Serodiagnosis of canine Babesia gibsoni infection by enzyme-linked immunosorbent assay with recombinant P50 expressed in Escherichia coli

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni-infected, Babesia canis-infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coil is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.     

Morphological changes of Babesia gibsoni grown in canine red blood cell-substituted severe combined immune deficiency mice

Canine red blood cell-substituted severe combined immune deficiency (Ca-RBC-SCID) mice were prepared for canine Babesia gibsoni infection. The Ca-RBC-SCID mice infected with B. gibsoni developed a high level of parasitemia, and showed clinical symptoms such as anemia and hemoglobinuria, which are similar to those observed in dogs infected with B. gibsoni. The B. gibsoni parasites grown in Ca-RBC-SCID mice showed marked morphological changes, including a significantly larger size of parasites than those in dogs and abundant RBCs containing 4, 8, 16, and 32 parasites. The multiple infection may have resulted from 1 parasite because the posterior end of each parasite in a multiply infected cell was connected. The parasites grown in SCID mice retained their infectivity and virulence to dogs and their morphology was dramatically restored to the original state when they were returned to dogs.     

Intratesticular injection of a zinc-based solution as a contraceptive for dogs

The objective of the present study was to evaluate, by light and transmission electron microscopy, the efficacy of a single intratesticular injection of a novel zinc-based solution, as a contraceptive for male dogs. Fifteen mongrel dogs were assigned to three groups (five dogs/group). Group 1, the control group, which consisted of animals ranging from 8 mo to 4 yr, was injected with saline solution. Group 2, which consisted of animals ranging from 8 mo to 1 yr old and Group 3, animals ranging from 2 to 4 yr old, were injected with a zinc-based solution (0.2-1.0mL; volume based on testicular width). There were no histopathological changes detected in testes from control dogs. Histological examination of treated groups revealed degeneration, vacuolation, fewer germ cells, formation of multinucleated giant cells, and a lack of elongated spermatids in atrophic seminiferous tubules. Leydig cells had varying degrees of lipid degeneration and necrosis. The majority of seminiferous tubules in all zinc-treated dogs were lined only by Sertoli cells, which were vacuolated. Ultrastructure of testis of treated groups had degenerate Sertoli and Leydig cells, characterized by numerous mitochondria with the lack of a matrix and agglomeration of lysosomal bodies. The cytoplasm of elongated spermatids was characterized by tubules of hyperplastic and hypertrophic smooth endoplasmic reticulum and numerous Golgi apparati. Round spermatids in Golgi phase had lysis of acrosomal vesicles. The degree of histological changes suggested irreversibility. In conclusion, intratesticular injection of a zinc-based solution effectively impaired spermatogenesis.     

Schistosoma mansoni: vaccination of mice with 10-krad-irradiated, cryopreserved schistosomules

Protection against a Schistosoma mansoni cercarial challenge was evaluated in mice immunized with a vaccine composed of 10-krad-irradiated, cryopreserved schistosomules. The level of resistance induced in C57B1/6 or NMRI (CV) mice increased with the number of schistosomules injected. Up to 83% reduction in challenge worm burden was achieved when 5000 schistosomules were injected per mouse. Intramuscular injection of the vaccine was superior to subcutaneous. Multiple immunizations, up to 3 at 4-week intervals, did not increase the resistance induced by a single immunization. A high level of protection developed in as little as 2 weeks and was maintained through at least 12 weeks postimmunization. The vaccine irradiated with 10 krad from either a 60-cobalt or 137-cesium source induced equivalent levels of resistance, and no differences were found in the immunogenicity of vaccines comprised of organisms irradiated as cercariae or as 1- to 3-hr-old schistosomules. These findings are basic to the development of a cryopreserved, live vaccine against schistosomiasis of humans or domestic animals.     

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP

The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.     

