Construction of a recombinant vaccinia virus expressing Babesia gibsoni thrombospondin-related anonymous protein and evaluation of its immunogenicity in mice.

Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band with a molecular mass of 80 kDa, which is similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/ BgTRAP-infected RK13 cells. Mice inoculated with VV/BgTRAP produced a specific antiBgTRAP response. The antiserum against VV/BgTRAP showed reactivity against the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.

Seroprevalence of Toxoplasma gondii infection in sheep from northern China.

Toxoplasma gondii is an important zoonotic parasite causing significant health problems to humans and animals. In recent years, a number of investigations about the seroprevalence of T. gondii in China have been reported, but little is known on the prevalence of toxoplasmosis in sheep in northern China. In the present study, a total of 288 sheep serum samples were collected from Inner Mongolia, Heilongjiang, Jilin and Hebei provinces of northern China for T. gondii antibody survey using a latex agglutination test (LAT). Of these, 87 (30.2%) serum samples were positive for antibodies to T. gondii, and the antibody titres ranged from 1:64 to 1:1,024. Seroprevalence of T. gondii infection in sheep was 17.1% in Inner Mongolia, 33.8% in Heilongjiang, 24.6% in Jilin and 46.3% in Hebei. Age and rearing system significantly affected seropositivity. The present survey indicates antibodies to T. gondii are widely prevalent in sheep in northern China, which may cause public health problems in these provinces.

Molecular Detection of Theileria species in Cattle from Jilin Province, China.

Bovine theileriosis is a tick-borne disease that is hampering the development of the domestic cattle industry in northern China. This study involved a molecular survey of bovine Theileria species in 137 blood samples from cattle in the Jilin province of China. The DNA samples were screened by species-specific 18S rRNA PCR. Results revealed that 19.7% (27/137), 17.5% (24/137) and 10.9% (15/137) were found to be infected with Theileria sinensis, Theileria orientalis, respectively. Mixed infection was found in 8.8% (12/137). The overall detection rates of Baishan, Yanji, Jilin and Liaoyuan districts was 60.0%, 17.5%, 5.3% and 0%, respectively. There is little information on the detection and distribution of bovine Theileria species in northern China. Therefore, this study provides important data for understanding the epidemiology of Theileria species and designing appropriate approaches for the diagnosis and control of bovine theileriosis in northern China.

First evidence of Mycoplasma haemocanis in China.

Previous studies show heamotropic Mycoplasma infection frequently occurs among splenectomized, immuno-suppressive or co-infected dog populations. However, in our study, the detection of 162 blood samples from dogs found 3 healthy, female dogs infected with Mycoplasma haemocanis in southeastern China. These infected dogs were grown in dog breeding center and had a history of tick infestation. This is the first molecular report of M. haemocanis in dogs from China. The 16S rRNA gene was partially sequenced and a phylogenetic tree constructed. Mycoplasma spp. was 99.9%-100% identical to the corresponding gene sequences of M. haemocanis and M. haemofelis available in GenBank. In this study, Mycoplasma spp. was identified as M. haemocanis because the bacterium was obtained from dogs.

Molecular detection and genotyping of Anaplasma spp. and Theileria spp. infections in sheep and cattle from the northeast region of China.

Anaplasmosis and theileriosis are significant tick-borne diseases threatening the livestock industry worldwide. In the present study, we screened 127 cattle and 115 sheep blood DNA samples from northeastern China for Theileria and Anaplasma pathogens by polymerase chain reaction (PCR) using species-specific primers. The result showed that only Theileria orientalis and Anaplasma ovis were detected, with a prevalence of 2.9% for T. orientalis in cattle and 57.4% for A. ovis in sheep. Fragments of Anaplasma ovis major surface protein 4 (AoMSP4) and Theileria orientalis major piroplasm surface protein (ToMPSP) genes were sequenced for phylogenetic analysis. Sequence analysis showed that the AoMSP4 gene was conserved, with 100% sequence identity value among sheep samples. However, the ToMPSP gene was relatively diverse, with sequence identity ranging from 87.6%-99l.0% among cattle samples. Phylogenetic analysis showed that the ToMPSP gene sequences isolated from 4 cattle samples were classified into type 1, type 2 and type 7, while the AoMSP4 gene sequences obtained from 66 sheep were classified into genotype I, according to the neighbour-joining distance method. This study provides important data for understanding the epidemiology of tick-borne diseases and genetic diversity of these pathogens in the northeast region of China.

