serodiagnosis of Leptospira infection in rodents

The applicability of the recombinant LipL32 for serodiagnosis of leptospiral infection in field rodents was assessed in this study. An immunodominant region of LipL32 was determined by monoclonal antibodies, and then, truncated LipL32 (tLipL32) was designed to contain the region (87–188th amino acid). The tLipL32 was compared between two recombinant expression hosts Escherichia coli and Pichia pastoris in ELISA. With field rat sera, tLipL32 expressed by P. pastoris (tLipL32p) had high antigenicity without background reactions, while tLipL32 expressed by E. coli (tLipL32e) showed high background reactions, which were reduced by pre-adsorption of sera with E. coli. To evaluate tLipL32-ELISA, field rat sera were tentatively divided into a Leptospira infection positive (12 sera) and a negative group (12 sera) based on the results from flaB gene PCR of kidney samples and WB with whole Leptospira cell. Consequently, the sensitivity of tLipL32p-ELISA for field rat sera was 83% . A similar result was obtained from tLipL32e-ELISA with adsorbed sera, (92%). However, sensitivity of tLipL32e-ELISA using sera without an adsorption treatment was 50%. Regardless of the expression host, tLipL32-ELISA had 100% specificity and sensitivity in experimentally infected laboratory rats. These results suggest that recombinant LipL32 expressed by P. pastoris is more applicable for serodiagnosis in field rats due to a lack of background reaction.     

Involvement of a host erythrocyte sialic acid content in Babesia bovis infection

Host sialic acid (SA) has recently been suggested to play an important role in erythrocyte (RBC) infection by Babesia spp. The present study attempted to further determine the specific type of SAs important in the RBC invasion. Bovine RBC was found to bear abundant alpha2-3-linked SA residues but not alpha2-6-linked SA in nature, confirmed by flow cytometric analysis of the neuraminidase (Nm)-treated RBCs. Lectin-blot analyses revealed the removal of alpha2-3-linked SAs from the 97-, 33-, and 31-kDa bands by the Nm treatment. Addition of the Nm-treated RBCs into an in vitro culture of B. bovis resulted in a decreased population of the parasitized RBCs. The thin smear samples from the cultures were then observed under a confocal laser scanning microscope after staining with the alpha2-3-linked SA-specific lectin: a selective invasion of B. bovis was found only in the intact RBCs bearing the SAs, but not in the desialylated RBCs. Furthermore, a significant reduction of the parasitized RBCs was also observed in the culture supplemented with exogenous 3'-sialyllactose containing the alpha2-3-linked SAs. However, the complete inhibition of parasite proliferation was not achieved in the culture. These findings indicate that while the alpha2-3-linked SA-dependent pathway is needed for highly efficient invasion of host RBCs by B. bovis, there might also be other potential alternative pathways.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Comparison of antibody titers in rabbits following immunization with inactivated influenza virus via subarachnoidal or subcutaneous route

Rabbits were immunized with inactivated influenza virus via the subarachnoidal (SA) or subcutaneous (SC) route, and the antibody titers in cerebrospinal fluid (CSF) and serum were assayed. There were no nervous signs or morphological lesions related to SA immunization. In the SC group, the antibody titer was elevated in serum, but not elevated in CSF. In the SA group, the antibody titer was significantly elevated in serum and even in CSF, and their antibody titers were much greater than in the SC group. The present results suggest that intrathecal immunization is more effective than SC immunization at inducing a protective immune response against the transneural spread of viruses.     

Expression of HSP70 in response to heat-shock and its cDNA cloning from Mediterranean blue mussel

Expression of HSP70 in response to heat-shock was investigated at the protein and mRNA levels in Mediterranean blue mussel. Western and Northern blot analyses revealed that HSP70 was expressed following heat-shock in the mantle at both protein and mRNA levels, suggesting that gene expression of HSP70 is implicated in the cellular response to heat-shock stress in mussel. It was then attempted to clone HSP70 cDNA in order to determine the primary structure of mussel HSP70. As a result, two full-length cDNA encoding HSP70 were isolated from a cDNA library prepared from the heat-shocked mantle. The isolated cDNA consist of single open reading frames of 2067 bp and 1911 bp which encode proteins of 689 amino acids and 637 amino acids, respectively. Both HSP70 cDNA encode an ATPase do main, and a substrate-binding do main in addition to a Glu-Glu-Val-Asp (EEVD) peptide motif that is specific for cytosolic HSP70. These findings suggest that the cDNA clones obtained in the present study encode cytosolic HSP70.     

Description and phylogeny of Ceratomyxa anko sp. n. and Zschokkella lophii sp. n. from the Japanese anglerfish, Lophius litulon (Jordan)

Two new species of myxozoans from the Japanese anglerfish, Lophius litulon, are described using myxospore morphology and small subunit rDNA sequences. Ceratomyxa anko sp. n. is a parasite of the gall bladder and had a prevalence of 57%. Mature spores of C. anko sp. n. are arcuate to crescent shaped with valves tapering to rounded tips. A prominent sutural line runs centrally between the round adjacent polar capsules containing the polar filament coiled two to three times. Spore measurements: length 10.8 (9.7-11.9) microm, width 41.9 (36.9-47.2) microm, polar capsule diameter 4.6 (4.1-5.3) microm. Ceratomyxa anko sp. n. can be distinguished from other Ceratomyxa spp. due to its spore dimensions and shape. Zschokkella lophii sp. n. is a parasite of the urinary bladder and had a prevalence of 70%. Mature spores are ellipsoidal to semicircular with bluntly pointed ends. The sutural line is curved or sinuous and the valves have no discernable surface ornamentation. Two almost spherical polar capsules are located separately in the ends of the spore, opening in almost opposite directions and contain the polar filament with five coils. Spore measurements: length 20.1 (16.8-24.0) microm, width 14.9 (12.7-16.8) microm, polar capsule diameter 5.1 (3.6-5.8) microm. Zschokkella lophii sp. n. can be distinguished from other Zschokkella spp. due to the terminal opening of the polar capsules within the spores and the site of infection within the host fish. In the phylogenetic analyses, C. anko sp. n. grouped with other members of the same genus forming a monophyly. Zschokkella lophii sp. n. forms a discrete clade with another Zschokkella sp. that infects the urinary bladder of marine fish. This grouping forms a sister clade to one containing members of the genus Parvicapsula, all of which are parasites of the urinary system in marine fish.