Search

Bovine anaplasmosis caused by Anaplasma marginale represents a serious threat to cattle farming worldwide, especially in the tropics and subtropics. In the present study, archived DNA samples from the blood of cattle (n=437) in the Nuwara Eliya, Galle, Ampara, Polonnaruwa, and Jaffna districts and buffalo (n=327) in the Galle, Polonnaruwa, Mannar, and Mullaitivu districts in Sri Lanka, were screened for A. marginale using a major surface protein 5 (msp5) gene-based PCR assay. The findings showed that 32.7 and 57.5% of cattle and buffalo, respectively, were A. marginale-positive. The rate of positivity differed significantly among geographical regions. In conclusion, the high rates of A. marginale infection in cattle and buffalo highlight the importance of effective control measures in Sri Lanka.

Serum and DNA from blood samples collected from Vietnamese yellow cattle (n=101) and cattle imported from Thailand (n=54) at a Vietnamese slaughter house were screened for Babesia bovis and Babesia bigemina infections by enzyme-linked immunosorbent assay (ELISA) and PCR. The positive rates determined by ELISA (B. bovis and B. bigemina) or PCR (B. bigemina) in the Vietnamese cattle were significantly higher than those found in Thai cattle. Some PCR-positive Vietnamese animals were ELISA-negative, whereas all PCR-positive Thai cattle were ELISA-positive, suggesting that the animals were infected in Thailand. Importing Babesia-infected cattle may lead to the introduction of new parasite strains, possibly compromising the development of anti-Babesia immune control strategies in Vietnam.

A PCR-based survey of hemoprotozoa parasites detected Babesia bigemina, Theileria orientalis and Trypanosoma theileri among cattle and water buffalo in Vietnam, and a new Babesia sp. closely related to Babesia ovata was detected in cattle only. In addition, Theileria annulata and Trypanosoma evansi were not detected in both cattle and water buffalo. Phylogenetic analysis detected T. orientalis MPSP genotypes 3, 5, 7 and N3 in cattle and 5, 7, N1 and N2 in water buffalo. Additionally, water buffalo-derived T. theileri CATL sequences clustered together with a previously reported cattle-derived sequence from Vietnam. This is the first report of a new Babesia sp. in cattle, and T. orientalis MPSP genotype 7 and T. theileri in water buffalo in Vietnam.

We examined the localization of connexin 32 (Cx32), a component of gap junctions, in 24-month-old male B6C3F1 mice with spontaneously occurring hepatocellular altered foci or tumors. Immunohistochemically, Cx32-staining intensity in cell-to-cell membranes of altered hepatocytes was decreased in eosinophilic foci and increased in basophilic foci as compared to those in intact hepatocytes. These alterations were enhanced in adenomas and carcinomas with both eosinophilic and basophilic cytoplasm. In cell membranes facing on the sinusoidal portions, the intensities increased in all lesions. Image analyses confirmed that the spot areas of Cx32 were decreased in eosinophilic foci, but increased in basophilic foci, adenomas and carcinomas. These results demonstrate that Cx32 shows different expression in different types of hepatic lesions.

The presence of anti-erythrocyte autoantibodies in animals infected with various Babesia species is well reported. However, the pathogenesis of autoantibodies in babesiosis is poorly understood. Here, we demonstrated that anti-erythrocyte immunoglobulin (Ig) M and IgG were present in B. rodhaini-infected mice at 6 and 8 days after infection, respectively. Furthermore, we generated monoclonal antibodies against erythrocyte antigen from B. rodhaini-infected mice. Five clones were generated. By Western blotting analysis using whole erythrocyte antigens, one clone reacted with a broad band around 90-150 kDa, and the 2 clones reacted with a band larger than 150 kDa. B. rodhaini-infected mice and/or autoreactive monoclonal antibodies established in this study might be a powerful tool for in vivo pathogenesis studies of autoantibody development in infectious diseases.