The Influence of Immunization with Live or Killed Staphylococcus aureus Vaccines on the Early Development of Opsonizing and Bactericidal Factors in Lymph and Blood of Sheep

In order to obtain sensitive measurements on the synthesis of opsonins following immunization with live or killed S. aureus vaccines, lymph was collected from the efferent popliteal lymphatic duct of sheep during the early phase of the immune response. Lymph and blood serum were assayed for opsonizing capacity using ³H-labeled S. aureus. Within 1 hr after vaccination there was a rapid, transitory decrease in uptake by neutrophils of bacteria opsonized with lymph from sheep given the killed vaccine (Group 2). These results were in contrast to the relatively constant uptake rates of bacteria opsonized with lymph from sheep given the live vaccine (Group 1) and non-vaccinated controls (Group 3) at this time. At 72, 96, and 120 hr post-injection mean uptake values for bacteria opsonized with lymph from either vaccinated group were significantly greater than comparable values for controls. Mean uptakes for organisms opsonized with blood serum from Group 1 at 72 and 96 hr post-injection were significantly greater than comparable values for the control group. The percentage of viable neutrophil-associated bacteria decreased when lymph collected from animals in Group 2 in the first hour post-injection was used to opsonize the organisms. Percentages of viable, neutrophil-associated S. aureus for assays in which blood serum was used to opsonize remained relatively constant at around 45% for Groups 2 and 3. In contrast, however, values of viable neutrophil-associated bacteria for Group 1 decreased during the 120 hr after immunization.     

Effect of autonomic blockade on tracheobronchial blood flow

Tracheobronchial blood flow increases two to five times in response to cold and warm dry air hyperventilation in anesthetized tracheostomized dogs. In this series of experiments we have attempted to attenuate this increase by blockade of the autonomic nervous system. Four groups of anesthetized, tracheostomized, open-chest dogs were studied. Group 1 (n = 5) were hyperventilated for 30 min with 1) warm humid [approximately 26 degrees C, 100% relative humidity, (rh)] air followed by bilateral vagotomy, 2) warm humid air, 3) cold (-22 degrees C, 0% rh) dry air, and 4) warm humid air. Groups 2, 3, and 4 (n = 3/group) were hyperventilated for 30 min with 1) warm humid (approximately 41 degrees C, 100% rh) air, 2) warm dry (approximately 41 degrees C) air, 3) warm humid air, and 4) warm dry air. Group 2 were controls. Group 3 were given phentolamine, 0.6 mg/kg intravenously, as an alpha-blockade, and group 4 were given propranolol, 1 mg/kg, as a beta-blockade after warm dry air hyperventilation (period 2). Five minutes before the end of each 30-min period of hyperventilation, measurements of vascular pressures, cardiac output, arterial blood gases, and inspired, body, and tracheal temperatures were measured, and differently labeled radioactive microspheres were injected into the left atrium to make separate measurements of airway blood flow. After the last measurements had been made animals were killed and their lungs were excised. Blood flow to the airways and lung parenchyma was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infection with and antibody response to Pasteurella multocida and Bordetella bronchiseptica in immature rabbits

At 2, 4, 6, 8 and 10 weeks of age rabbits were cultured for Pasteurella multocida and Bordetella bronchiseptica from the paranasal sinuses, trachea, middle ears, lungs and liver. Sera were tested for antibodies (IgG) against P. multocida and B. bronchiseptica. Pasteurella multocida was isolated from all ages of rabbits, and B. bronchiseptica was isolated from rabbits 4 to 10 weeks old. The sinuses were colonized most often, followed by the trachea, middle ears and lungs. No bacteria were isolated from the liver. The number of rabbits with antibodies against both bacteria decreased between 2 and 6 weeks of age, indicating a fall in maternal antibodies, and increased between 6 and 8 weeks of age, suggesting an active humoral response.     

The effects of 5% carbon dioxide on the quantitative analysis of long-term pathology of the brain after surface hypothermia

To evaluate the effects of 5% carbon dioxide (CO2) administration for hypothermic circulatory arrest, neurological evaluation and pathological studies were carried out on the canine brain. Twenty-two dogs were assigned to five groups: Group 1: Three dogs without hypothermia were sacrificed as the control group. Group 2: Nine dogs were subjected to surface hypothermia (20 degrees C) under deep ether anesthesia with 100% oxygen (O2) and hyperventilation. Circulatory arrest time was 30 min in Group 2A and 60 min in Group 2B. Group 3: Ten dogs were surface cooled (20 degrees C) under deep ether anesthesia with a 95% O2 and 5% CO2 mixture. Thirty minutes of circulatory arrest was instituted in Group 3A and 60 min in Group 3B. Dogs in Groups 2 and 3 were surface rewarmed and kept alive until they were sacrificed electively 6 or more months later. Results were as follows: (i) Postoperative neurological disturbance was detected in only two dogs in Group 2B. (ii) The percentage of damaged nerve cells among the total nerve cells counted in the cerebral cortex of the frontal lobe was significantly greater in Groups 2A (22.4%), 2B (30.1%), 3A (19.6%), and 3B (22.2%) compared with Group 1 (7.1%). (iii) The number of glia cells per nerve cell in the cerebellar dentate nucleus was significantly higher in Group 2B (27.2) than in Groups 1 (11.8), 2A (16.7), 3A (17.9), and 3B (18.6). (iv) The number of Purkinje cells in a 10-mm length of the cerebellum was markedly reduced to 89 in Group 2B compared with 122, 134, and 117 in Groups 1, 2A, and 3A, respectively. In conclusion, the results of quantitative pathological brain analysis reflected the incidence of postoperative neurological disturbance and suggested that the administration of 5% CO2 could prolong the time limit for circulatory arrest.     

