Serological survey of Encephalitozoon cuniculi infection in Japanese dogs.

Antibodies to Encephalitozoon cuniculi were examined by enzyme-linked immunosorbent assay using E. cuniculi PTP2 recombinant protein and by Western blot analysis on a total of 472 dog serum samples that had been collected in Japan. Of these samples, 21.8% (103/472) had antibodies against E. cuniculi. Each of 5 serum samples that showed high (>1.0) or low (<0.1) OD value was selected randomly and further examined by Western blot using E. cuniculi-native antigens. All samples with high OD values reacted with specific E. cuniculi proteins, including an antigen of approximately 35 kDa corresponding with PTP2; sera with low OD values did not recognize this E. cuniculi band. This study is the first to demonstrate the prevalence of E. cuniculi infection in dogs in Japan.

Morphology of the cockerel's comb after androgen administration.

1. Androgen receptor (AR) expression and morphological changes in blood capillaries were investigated in the comb of cockerels, both untreated controls and after the administration of testosterone (T) or the androgen antagonist flutamide (F) for 7 weeks. 2. Twenty-six male Single Comb White Leghorn roosters were divided into T-treated, F-treated and untreated groups. Tissue sections were examined histologically, immunohistochemically, and comb blood vessel castings were analysed by scanning electron microscopy. 3. Histologically, the capillaries of the peripheral dermis layer in the T-treated group were dilated compared with controls. Many red blood cells were seen in the lumen. Although the capillary diameter in the F-treated group did not show a significant difference as compared with control, blood capillaries with small diameters were often observed, and there were few red blood cells in the capillaries. Some capillary castings were extended markedly in the T treated group, and small blood vessels were observed arborising from the extended blood capillaries. In contrast, all capillaries were slender in the F-treated group, and the casting surface was rough. 4. Immunoreactivity for AR was found in capillary endothelial cells in the peripheral dermis layer of the comb. The intensity of staining in these cells was increased in the T-treated group but was reduced in the F-treated group. 5. It is concluded that immunoreactivity for AR was found in capillary endothelial cells in the peripheral dermis layer of the rooster's comb. The intensity of staining in these cells was increased in the T-treated group but reduced in the F-treated group. Thus, the capillary endothelial cells in the peripheral dermis layer of the comb are androgen targets.

Cutaneous toxoplasmosis in a female Japanese cat.

A 16-year-old female Japanese cat was presented with a single mammary-gland nodule approximately 3 cm in diameter. Histologically, the nodule consisted of necrotizing granulomatous panniculitis, vasculitis, and mastitis, and contained free and clustered protozoal organisms. The organism was present in the cytoplasm of macrophages, fibroblasts, endothelial cells, and mammary-gland epithelia. The organism was positive for anti- Toxoplasma gondii and anti- Neospora caninum antibodies. Electron microscopy showed single and grouped tachyzoites, with morphologic features similar to those of T. gondii. Polymerase chain reaction and deoxyribonucleic acid sequence analysis was consistent with T. gondii infection. This is the first report of cutaneous toxoplasmosis in a Japanese cat.

Molecular characterization of a putative protein disulfide isomerase from Babesia caballi.

We produced a mAb against the Babesia caballi extracellular merozoite termed mAb 2H2 and used it to screen a cDNA expression library prepared from B. caballi merozoite mRNA for highly expressed proteins. The complete nucleotide sequence of the cloned gene had 1547 nucleotides and contained a 36-nucleotide intron. The 1398 nucleotide open reading frame predicts a 51 kDa protein showing similarity to protein disulfide isomerase (PDI) from other species. The PDI gene had a predicted N-terminal signal sequence of 19 amino acids and a C-terminal tetrapeptide sequence (His-Thr-Glu-Leu; HTEL) for retention in lumen of the endoplasmic reticulum (ER). The recombinant protein expressed in baculovirus showed an apparent mass of 51 kDa, identical to that the native B. caballi protein. Moreover, the ER retention signal site (HTEL) of the recombinant protein retained its function in ER of insect cells. This 51 kDa protein was strongly expressed by extracelluar B. caballi merozoites in indirect immunofluorescence antibody tests, and was not expressed in the early phase of trophozoite development. Interestingly, detailed observation showed that the reaction of anti-P51 antibody and mAb 2H2 against pear-shaped forms was very erratic, some displaying one or two brightly fluorescent patterns.