Trypanosoma congolense is a major livestock pathogen in Africa, causing large economic losses with serious effects on animal health. Reliable serodiagnostic tests are therefore urgently needed to control T. congolense infection. In this study, we have identified one T. congolense protein as a new candidate serodiagnostic antigen. The 46.4 kDa protein (TcP46, Gene ID: TcIL3000.0.25950) is expressed 5.36 times higher in metacyclic forms than epimastigote forms. The complete nucleotide sequences of TcP46 contained an open reading frame of 1,218 bp. Southern blot analysis indicated that at least two copies of the TcP46 gene were tandemly-arranged in the T. congolense genome. The recombinant TcP46 (rTcP46) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Western blot analysis and confocal laser scanning microscopy revealed that the native TcP46 protein is expressed in the cytoplasm during all life-cycle stages of the parasite. Moreover, an enzyme-linked immunosorbent assay (ELISA) based on rTcP46 detected the specific antibodies as early as 8 days post-infection from mice experimentally infected with T. congolense. No cross-reactivity was observed in the rTcP46-based ELISA against serum samples from cattle experimentally infected with Babesia bigemina, B. bovis and Anaplasma marginale. These results suggest that rTcP46 could be used as a serodiagnostic antigen for T. congolense infection.

The objective of this study is to investigate the seroprevalence of equine piroplasmosis in China. A total of 1990 sera were collected from clinically healthy horses in various districts located in ten different provinces of China and examined by using indirect enzyme-linked immunosorbent assays (ELISAs) with recombinant Theileria equi (T. equi) merozoite antigen 2 (rEMA-2) and Babesia caballi (B. caballi) 48-kDa rhoptry protein (rBc48), respectively. The results showed that 1,018 (51.16%) and 229 (11.51%) samples were positive for B. caballi and T. equi infection, respectively. The number of samples with mixed infection was 152 (7.64%). These results indicated that equine piroplasmosis was widespread in China.

The emergence of drug resistance and adverse side effects of current bovine babesiosis treatment suggest that the search of new drug targets and development of safer and effective compounds are required. This study focuses on dihydroorotate dehydrogenase (DHODH), the fourth enzyme of pyrimidine biosynthesis pathway as a potential drug target for bovine babesiosis. Recombinant Babesia bovis DHODH protein (rBboDHODH) was produced in Escherichia coli and used for characterization and measurement of enzymatic activity. Furthermore, the effects of DHODH inhibitors were evaluated in vitro. The recombinant B. bovis DHODH histidine fusion protein (rBboDHODH) had 42.4-kDa molecular weight and exhibited a specific activity of 475.7 ± 245 Unit/mg, a Km = 276.2 µM for L-dihydroorotate and a Km= 94.41 µM for decylubiquinone. A 44-kDa band of native BboDHODH was detected by Western blot analysis and found in parasites mitochondria using a confocal microscope. Among DHODH inhibitors, atovaquone (ATV) and leflunomide (LFN) significantly inhibited the activity of rBboDHODH as well as the growth of B. bovis in vitro. The half maximal inhibitory concentration (IC50) of ATV and LFN was 2.38 ± 0.53 nM and 52.41 ± 11.47 µM, respectively. These results suggest that BboDHODH might be a novel target for development of new drug for treatment of B. bovis infection.

Hemoprotozoan infections often cause serious production losses in livestock. In the present study, we conducted a PCR-based survey of Babesia bovis, Babesia bigemina, Theileria annulata, Theileria orientalis, Trypanosoma evansi and Trypanosoma theileri, using 423 DNA samples extracted from blood samples of cattle (n=202), water buffaloes (n=43), sheep (n=51) and goats (n=127) bred in the Hue and Hanoi provinces of Vietnam. With the exception of T. annulata and T. evansi, all other parasite species (B. bovis, B. bigemina, T. orientalis and T. theileri) were detected in the cattle populations with B. bovis being the most common among them. Additionally, four water buffaloes and a single goat were infected with B. bovis and B. bigemina, respectively. The Hue province had more hemoprotozoan-positive animals than those from the Hanoi region. In the phylogenetic analyses, B. bovis-MSA-2b, B. bigemina-AMA-1 and T. theileri-CATL gene sequences were dispersed across four, one and three different clades in the respective phylograms. This is the first study in which the presence of Babesia, Theileria and Trypanosoma parasites was simultaneously investigated by PCR in Vietnam. The findings suggest that hemoprotozoan parasites, some of which are genetically diverse, continue to be a threat to the livestock industry in this country.