Estrous cycle characteristics and response to estrus synchronization in mammoth asses (Equus asinus americanus)

Breeding records from a herd of mammoth asses (Equus asinus americanus) maintained on pasture in southeast Texas from 1990 to 1998 were reviewed. Jennies were pasture or hand mated, and estrus was either observed while the jennies were on pasture or when exposed to a jack after being penned. Eighty-one estrus periods and 43 diestrus intervals were recorded in 33 jennies over 4 seasons of the year (January-March, April-June, July-September, and October-December). Estrous cycle length and the duration of estrus were similar among seasons. Over all seasons, estrous cycle length was 23.3 +/- 2.6 d, duration of estrus was 5.9 +/- 2.1 d, and diestrus length was 17.4 +/- 2.6 d (mean +/- SD). During these same 9 yr, 58 injections of PGF2 alpha (5 mg, i.m.) were administered to 38 jennies without regard to stage of estrous cycle. Seventy-six percent (44/58) of the jennies showed signs of estrus after PGF2 alpha treatment, with an interval to estrus of 4.4 +/- 1.6 d and a duration of estrus of 5.6 +/- 1.7 d. Two estrus synchronization schemes were also assessed. Trial 1 was performed in October to November 1996, and Trial 2 was performed in February to March 1998. In Trial 1 (Group PE + PGF, n = 10), each jenny was injected intramuscularly once daily for 10 d with 150 mg progesterone and 10 mg estradiol-17 beta in sesame oil, and PGF2 alpha (10 mg) was injected intramuscularly on the last day of treatment. In Trial 2 (Group PGF-2X, n = 11), each jenny was injected intramuscularly twice, 16 d apart, with 10 mg PGF2 alpha. All Group PE + PGF jennies responded to treatment. One jenny in Group PGF-2X did not respond to either injection of PGF2 alpha, while 2 jennies responded to the first but not the second PGF2 alpha injection (8 of 11 jennies returned to estrus and ovulated after the second PGF2 alpha injection). Duration of estrus was 6.8 +/- 1.9 d for Group PE + PGF and 7.1 +/- 1.8 d for Group PGF-2X jennies. Interval to estrus and interval to ovulation following the last treatment were 9.0 +/- 0.9 d and 14.5 +/- 1.7 d, respectively, in Group PE + PGF jennies, and 4.5 +/- 0.9 d and 10.4 +/- 1.8 d, respectively, for Group PGF-2X jennies. In summary, estrous cycle characteristics of mammoth asses are similar to those reported for standard jennies, and estrus synchronization schemes used in horses are effective in mammoth asses.     

Transport of univalent cations across the erythrocyte membrane of hypertensive rats of various ages

In the erythrocytes incubated at low temperature (3-6 degrees C), the uptake of Li+ in 6- and 16-week old spontaneously hypertensive rats (SHR) was significantly higher than in the normotensive rats (WKY) of the same age. During the incubation of cells at 37 degrees C no difference occurred in either ouabain-sensitive or ouabain-resistant fluxes of Rb+, Na+ and Li+ between the 16-week old SHR and the WKY. K+ efflux from the erythrocytes at 3 degrees C was consistently stimulated after addition to the incubation medium of 1 mmol/l Ca2+. The value of Ca2+-dependent K+-transport was significantly elevated in 16-week old SHR than in the WKY, but there was no difference in 6-week old rats. Propranolol-induced Ca2+-dependent K+ efflux from the cells at 22 degrees C was markedly higher in 6- and 16-week old SHR as compared with the WKY. The results provide a further evidence in favor of the hypothesis on the existence of a "membrane defect" in red blood cells in the SHR.     