Characterization of monoclonal antibodies against Gnathostoma nipponicum.

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

Detection of Babesia caballi and Babesia equi in Dermacentor nuttalli adult ticks.

Ticks play an important role in human and veterinary medicine particularly due to their ability to transmit protozoan pathogens. In this study we have demonstrated that polymerase chain reaction (PCR) and nested PCR methods enabled detection of Babesia caballi and Babesia equi in field isolates of Dermacentor nuttalli adult ticks from Mongolia. Primers specific for 218 bp fragment merozoite antigen 1 (EMA-1) gene of B. equi successfully amplified products from all samples of D. nuttalli adult ticks while primers for the 430 bp fragment product from BC48 gene of B. caballi amplified products from seven of the 54 samples. Using PCR and nested PCR methods we have found mixed infections with B. equi and B. caballi in the tick vector. The amplified DNA fragment from D. nuttalli ticks was inserted into the EcoRV site of pBluescript SK and sequenced. The sequence of the 430 bp fragment was completely identical to the nucleotide sequence of the USDA strain of B. caballi. These results suggest that D. nuttalli may play an important role as a vector of both B. caballi and B. equi and also may be important in maintaining endemicity of equine piroplasmosis in Mongolia.

Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiated B. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to B. equi.

Molecular evidence of Babesia caballi (Nuttall and Strickland, 1910) parasite transmission from experimentally-infected SCID mice to the ixodid tick, Haemaphysalis longicornis (Neuman, 1901).

Molecular evidence that suggests the possible role of the ixodid tick, Haemaphysalis longicornis and its eggs in the transmission of equine Babesia caballi parasites is presented herein. Using polymerase chain reaction (PCR) to assay for DNA in parasites, presumably acquired by ticks that were allowed to feed on splenectomized-SCID mice, experimentally exposed to in vitro-cultivated B. caballi, we have obtained positive bands that corresponded to the expected B. caballi-specific 430bp gene fragment in 50% of female ticks used, and in 75 and 25% of eggs and larval progeny, respectively. Also, parasite DNA was detected in ticks, eggs and larvae as late as the 16th to the 20th day post-host infestation. Present findings support to the potential role of H. longicornis in the transmission of B. caballi parasites. Its capability, however, to successfully transmit the infection to horses under natural conditions in the field needs to be further ascertained. To our knowledge, this is the first documented study incriminating H. longicornis as a most and likely biological vector of equine babesias.

Analysis of a growth-promoting factor for Babesia caballi cultivation.

Serum-free media were examined to culture Babesia caballi. Daigo's T (DT) basal medium supplemented with Daigo's GF21 (GF21) or GIT medium, which already contains GF21, supported the parasite propagation at 37 C in a humidified atmosphere under 5% CO2 in air. Growth of B. caballi was dependent of the suitable concentration (10-20%) of GF21. Therefore, GF21 was suggested as the growth-promoting factor for B. caballi. However, GIT medium did not support the growth of parasites from cryopreserved stabilates, and serum supplementation was essential for the retrieval of parasites.

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein.

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.

Protein analysis of Babesia caballi merozoites by two-dimensional polyacrylamide gel electrophoresis and western blotting.

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.

Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay.

A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.

Ultrastructural characteristics of Babesia caballi in equine erythrocytes in vitro.

Babesia caballi cultured continuously in equine erythrocytes was examined by transmission electron microscopy. The use of cultured B. caballi permitted examination of a large number of parasitized cells with various stages of intra erythrocytic development. The piriform merozoites of B. caballi were composed of an outer membrane and an inner double-membrane complex. Numerous micronemes and three rhoptries were found in the pellicle of the merozoite, and a spherical body was seen in the anterior part of the merozoite which usually lay adjacent to the nucleus and the pellicle. These findings were similar to those for merozoites of bovine Babesia parasites such as B. bigemina. The trophozoites were surrounded by a single membrane, were continuously changing their body shape with extension and retraction of the pseudopod. A long pseudopod extended far into the host cell cytoplasm, and was finally completely enclosed in a cell, but did not have hemozoin pigment, the breakdown product of hemoglobin digestion.