In the present study, we screened blood DNA samples obtained from cattle bred in Brazil (n=164) and Ghana (n=80) for Babesia bovis using a diagnostic PCR assay and found prevalences of 14.6% and 46.3%, respectively. Subsequently, the genetic diversity of B. bovis in Thailand, Brazil and Ghana was analyzed, based on the DNA sequence of merozoite surface antigen-1 (MSA-1). In Thailand, MSA-1 sequences were relatively conserved and found in a single clade of the phylogram, while Brazilian MSA-1 sequences showed high genetic diversity and were dispersed across three different clades. In contrast, the sequences from Ghanaian samples were detected in two different clades, one of which contained only a single Ghanaian sequence. The identities among the MSA-1 sequences from Thailand, Brazil and Ghana were 99.0-100%, 57.5-99.4% and 60.3-100%, respectively, while the similarities among the deduced MSA-1 amino acid sequences within the respective countries were 98.4-100%, 59.4-99.7% and 58.7-100%, respectively. These observations suggested that the genetic diversity of B. bovis based on MSA-1 sequences was higher in Brazil and Ghana than in Thailand. The current data highlight the importance of conducting extensive studies on the genetic diversity of B. bovis before designing immune control strategies in each surveyed country.

Babesia ovata is a tick-transmitted hemoprotozoan parasite that infects cattle. In our study, bovine blood samples (n=2,034) were collected from 10 different countries (Brazil, China, Ghana, Japan, Mongolia, the Philippines, South Africa, Sri Lanka, Thailand and Vietnam) and DNA extracted. The DNA samples were screened using an established and specific polymerase chain reaction (PCR) assay targeting the Apical membrane antigen 1 (AMA-1) gene. Parasite DNA was detected among samples collected from Japan, Mongolia and Thailand. Sequence analyses confirmed that the PCR assay detected only B. ovata AMA-1, and that amplicons from different geographical locations were conserved. Our findings highlight the importance of designing adequate strategies to control B. ovata infection in Japan, Mongolia, and Thailand.

Connexin 32 (Cx32) is a major gap junction protein in the liver. Neoplastic and non-neoplastic lesions were examined in Cx32-deficient (Cx32KO) mice maintained for 24-month, and compared with those in wild-type mice as a corresponding control. In neoplastic lesions, hepatocellular carcinoma increased significantly only in male Cx32KO mice, suggesting that Cx32 deficiency may be related to their pathogenesis. For females, the incidence of pituitary adenoma in the pars distalis of Cx32KO mice was lower than that of wild-type mice. No non-neoplastic lesions related to Cx32-deficiency were observed in the Cx32KO mice. In conclusion, these results demonstrate that the incidence of hepatocellular carcinoma increases only in male Cx32KO mice, presumably due to enhanced tumor promotion and progression signals associated with Cx32 deficiency.

To investigate effects of Babesia infection on drug metabolism, we intraperitoneally inoculated B. microti into ICR mice and measured the expression and activity of hepatic cytochrome P450 (CYP) 3A, a major drug-metabolizing enzyme. Twelve days after infection, CYP3A11 mRNA, CYP3A protein and activity and mRNAs of nuclear receptors, which participate in CYP3A expression, were significantly reduced. These results suggest that B. microti infection suppresses CYP3A-dependent drug metabolism. Additionally, tumor necrosis factor (TNF)-α and nitric oxide synthase (NOS) 2 mRNAs were induced in the infected mouse liver. Since TNF-α is one of the potent mediators that induce NOS2 and repress CYP3A transcription, the possible involvement of TNF-α in this downregulation of CYP3A was discussed.

Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).