Mechanism for increase in tracheobronchial blood flow induced by hyperventilation of dry air in dogs

To test whether the consistent increase in tracheal and bronchial blood flow observed in dogs during hyperventilation of dry air might be the result of release of mediators such as vasodilatory prostaglandins or neuropeptides, we studied two groups of anesthetized mechanically ventilated dogs. Group 1 (n = 6) was hyperventilated for four 30-min periods with 1) warm humid air (38-40 degrees C, 100% relative humidity), 2) warm dry air (38-40 degrees C, 0% relative humidity), 3) warm humid air, and 4) warm dry air. After period 2, a loading dose of indomethacin (4 mg/kg iv) was given over 15 min followed by a constant infusion (4 mg.kg-1.h-1). Group 2 (n = 10) was hyperventilated for four 15- to 20-min periods by use of the protocol described above. After period 3 (group 2a) or period 2 (group 2b), topical 4% lidocaine hydrochloride solution was instilled into the trachea and main stem bronchi. Five minutes before the end of each period of hyperventilation, cardiac output and vascular pressures were measured. To determine airway blood flow, differently labeled radioactive microspheres were injected into the left atrium. After the last measurements, dogs were killed and the lungs excised. Blood flow to the trachea, main stem bronchi, and parenchyma (group 1 only) was calculated. Results showed that hyperventilation of dry air produced a significant increase in blood flow to the trachea and bronchi (period 2). In group 1, this increase was attenuated (P less than 0.02) after administration of indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in ovulation rate by active immunization against bovine inhibin-based synthetic peptides in a non-prolific sheep breed

The objective of this study was to explore the possibility of a recurrent increase in the ovulation rate of Malpura sheep, a non-prolific breed, by immunization against inhibin-based peptide immunogens over a period of 3 years. Adult ewes (4–7 years of age) and weighing between 28 and 38 kg were randomly allocated equally to three treatment groups. The immunization of the ewes was initiated during the autumn breeding season. Ewes were divided into three groups (n = 5 ewes/group) and actively immunized against the synthetic peptides from the α_C [bIα(1–29)-Tyr³⁰] (Group I) or α_N [bI-₄₃-Tyr¹⁵²(153-167)Cys¹⁶⁸] (Group II) area of the bovine inhibin α-subunit, conjugated to ovalbumin or against ovalbumin (control). Each ewe received a primary immunization of 400 μg immunogen and 3 booster injections, 200 μg immunogen each at 4-week intervals. Estrous was synchronized in all the ewes by administering two doses of PGF₂α at 10-day intervals for three consecutive years. Ovaries of ewes were examined each year between days 4 and 6 of the synchronized cycle, with the aid of the laparoscope to determine the ovulation rate. Active immunization significantly (p < 0.05) increased the ovulation rate. The overall ovulation rate, irrespective of the treatment period was 5.2 ± 0.44, 2.3 ± 0.38 and 0.9 ± 0.11 in Group I, Group II and the control, respectively. Although the beneficial effect of immunization on ovulation rate persisted for the entire period of the study, the interaction between immunization treatment and the time period was non-significant. The results clearly indicate that the active immunization against inhibin peptides can induce multiple ovulations in Malpura ewes and its effect on multiple ovulations is sustained for a prolonged period of time after the initial immunization.     

Antibacterial immunity of lower airways: local or localized?

Clearance of bacteria in the bronchoalveolar lavage, the level and functional activity of IgA and changes in the cellular composition of BAL were examined in mice after supralaryngeal immunization and subsequent challenge with Klebsiella pneumoniae. More than 60% of the bacterial inoculum was removed by nonspecific mechanisms within 90 min after inoculation; within the time interval 1.5-3.5 h, clearance was significantly accelerated in locally immunized mice. The enhancement of clearance effectiveness is specific and increases proportionally with the length of immunization (1 less than 2 less than 4 weeks); it is of short duration and towards the end of the 3rd week after immunization, in 73% of immunized animals, the clearance values did not differ from values found in controls. The local immunization did not influence the total level of IgA in BAL, the formation of specific IgA antibody was minimal, in vivo binding of IgA to klebsiella could not be demonstrated. In immunized mice, a significant increase in the numbers of PMN and lymphocytes, as well as an increased activity of phagocytic cell (PMN, MP) was found in BAL. The time interval of 1.5-3.5 h after challenge bounds the space for mechanisms, activated by local immunization in lower airways. The actual participation of individual factors in the accelerated elimination of bacteria from the lumen of airways, remains unclear so far.     