A genetic linkage map of rat chromosome 9 with a new locus for variant activity of liver aldehyde oxidase.

A genetic linkage map of rat chromosome 9 consisting of five loci including a new biochemical marker representing a genetic variation of the activity of the liver aldehyde oxidase, (Aox) was constructed. Linkage analysis of the five loci among 92 backcross progeny of (WKS/Iar x IS/Iar)F1 x WKS/Iar revealed significant linkages between these loci. Minimizing crossover frequency resulted in the best gene order: Aox-D9Mit4-Gls-Cryg-Tp53l1. The homologues of the Cryg, Gls, and Aox genes have been mapped on mouse chromosome 1 and human chromosome 2q. The present findings provide further evidence for the conservation of synteny among these regions of rat, mouse, and human chromosomes.

Tubular structures associated with Babesia caballi in equine erythrocytes in vitro.

In-vitro-propagated Babesia caballi parasites were examined by scanning and transmission electron microscopy. Many small pores were observed over the entire surface of infected erythrocytes on scanning electron microscopy, and on transmission electron microscopy these small pores were found to be openings of tubular structures. By the examination of a number of infected cells the tubular structures were found to be connected with the parasite, and this observation might indicate that the tubular structures arose the edge of the parasite and terminated at an Invagination on the surface of the erythrocyte. These findings suggest that intraerythrocytic stages of B. caballi come into direct contact with culture medium.

Inhibitory effect of monoclonal antibodies on the growth of Babesia caballi.

Monoclonal antibodies (mAbs) were produced against Babesia caballi (USDA strain) to define a species-specific antigen for use in diagnosis and vaccine development. Eight positive clones of B. caballi mAbs determined by indirect immunofluorescent antibody test were selected for purification and further characterisation. Confocal laser microscopy showed that the antigens recognised by the mAbs were located on the surface/cytoplasm, central part, and/or anterior end of B. caballi parasites, with five different reactive patterns. These mAbs seemed to be species-specific, since they did not cross-react with Babesia equi-infected erythrocytes or uninfected erythrocytes. In Western blotting analysis, 18, 20, 34, 36, 48, and 155 kDa proteins of B. caballi merozoites were recognised by six different mAbs. When added to in vitro cultures, four of the mAbs significantly inhibited the in vitro growth of B. caballi parasites. These results provide a rationale for evaluating antigens for the development of diagnostic methods or vaccines.

Expression of Babesia equi merozoite antigen-2 by recombinant baculovirus and its use in the ELISA.

In this study, the gene encoding Babesia equi merozoite antigen-2 was inserted into a baculovirus transfer vector, and a recombinant virus expressing B. equi merozoite antigen-2 was isolated. Two B. equi merozoite antigen-2-related recombinant baculovirus-expressed peptides of 25 and 30 kDa were detected with a murine anti serum against B. equi merozoite antigen-2; these corresponded to the native B. equi merozoite antigen-2 and were secreted into the supernatants of insect cell cultures. Recombinant B. equi merozoite antigen-2 was not effected by tunicamycin treatment, indicating that B. equi merozoite antigen-2 was not glycosylated. The potential of recombinant B. equi merozoite antigen-2 for use in the ELISA was evaluated by measuring the antibody response to B. equi merozoite antigen-2 in horses experimentally infected with B. equi.

Susceptibility of several species of Cyprinidae and Salmonidae freshwater fish to larval Gnathostoma nipponicum infection.

Susceptibility of five species of Cyprinidae and Salmonidae freshwater fish to the early third-stage larvae (EaL3) and advanced third-stage larvae (AdL3) of Gnathostoma nipponicum infection were examined. Two fish species inoculated orally with EaL3 were infected, and AdL3 were recovered from them with rate of 21.0% in Tribolodon hakonensis and 0.5% in Cyprinus carpio at 30 days postinoculation (PI). Attempts to infect five fish species with AdL3 were all successful. The recovery rate of AdL3 was 69.0% in T. hakonensis, 47.5% in Carassius auratus subsp., 35.0% in C. carpio, 53.0% in Oncorhynchus masou, and 32.0% in O. mykiss at 10 days PI. These results confirmed that the Cyprinidae and Salmonidae fish species reported here were susceptible to larval G. nipponicum infection and AdL3 had higher infectivity to them than the EaL3.