Theileria orientalis is a benign protozoan species that is widely distributed in Japan, yet sometimes causes serious economic losses in the livestock industry. In this study, we conducted a molecular survey based on genes encoding the major piroplasm surface protein (MPSP) and p23 for T. orientalis detected in cattle grazing in southern areas of Japan, consisting of 2 farms in Kumamoto prefecture (Aso and Kuma districts) and 3 farms in Okinawa prefecture (Ishigaki, Iriomote, and Yonaguni Islands). High prevalence rates of T. orientalis infection were shown in all the cattle populations using the diagnostic MPSP- and p23-PCR assays. Phylogenetic analyses revealed 4 MPSP genotypes and 3 p23 genotypes. Furthermore, MPSP genotype-specific PCR methods were developed in this study and wide distributions of 5-district genotypes of T. orientalis were observed for the examined farms. Our results indicate that at least 5 types of T. orientalis exist in Kumamoto and Okinawa prefectures of Japan and that genotype-specific PCR assays are highly applicable for the quarantine of transported cattle and for epidemiological surveys of bovine theileriosis in Japan.

Theileria orientalis is one of the benign species of Theileria that is widely distributed in Japan and is sometimes responsible for serious economic losses in the livestock industry. In the present study, we surveyed the current status of T. orientalis infection in grazing cattle in the eastern areas of Hokkaido (Taiki, Otofuke, Shintoku, and Shin-Hidaka districts) using molecular methods, as well as traditional methods, of diagnosis. The genes encoding the major piroplasm surface protein (MPSP) and p23 of T. orientalis were identified using highly detectable polymerase chain reaction (PCR). Results of the MPSP-PCR assay indicated that grazing cattle in these districts, after about 1.5 months pasturage, showed high rates of infection, ranging from 10.0-64.8%. Although the main MPSP and p23 genotypes detected were the Ikeda- or Chitose-types, an MPSP gene closely relating to that found in Okinawa prefecture, and a p23 gene closely relating to the Australian (Warwick) Buffeli-type gene, were found in the cattle in Shintoku and Shin-Hidaka districts. The present survey indicated that there were at least five types of T. orientalis classified by their MPSP genes in Hokkaido, Japan, and that T. orientalis infection rates are still high in this region.

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.

Serological analysis was performed to detect Toxoplasma gondii and Neospora caninum infection in seals in Hokkaido. Serum samples were collected from 322 Kuril harbor seals (Phoca vitulina stejnegeri) at Nosappu, Akkeshi and Erimo, from 46 spotted seals (P. largha) at Nosappu, Erimo, Yagishiri Island, Hamamasu and Syakotan, and from 4 ribbon seals (P. fasciata) and a bearded seal (Erignathus barbatus) at Nosappu between 1998 and 2006. Recombinant surface antigen of T. gondii (SAG2t) and N. caninum (NcSAG1t) were used as antigens for ELISA to detect antibodies. Antibodies against SAG2t were detected from 4% of 77 Kuril harbor seals at Nosappu in 2005. Antibodies against NcSAG1t were detected from 2% (1/66) in 2003, 5% (4/79) in 2004 and 10% (8/77) in 2005 of Kuril harbor seals and 11% of 9 spotted seals in 2004 sampled at Nosappu. Eight percent of 12 Kuril harbor seals from Akkeshi and 25% of 4 spotted seals from Erimo in 2005 also contained antibodies against NcSAG1t. These suggest sporadic infection of T. gondii and N. caninum in Kuril harbor seals and spotted seals in Hokkaido. Of the ELISA-positive seals, 2 seals having anti-SAG2t antibodies and 3 seals having anti-NcSAG1t antibodies in 2005 were judged to be juveniles that have no maternal antibodies. These suggest that the protozoan infections have occurred in recent years. Infection of terrestrial protozoa such as T. gondii and N. caninum in seals indicates that the sea environment has been contaminated with protozoa.

The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-length), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliable reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.