Removal of Pu and am from beagles and mice by 3,4,3-LICAM(C) or 3,4,3-LICAM(S)

Decorporation of Pu and Am by tetrameric catechoylamide (CAM) ligands has been investigated in beagles and mice. Eight dogs were injected intravenously (iv) with 237 + 239Pu(IV) + 241Am(III) citrate, and 30 min later, pairs of dogs were injected iv with 30 mumole/kg of 3,4,3-LICAM(C) [N1,N5,N10,N14-tetrakis(2,3-dihydroxy-5-sulfobenzoyl)tetr aazatetradecane, tetrasodium salt], 3,4,3-LICAM(S) [N1,N5,N10,N14-tetrakis(2,3-dihydroxy-4-carboxybenzoyl)te traazatetradecane, tetrasodium salt], CaNa3-DTPA, or each of the latter two ligands. Blood was sampled, and excreta were collected for 7 days, at which time the dogs were sacrificed and nuclide retention in liver and nonliver tissue was measured. Groups of five mice were each given 238Pu(IV) or 241Am(III) citrate iv; 3 min later 30 mumole/kg of a CAM ligand was injected intraperitoneally, mice were killed at 24 hr, and separated excreta and tissues were analyzed. In the dogs, average retention at 7 days of the injected Pu and Am, respectively, was as follows: 12 and 70% after treatment with a CAM ligand alone; 30 and 20% after DTPA; 12 and 20% after LICAM(S) plus DTPA; 90 and 89% without a ligand. In the mice, mean retention of the injected Pu and Am, respectively, was as follows: 14 and 66% after treatment with LICAM(C); 21 and 54% after LICAM(S); 91 and 87% without a ligand. In both species, about 99% of net Pu excretion (excretion with ligand - excretion without ligand) promoted in 24 hr by DTPA or LICAM(S) was in the urine, whereas about 10% of net Pu excretion promoted by the less hydrophilic LICAM(C) was in feces. Delayed excretion of both Am and Pu was significant in all ligand-treated dogs. Comparison of the nuclide content of tissues of ligand-treated mice with those of mice killed 3 min after nuclide injection indicated that the CAM ligands chelated circulating Pu and Am and prevented further deposition. In addition, the CAM ligands removed much of the presumably loosely bound Pu present in liver and skeleton at the time of ligand injection. LICAM(C) was more effective in removing Pu from liver and LICAM(S) was more effective in the skeleton. Moderate to severe uremia and histological evidence of cell killing in the distal tubules of the kidney were observed in the four dogs injected once with 30 mumole/kg of LICAM(S).(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental Babesia gibsoni infection in coyotes (Canis latrans)

Four 5 mo old captive raised coyotes (Canis latrans) were experimentally inoculated with approximately 1 x 10(6) Babesia gibsoni organisms. Parasites were detected 1 wk post-inoculation in all coyotes with maximum parasitemia of 8-11% occurring at 34 wk. Parasitemias remained at or above 1% for at least 12 wk and were still detectable 20 wk post-inoculation. All experimentally infected coyotes developed pale mucous membranes, splenomegaly, and a positive heme reaction in urine while one coyote exhibited mild depression and inappetence. Infected coyotes also developed a regenerative anemia, thrombocytopenia, and neutropenia. The mild clinical signs coupled with the high level and long duration of parasitemia indicate that coyotes could serve as reservoirs for B. gibsoni. Entrance of this foreign parasite into the United States suggests the need for strict quarantines and thorough health and blood film examinations for imported animals.     

Early colonization of the rat upper respiratory tract by temperature modulated Bordetella bronchiseptica.

The ability of nonmodulated Bvg+ phase cultures, temperature modulated Bvg- phase cultures, and a Bvg- phase-locked mutant of Bordetella bronchiseptica to colonize the rat upper respiratory tract was investigated. Initially, greater numbers of the temperature modulated Bvg- phase bacteria adhered to the nasal cavity of the rats. The temperature modulated Bvg- phase bacteria appeared to be quickly cleared to levels lower than the Bvg+ phase bacteria by 4 h after inoculation and stayed lower until 48 h after inoculation when colonization levels were equal to the Bvg+ phase bacteria. The level of colonization with the Bvg- phase-locked mutant of B. bronchiseptica was lower than both the nonmodulated Bvg+ phase and temperature modulated Bvg- phase cultures and declined over time during the experiment. These findings suggest that there may be increased adherence from an environmental phase to ensure bacteria survive initial clearance mechanisms until the switch to the Bvg+ phase occurs.     