Induction of antibodies to canine herpesvirus in mice by immunization with anti-idiotypic antibodies.

Anti-idiotypic antibodies (anti-Id Abs) were produced in rabbits after inoculation with two mouse monoclonal antibodies (mAbs) directed against canine herpesvirus (CHV) glycoproteins (gps). One of the mAbs, 12H11, was directed against an epitope on gp 145/112 of CHV which induced virus neutralizing (VN) antibodies and against a cross-reacting epitope on the gp 143/108 of feline herpes-virus type 1 (FHV-1). The other mAb, 11F7, was directed against epitopes on CHV gp47 which induce VN and hemagglutination-inhibition (HAI) antibodies. Using VN-inhibition and HAI-inhibition assays with CHV and FHV-1, the anti-Id Abs obviously inhibited the activities of autologous mAbs, suggesting that anti-Id Abs mimic the epitopes of CHV gp 145/112 or FHV-1 gp 143/108 and CHV gp47 by binding the anti-combining site of the mAbs. These anti-Id Abs, when injected into mice, elicited specific CHV-neutralizing and HAI antibody responses, and one of them also elicited a specific FHV-1-neutralizing antibody response. These data supported the idea that immunization with anti-Id Ab can induce specific VN antibody response, as has been theorized by other workers.

Homologous and heterologous antibody responses of mice immunized with purified feline herpesvirus type 1 and canine herpesvirus glycoproteins.

The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.

Restriction endonuclease analysis of canine herpesviruses isolated in Japan.

Eighteen canine herpesvirus (CHV) isolates from Japan and two reference strains were compared by restriction endonuclease analysis technique using total DNA extracts from cells infected with the viruses. In order to select the suitable restriction endonucleases for differentiation of CHV isolates, ten enzymes were used and three of them, HindIII, XbaI, and PvuII, were found to be useful for strain differentiation. With these enzymes, CHV isolates from unrelated individuals were readily differentiated from each other. In contrast, all the isolates derived from the same litter were not distinguishable on the basis of restriction cleavage patterns. However, slight mobility shifts were observed among the isolates from the same litter or the same individual. The results showed that this method provides a powerful tool for epidemiological surveys of CHV infection.

Canine herpesvirus 47 kD and mutated 41 kD glycoproteins are responsible for hemagglutination.

Monoclonal antibodies (MoAbs) were used to identify the hemagglutinin of canine herpesvirus (CHV). The inhibition of viral hemagglutination (HA) activity was observed with MoAbs against 41 kD glycoprotein, while no hemagglutination-inhibition (HI) activity was observed with those against 145/112 kD and 80 kD glycoproteins, suggesting that the 41 kD glycoprotein is the hemagglutinin of plaque-selected virus of CHV YP11 strain used as immunogen for MoAb production. All of the HI MoAbs also showed HI activities against HA antigens which were prepared from cells infected with other CHV strains, namely, F-205 V and Glasgow CHV2 reference strains, eight Japanese isolates, and the original YP11 strain. However, on immunoblotting analysis, a 47 kD protein band was detected in these strains by the HI MoAbs. These data suggest that the 47 kD glycoprotein is the common molecule of the hemagglutinin among CHV strains and the plaque-selected virus of YP11 strain appears to be a mutant whose molecular weight of the hemagglutinin changed into 41 kD.

Serodiagnosis of canine herpesvirus infection--development of an enzyme-linked immunosorbent assay and its comparison with two improved methods of serum neutralization test.

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.