Trypanosoma brucei, the causative agent of sleeping sickness in humans, requires transferrin (TF) for growth. Therefore, T. brucei has a TF receptor that allows it to obtain iron from TF. Lactoferrin (LF), a member of the TF family protein, is an iron-binding protein that is found in most biological fluids of mammals. LF has been shown to interact with some bacteria species by specific receptor-ligand binding. We examined the ability of T. brucei to bind bovine LF (bLF) by using a fluorescence test, streptavidin-biotin (SAB) microplate analysis, and far Western blotting using a biotin-streptavidin system. We found that bLF bound to components of T. brucei, and that bLF hydrolysate disrupted the sites responsible for binding to parasite proteins. Furthermore, bLF, human LF, bovine TF, and ovotransferrin bound same proteins of T. brucei, which exhibited molecular masses of 40 and 43 kDa. The N-terminal amino acid sequence of the 40 kDa bLF binding protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Lactoferrin (LF), a member of the transferrin (TF) protein family, is an iron-binding protein that is known to interact with bacteria through a specific receptor. We examined the binding of bovine LF (bLF), bovine TF (bTF), and ovotransferrin (OTF) by Toxoplasma gondii using a fluorescence test and the streptavidin-biotin (SAB) method using biotin-streptavidin, and found that bLF, bTF, and OTF bound to the protein components of T. gondii. Furthermore, we confirmed that bLF, bTF, and OTF bound a 42 kDa soluble protein of T. gondii by far Western blot method. These results demonstrated that bLF binding proteins are present on T. gondii.

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.

Babesia equi (EMA-1) and Babesia caballi (BC48) gene fragments were amplified by polymerase chain reaction (PCR), in blood samples, and partially fed-females and egg and larval progenies of Dermacentor nuttalli, collected from horses in Altanbulag, Tuv Province, Mongolia. While Babesia parasite DNA was detected in some horse blood samples during the first PCR, all positive cases in partially fed-female ticks, eggs and larvae were confirmed by nested PCR. Present study reinforces earlier similar findings in unfed D. nuttalli ticks collected from an open space vegetation in Bayanonjuul, Tuv Province in Central Mongolia, pointing to the most likely important role of D. nuttalli in the transmission of equine babesiosis in Mongolia. The detection of parasite DNA in eggs and larval progenies is likewise suggestive of transovarial parasite transmission in this tick species.

Antibodies to Babesia caballi and Babesia equi were examined on a total of 2,019 horse serum samples that had been collected in 1971-1973 by the National Institute of Animal Health by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins and by Western-blot analysis. Based on the criterion for positivity by ELISA, 5.4% (109/2,019) and 2.2% (44/2,019) had antibodies against B. caballi and B. equi, respectively. The ELISA-positive sera were further examined by Western blot; 30/109 for B. caballi and 2/ 44 for B. equi were positive for native B. caballi or B. equi, but none of them was seropositive for both infections. Based on the results of this study, further investigations should be required to survey horses that have arrived in Japan relatively recently and tick vectors of equine Babesia using ELISA with some recombinant protein, a parasite detection method in an in vitro culture of equine Babesia, and PCR testing.

Using macrophage scavenger receptor-A knockout (SRKO) mice, we examined the role of macrophage class A scavenger receptors (MRS-A) on the immune response and acquisition of host resistance against repeated infestation with Haemaphysalis longicornis. Except for one batch of nymphs that infested one of the SRKO (SR-/-) mice and showed no appreciable reduction in body weight, all the other groups of nymphs manifested significant decrease in body weight. Both SR-/- and wild type (SR+/+) mice showed a sustained increase in anti-tick antibody titers, but SR+/+ mice showed significantly higher titers. The IFN-gamma assayed in SR-/- mouse immune sera was substantially less compared with that in SR+/+ mice. Immune sera from SR-/- and SR+/+ mice recognized the 51 and 44 kDa, and 44 kDa proteins, respectively, of the salivary gland antigen. The difference in the level of anti-tick resistance manifested by both groups of mice may be influenced by less efficient trapping and processing of tick antigens by macrophages in mice lacking for the macrophage scavenger receptors, and consequently affected the cascade of Th1 and Th2 responses. We have thus obtained valuable data that strongly infer the role of MSR-A in enhancing host defense against repeated infestation with H. longicornis.

