Production of recombinant extracellular domains of canine desmoglein 1 (Dsg1) by baculovirus expression

The aim of this study was to generate a recombinant protein to represent the entire extracellular domain of canine desmoglein 1 (Dsg1), a desmosomal cell-cell adhesion molecule, by the baculovirus expression system. Cotransfection of a baculovirus transfer vector containing the cDNA for the entire extracellular domain of canine Dsg1 with baculovirus DNA into insect cells resulted in the secretion of soluble canine Dsg1 into insect culture supernatants. Immunoreactivity of 11 human pemphigus foliaceus (PF) sera against the cell surface of canine keratinocytes was completely removed when the sera were preincubated with the canine Dsg1 baculoprotein. This recombinant canine Dsg1 produced by baculovirus shares the major epitopes of the authentic canine Dsg1 recognized by human PF sera, and will be useful in studying the molecular pathophysiological mechanisms in PF and impetigo in canine patients.     

Utility of the Cryptosporidium oocyst wall protein (COWP) gene in a nested PCR approach for detection infection in cattle

A preliminary molecular epidemiological study was carried out to investigate the utility of the Cryptosporidium oocyst wall protein (COWP) gene in the detection of Cryptosporidium oocysts in fecal samples. A nested polymerase chain reaction (PCR) approach using COWP gene primers was adopted for this purpose. Fecal samples were spiked with each of 1, 10, and 100 oocysts of C. parvum, four samples for each number, and the DNA was extracted from each sample using a glassbead method. The presence of oocysts was determined using the nested PCR with COWP gene primers, and the limit of detection of oocysts by the PCR was determined. The limit of detection was 100 oocysts spiked in 1 ml of fecal material (50% sold material) (four positives/four samples tested). Seventy-five percent of DNA extracted samples spiked with 1 and 10 oocysts was positive by the PCR (three positives/four samples tested). Based on this, small sample size using the COWP gene primers with a nested PCR analysis could reliably identify infected animals rather conveniently and accurately.     

Vaccine development against Neospora caninum infection

Neospora caninum is a recognized protozoan parasite of a wide range of mammalian hosts, and was reported for the first time in 1988. The isolation of its oocysts in dog's faeces in 1998 led to its establishment as a parasitic species undergoing typical coccidian life cycle. Infection with N. caninum causes paralysis and death in young livestock and companion animals, and is associated with abortions and stillbirth in cattle, and neurologic disease in calves. Considering the economic and agricultural importance of neosporosis, there is the urgent need to develop biological control measures aimed at preventing its transmission, infection, as well as reducing severity of the disease. In this paper, we have reviewed the progress made to date on the parasite-host immunology and on vaccine development including its prospects, and discussed possible strategies in the formulation of vaccine(s) against neosporosis.     

Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni

The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.     

Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells

We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2.     

Isolation and characterization of Shiga toxin-producing Escherichia coli O157:H7 from beef,pork and cattle fecal samples in Changchun,China

Meat samples and fecal specimens from adult cattle were collected in Changchun, China and were examined for presence of Shiga toxin-producing Escherichia coli (STEC) serogroup O157. STEC O157 strains were isolated from 2 (5%) of 40 beef, 1 (3.3%) of 30 pork, and 3 (1.7%) of 176 adult cattle fecal samples. The strains belonged to phage types (PT) 4, 8, or 47. Two beef strains and a strain previously isolated from a patient in Shandong, China, were PT-4 and showed a similar PFGE pattern, suggesting the possibility of food-borne transmission. It is suggested that cattle are a reservoir of STEC O157:H7 and meat products are contaminated by this pathogen in Changchun, China as well as in other countries.     

Comparison of changes in urinary and blood levels of biomarkers associated with proximal tubular injury in rat models

To investigate useful biomarkers associated with proximal tubular injury, we assessed changes in levels of a focused set of biomarkers in urine and blood. Male rats administered a single dose or four doses of gentamicin (GM, 240 mg/kg/day) or a single dose of cisplatin (CDDP, 5 mg/kg) were euthanized on days 2 (the day after initial dosing) 5, or 12. At each time point, histopathological examination of the kidney and immunohistochemistry for biomarkers, kidney injury molecule-1 (Kim-1), lipocalin (NGAL), clusterin (CLU), cystatin C (CysC) and β2-microglobulin (β2M) were performed. Biomarker levels were measured in urine and blood. In both treatment groups, degenerated/necrotic proximal tubules and regenerated tubules were mainly observed on days 5 and 12, respectively. At the same time as these tubular injuries, urinary Kim-1, CysC and β2M levels were increased. Moreover, urinary levels of CysC and β2M in GM-treated animals and Kim-1 in CDDP-treated animals increased (on day 2) prior to tubular injury on day 5. This was considered to reflect the characteristics of drug toxicity. Although almost all of the biomarkers in blood were not sufficiently sensitive to detect proximal tubular injury, urinary and plasma β2M levels simultaneously increased. Therefore, in addition to urinary Kim-1, CysC and β2M levels, plasma β2M levels were also considered useful for detecting proximal tubular injury.     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.     

Controlling highly prevalent Staphylococcus aureus mastitis from the dairy farm

In 57 Holstein cows where the dairy farm uses a milking parlor system, the somatic cell count (SCC) increased persistently in the bulk milk (monthly mean 52.3 x 10(4) cells/ml; range 21 to 94 x 10(4) cells/ml). We detected S. aureus in 24 (41.2%) of the 54 lactating cows and in 29 (12.8%) of 227 quarters of the 57 milking cows in the herd. A control program was implemented in an effort to eradicate S. aureus mastitis from this dairy farm. The control plan established improved handling of the lactating cows, improved milking procedures, dry-cow therapy, and culling of infected cows. The program was monitored for 3.5 years by frequent checkups on the rate of S. aureus infection, the SCC, and the changes in milk composition. Eighteen months after the control program was started, the rate of S. aureus infection in the quarter milk decreased dramatically, and no S. aureus isolates were found in the milk of the remaining cows. The SCC in the bulk milk of the herd dropped to a monthly mean of <20 x 10(4) cells/ml. In conclusion, the control program was effective for controlling persistent S. aureus mastitis in this dairy herd.