Survey of mycotic mastitis in dairy cows from Heilongjiang Province, China.

A survey of the prevalence rate, pathogenic subspecies, and risk factors of mycotic mastitis in dairy cows from Heilongjiang Province, China, was conducted. Milk samples from 412 cows with chronic mastitis were collected and cultured on 8 % sheep blood agar, MacConkey agar, and Sabouraud agar with chloramphenicol. Counting of the morphologically distinct colonies was performed, as well as the isolation and identification of organisms through phenotypical and physiological criteria. Four hundred seventy-eight aerobic microorganisms were isolated. Yeasts and yeast-like fungi 35.6 % (170/478) and bacteria 64.4 % (308/478) were isolated. The fungal isolates were identified as Candida (79.4 %), Trichosporon (5.9 %), Aspergillus (7.1 %), Cryptococcus (2.4 %), and Rhodotorula (4.1 %). More than ten species of yeast were isolated including Candida krusei 50/135 (37 %), Candida rugosa 16/135 (11.9 %), and Candida lusitaniae 15/135 (11.1 %). A higher positivity (18.5 and 56.3 %) (P ≤0.05) was observed in cows from environmental temperatures of 0-15 and 15-35 °C than those at <0 °C and in cows affected by the disease for >45 and 30-45 days compared with cows suffering 10-30 days. Meanwhile, a statistically significant difference (44.9 vs. 31.4 %) (P ≤0.05) was observed under extensive raising systems vs. intensive raising systems. It appears that Candida is a major pathogen of mycotic mastitis of dairy cows. Extensive raising system, high environmental temperature (15-35 °C), and the duration of the disease (>30 days) were important risk factors of the incidence of mycotic mastitis. Here, we provide a theoretical foundation for research into preventing and treating mycotic mastitis of dairy cows in China.

[Cloning, expression and immunogenicity analysis of cathepsin L-like protease of Fasciola hepatica].

OBJECTIVE: To clone and express the cathepsin L-like protease gene of Fasciola hepatica (FhCL) and investigate the immunogenicity of the recombinant FhCL protein. METHODS: Specific primers were designed according to the reported FhCL gene in GenBank. Using total RNA from adult worms of F. hepatica, FhCL gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and then subcloned into pET30a(+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The expression situation of recombinant FhCL was analyzed by SDS-PAGE. Its immunoresponse to the sera of infected goat and the antisera of SD rats against FhCL was examined by Western blotting analysis. RESULTS: PCR and double enzyme digestion showed that the FhCL gene fragment was about 1,000 bp in length. The constructed recombinant plasmid pET30a (+)-FhCL was identified by sequencing. The recombinant protein (Mr 42,000) was expressed in the form of inclusion body. The protein was recognized respectively by the sera of infected goat and the sera from rat immunized with FhCL. CONCLUSION: The recombinant plasmid pET30a(+)-FhCL has been constructed, which shows high antigenicity.

[Investigation on bovine leukocyte adhesion deficiency].

Bovine leukocyte adhesion deficiency (BLAD) is autosomal recessive disease. The pathogeny of BLAD is genic mutation of CD18-integrins on the leukocyte. In order to know the carrier and occurrence of bovine leukocyte adhesion deficiency (BLAD) among cows age from one to six years old in China, 1,000 cows were investigated by means of amplifying a CD18 gene fragment via reverse transcriptase-PCR followed by restriction digestion with Taq I. Results showed that 19 cows were BLAD carriers, indicating that the BLAD carrier rate was 1.9 percent. In addition, one cow was found to have BLAD.

[Prokaryotic expression of HN gene of bovine parainfluenza virus type 3 and the establishment of indirect ELISA method].

The prokaryotic expression plasmid pQE30-HN of hemagglutinin-neuraminidase (HN) protein gene of bovine parainfluenza virus type 3 (BPIV3) strain HJ-1 was expressed by IPTG induction in E. coli XL1Blue. The recombinant HN protein(rHN) was purified by electroeluting method, and used as coated antigen. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody valence of BPIV3. The best working conditions of ELISA were as follows: the antigen concentration was 6 microg/mL; the serum dilution was 1:50; the blocking reagent was 5% skimmed milk; the blocking time was 60 min at 37 degrees C; the second antibody concentration was 1:10 000; The cut-off value was 0.30. The method revealed a good specificity, no cross-reaction to the positive sera of BCV, IBRV or BRSV was observed. We applied the method to detect 323 serum samples of dairy cow in Heilongjiang Province, the seropositivity rate of BPIV3 was about 58%. The indirect ELISA established provided a technological basis for the development of ELISA kit.

[Cloning and expression of Staphylococcus aureus surface protein Isdb and its immune experiment in mice].

In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface Isdb, we amplified Isdb gene from S. aureus Wood46 strain. The isdb gene was subsequently inserted into pET32a(+) vector and the recombinant plasmid was transformed into E. coli strain BL21. The recombinant Isdb was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level was measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus strains Wood46 and HLJ23-1. These results showed that isdb gene sequences were highly conserved, and the recombinant Isdb was successfully expressed. The antibody titer in the immunized groups was increased significantly (P < 0.05) compared with the control, the protective rate of Isdb protein inducted by challenge with the two S. aureus stains Wood46 and HLJ23-1 was 62.5% and 75%, respectively. These results showed that the Isdb protein had high immunogenicity and immunoprotective capacity.

[GAPDH activity and immunogenicity of Staphylococcus aureus recombinant GapC protein].

In order to characterize the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein GapC, gapC gene of S. aureus was amplified from strain BMSA/855/23-1 by PCR, and was inserted into pQE-30 vector subsequently. The recombinant plasmid, designated as pQE/gapC, was transformed into E. coli strain M15 (pREP4). The recombinant GapC fusion proein was successfully expressed in E. coli M15 induced with IPTG and its GAPDH activity was confirmed by GAPDH activity assay. Then, the recombinant GapC protein, inactivated S. aureus whole cell and placebo (PBS) were administrated to healthy rabbits respectively. The IgG antibody titers, concentration of IFN-gamma and IL-4 cytokines in immunized rabbit sera were measured with Enzyme-Linked Immunosorbnent Assay (ELISA). Finally, immunized rabbits were challenged with S. aureus strain Wood46 to evaluate the immunoprotection. The IgG antibody titers against GapC and whole cell in rabbit sera reached their peaks at day 28 after boost immunization (1:64,000). The concentration of IL-4 and IFN-gamma in GapC groups rabbit sera increased significantly (P<0.05) at day 14 after boost immunization, while the concentration of those in whole cell group did not increase (P>0.05) compared with the placebo group. 4 rabbits in 5 of the protein immunized group were protected against challenge with 1 x 10(8) CFU S. aureus. The results above indicate that the expressed recombinant GapC protein have high GAPDH activity and immunogenicity, can also protect against S. aureus challenge to some extent. S. aureus GapC protein could be an attractive target for further genetic engineering vaccine.