A detailed comparison of the accessory cell activities was carried out among murine peritoneal cavity macrophages (PEC-Mphi), peritonea] cavity macrophages stimulated with granulocyte-macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4), the most popular cytokine combination widely used to generate dendritic cells (DC) and peritoneal cavity macrophage-derived DC (PEC-DC) using a two-way mixed lymphocyte reaction (MLR). All the cell types used efficiently induced statistically significant naïve T cell proliferation at all culture time points and responder:stimulator ratios used. However, marked differences were noted in the magnitude of the proliferative responses. These variations may be attributed to the intensity of expression of MHC class II glycoproteins, as well as the actual numbers of MHC class II+ cells.

Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were 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 the States of Sao Paulo and Mato Grosso do Sul, Brazil.

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.

Leukocyte populations present in the discrete Peyer's patches (PP) of the pig were characterized from birth (Day 0) to day 35 after birth by immunohistochemistry and image analysis. Immediately after birth, cell membrane expression of CD2 and CD3, major histocompatibilty complex (MHC) class 11 (both SLA (swine leukocyte antigen) -DQ+ and SLA-DR+), CD21, 74-22-15 and surface immunoglobulin (sIg) were all demonstrable. Computer assisted morphometric techniques were used to confirm the significant expansion of these cell populations from birth onwards. The distribution of the cell types was not random but suggested a preferential retention of cells at specific sites. This implies a degree of organization of immunological cells within the discrete PP, enhancing the potential to mount immune responses in the most efficient manner.

Control of cryptosporidiosis is important in public health. Rivers that are polluted with Cryptosporidium and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptosporidiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptosporidium oocysts in environmental water, the filtration method was used for 28 samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency (U.S. EPA) filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by discontinuous sucrose gradient method. Twelve samples were collected from 10 rivers and parasites were purified by iron (III) flocculation method. Cryptosporidium parvum oocysts were identified with the immunofluorescence antibody technique using DIF kit (Cellabs Pty. Ltd., Sydney/Australia). We detected Cryptosporidium oocysts in 6 out of 10 rivers sampled. Fifty percentage (14/28) of the samples were Cryptosporidium-positive. The average number of Cryptosporidium oocysts was 16.73/100 L (max. 80/100 L).

The surface proteins of Babesia rodhaini have previously been shown to induce a high degree of protective immunity. In the present study, one of those proteins, B. rodhaini antigen p26 was expressed in Escherichia coli and in insect cells infected with a recombinant baculovirus. These proteins were recognized by immune serum from a drug-cured BALB/c mouse. While BALB/c mice immunized with both recombinant antigens and Freund's adjuvants showed 40-100% survival rate against challenge infection with B. rodhaini, saponin failed to induce protection, although significant levels of B. rodhaini-specific antibodies were produced in both immunized mice (1:1,000-2,000 by indirect immunofluorescent antibody test). The immunization of IFN-gamma-deficient mice with the recombinant proteins was not protective against B. rodhaini infection, indicating that IFN-gamma is one of the important factors for the survival against lethal B. rodhaini infection.

Babesia caballi merozoites were prepared by combining two improved methods of cultivation and purification of merozoites using Percoll-gradiation, and the protein compositions of merozoites were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The relative molecular masses of the major proteins and protein masses separated by electrophoresis were >94, 80-70, 50-45, 34-30, 30-28 and 18 kDa. By Western blotting, twelve proteins or protein groups were recognized by pooled sera from two horses experimentally infected with B. caballi. Among twelve proteins, five new proteins (54, 30-26, 24, and two 18 kDa) were identified, and the 48 kDa protein was revealed to consist of 2 components in the B. caballi merozoite. One protein (54 kDa) of B. caballi was also recognized by the pooled sera from two horses experimentally infected with B. equi.

Babesia caballi infected erythrocytes were collected from the blood of an experimentally infected horse and could be continuously cultivated in vitro with parasitemia ranging from 2-4% in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2% O2, 5% CO2 and 93% N2). All attempts to increase parasitemia failed using other culture media, serum concentrations and culture vessels. However, parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO2 in air, with parasitemia ranging from 8-10%.

Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR".

Lactoferrin (LF) is known to have broad spectrum antimicrobial properties. In regards to its defense mechanism against parasitic infection, it has shown phagocytic activity in the destruction of amastigotes, an intracellular parasitic form of Trypanosoma cruzi in macrophages. The effect of bovine lactoferrin on the intracellular growth Toxoplasma gondii parasites was examined in murine macrophage and embryonal cells. Co-cultures of host cells with the parasites were supplemented with either lactoferrin, apo-lactoferrin, holo-lactoferrin or transferrin in the culture media for varying periods. The growth activity of intracellular parasites in the host cells was determined by the measurement of selective incorporation of 3H-uracil. Supplement of lactoferrin had no effect on the penetration activity of the parasites, while development of intracellular parasites was inhibited linearly in concentration of lactoferrin. Supplement of apo-lactoferrin and holo-lactoferrin, but not transferrin showed similar effects. These suggest that lactoferrin induces the inhibitory effects on the development of intracellular parasites. Pretreatment of lactoferrin to the macrophages, however, did not show any inhibitory effects. Whereas, mouse embryonal cells preincubated with lactoferrin suppressed the intracellular growth. Thus, the action of lactoferrin to macrophages would be different from that of mouse embryonal cells.

The prevalence of Sarcocystis infection was examined in older cows and imported cattle slaughtered in East Hokkaido. Samples of myocardial tissues were examined for Sarcocystis microscopically. Sarcocystis cysts were detected in 15.7% of 83 older cows, 48.4% of 91 imported cattle which were kept in East Hokkaido prior to slaughter and 51.1% of 94 imported cattle slaughtered immediately after quarantine check. Based on the morphology of the cyst wall and the establishment of infection in experimentally inoculated dog, the Sarcocystis species was identified as Sarcocystis cruzi.

Protective response against Babesia microti was studied in Isospora felis-infected mice. Isospora felis-infected mice which were exposed to B. microti on the 28th day post-infection showed absolute resistance against Babesia microti. Interestingly, these mice showed no anti-B. microti antibodies. Mice that received spleen cells from I. felis-infected donors that were subsequently exposed to B. microti showed lower peak parasitemia (10.3% +/- 2.6) compared to those mice that received normal spleen cells (60.9% +/- 15.0), and no spleen cells at all (47.3% +/- 8.5). Treatment of I. felis-infected mice with monoclonal antibodies against L3T4+ cells resulted to a depression of their resistance to B. microti, as clearly manifested by high levels of parasitemia. Findings of the present study demonstrate the role of cell mediated immunity, specifically by L3T4+ T-cells induced by I. felis infection, in providing mice protection against B. microti.

In order to identify mechanisms responsible for the anti-tumor effects of Toxoplasma lysate antigen (TLA), we used an in vitro 51Cr release assay to study the functional properties of plastic-adherent cells during induction of splenic cytotoxic activity by TLA. Cytotoxic activity of non-adherent cells was measured in all experiments after a 6 days incubation. Induction of cytotoxic non-adherent cells by TLA required the presence of plastic-adherent spleen cells. In contrast, rhIL-2 alone was able to induce transformation of cytotoxic non-adherent cells from non-adherent spleen cells. Contact between adherent and non-adherent spleen cells was necessary for successful induction of cytotoxic non-adherent cells by TLA. Treatment of spleen cells with anti-macrophage serum prevented induction of cytotoxic activity by TLA. Biologically active IL-2 was not detected in culture supernatants of spleen cells exposed to TLA. These findings suggest that contact between TLA-sensitized non-adherent cells and macrophages is necessary for induction of cytotoxic cells in the presence of TLA. This contact, however, is not necessary for generation of IL-2-induced killer cells.

Growth of the tumor autoinduced by 20-methylcholanthrene (MC) in rats was inhibited after administration of Toxoplasma lysate antigen (TLA). The antitumor activity of TLA was most obvious in the early stage of tumoral growth. When TLA was administered to rats before the appearance of tumour, tumor formation was delayed slightly. Histopathological studies revealed dense growths of spindle tumor cells in untreated control rat, while enlarged central necrosis with the infiltration of lymphocytes and neutrophils was apparent in TLA-treated rats. According to the immunohistological examination of tumor tissue with anti-Thy-1 antibody, the rats treated with TLA showed large Thy-1 positive granular cells, whereas the untreated rats indicated only a few small Thy-1 positive cells. These observations indicate that TLA is a useful modifier of biological responses to MC-induced tumors.

Spleen cells from Toxoplasma lysate antigen (TLA)-sensitized BALB/c mice showed the strong cytotoxic activity against both natural killer (NK)-sensitive cells (YAC-1 and RL male-1) and NK-insensitive cells (P-815), when incubated with TLA or recombinant human IL-2 (rhIL-2). The increment of TLA concentration in culture medium increased the cytotoxic activity. Treatment of effector cells; spleen cells from TLA-sensitized mice incubated with TLA, with anti-asialo GM1 or anti-Thy-1 plus complement inhibited the cytotoxic activity of effector cells, whereas treatment with anti-mouse Lyt-2.2 serum plus complement had no effect on the cytotoxic activity. Treatment of spleen cells from TLA-sensitized mice with anti-asialo GM1 and/or anti-Thy-1 plus complement inhibited cytotoxic activities of effector cells. These results suggested that spleen cells sensitized with TLA both in vivo and in vitro were asialo GM1 positive and Thy-1 positive, and the majority of cytotoxic cells induced by TLA were similar to lymphokine-activated killer (LAK) cells induced by IL-2.

Therapeutic effects of Toxoplasma lysate antigen (TLA) were studied in mice bearing the tumor in the second passage of 20-methylcholanthrene (MC)-induced tumor cells. Intramuscular administration of TLA 7 days after the tumor-cell inoculation caused apparent inhibition of the tumor growth on day 14. The second treatment facilitated the therapeutic effects. Intravenous transfer of spleen cells prepared from TLA-sensitized mice into tumor-bearing mice also represented the growth inhibitory effects. Prominent effects were seen when the transferred cells were prepared 5 days after sensitization of donor animals. The inhibitory effects were absent in the groups transferred only the adherent cells or the non-adherent cells prepared from sensitized mice. The strongest inhibitory effect was observed in the group to which both adherent and non-adherent spleen cells were transferred simultaneously from sensitized mice. In in vitro experiments, spleen cells obtained from sensitized mice showed cytolytic effect on P-815 or YAC-1 cells after the secondary stimulation in vitro with TLA. Large non-adherent cells containing densely packed granules were induced when cultured with the adherent cells obtained from sensitized mice. These results revealed that TLA can inhibit the growth of the chemically-induced transplantable tumors by activation of adherent and non-adherent spleen cells.

Novel synthetic Obiopeptide (Obi-1), Glycil-penta-Glutamate, as an immunoregulator, injected intramuscularly to methylcholanthrene (MC) induced tumour bearing mice at 30-100 micrograms per mouse weekly, significantly inhibited tumour growth, compared to that of the untreated control. Numbers of mononuclear cells (MNC) in the spleen obtained from Obi-1 treated and untreated mice were reduced to 64% and 10% of the normal levels, respectively. In histopathological findings, variations of MNC in the spleen was observed in colonies of large MNC and lymphocytes in Obi-1 treated red marrow, while untreated one showed only scattered number of small lymphocytes. A significant O2- production was noted in monolayers of normal macrophages incubated with 0.5 mg Obi-1/ml for 48 hr. Cytotoxicity of spleen MNC cultured with Obi-1 against MC-tumour, Meth A, P-815 and YAC-1 target cells showed less than 10% cytotoxic activity in almost all experiments.