Identification of moderately halophilic bacteria from Thai fermented fish ( pla-ra ) and proposal of Virgibacillus siamensis sp. nov

Forty-one isolates of moderately halophilic bacteria were isolated from fermented fish (pla-ra) in Thailand. On the basis of their phenotypic and chemotaxonomic characteristics, DNA-DNA relatedness and 16S rRNA gene sequences analyses, they were divided into six groups. The isolates in Group I to V were Gram-positive rod-shaped bacteria. They contained meso-diaminopimelic acid in the cell-wall peptidoglycan and menaquinone with seven isoprene units (MK-7). An isolate in Group VI was a Gram-negative rod-shaped bacterium. The DNA G+C contents of tested strains ranged from 36.5-63 mol%. Ten strains (Group I) were identified as Virgibacillus dokdonensis, 13 isolates (Group II) as V. halodenitrificans, 14 isolates (Group III) as V. marismortui, 1 isolate (Group IV) as Virgibacillus sp., 2 isolates (Group V) as Bacillus vietnamnensis, and 1 isolate (Group VI) as Chromohalobacter salexigens. Isolate MS3-4 in Group IV was closely related to V. carmonensis KCTC 3819(T) (95.9%). This strain contained anteiso-C(15:0) (55.8%) and anteiso-C(17:0) (17.7%) as major cellular fatty acids and had phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid as polar lipids. The DNA G+C content of MS3-4 was 38.0 mol%. The strain from Group IV is proposed as Virgibacillus siamensis sp. nov. and MS3-4(T) is the type strain (JCM 15395(T) =PCU 312(T) =TISTR 1957(T)).     

Animal age-, dose- and cell line-dependent growth of human neuroblastoma in nude mice. A statistical analysis.

Cells lines from human neuroblastoma (NB) and T/lymphoma (T-L) were injected subcutaneously (sc) in female CD1 nu/nu athymic nude mice. Results obtained after the observation of tumour growth were statistically analyzed by SAS. The following four parameters were considered: 1) dose of injected cells, 2) type of injected tumour (NB or T-L), 3) age of mice after individuation of three groups of animals (group A, 4-9 weeks old, group B, 9-20 weeks old, group C, > 20 weeks old), 4) injected cell line within the same tumour type. Latency time (LT), corresponding to the interval between cell inoculum and the appearance of a 5 mm diameter subcutaneous mass, and survival time (ST), corresponding to the interval between cell inoculum and the appearance of a 20 mm diameter subcutaneous tumour mass, were considered to evaluate tumour growth. Results showed that mass progression is affected by the number of injected cells and both LT and ST are age- and dose-dependent; furthermore, significant differences were recorded by using different NB and T-L cell lines. Group C showed longer LT than other groups; group B animals showed a statistically significant longer ST than groups A and C (p < 0.001). Our results indicate that growth of human NB in athymic mice is faster in young animals, which also show a significantly poorer prognosis, while better ST was observed in old and middle-aged animals. Results show statistically significant differences of both LT and ST in animals differing in age and in animals inoculated with different cell amounts. These results seem not to be related with biological properties of NB cells too, since neither the occurrence of MYCN amplification nor chromosome 1p deletion significantly modified such behaviour.     

Babesia gibsoni: An apical membrane antigen-1 homologue and its antibody response in the infected dogs

A cDNA encoding the apical membrane antigen-1 (AMA-1) homologue was obtained by immunoscreening a cDNA expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 2062bp. Computer analysis suggested that the sequence contains an open reading frame of 1794bp with a coding capacity of approximately 66kDa. Based on the homology analysis, this putative protein was designated as B. gibsoni AMA-1 (BgAMA-1). The BgAMA-1 gene was expressed in the Escherichia coli BL21 strain and used as the antigen in Western blotting and the enzyme-linked immunosorbent assay (ELISA). The results indicated that BgAMA-1 was recognized as an immunodominant antigen by the host immune system and that it induced a strong antibody response only in chronic B. gibsoni infection in dogs; however, the antibody response could not be detected in the early infection stage (within 15 days). This phenomenon might be explained by the limited stimulation with the low-abundance protein in the early infection stage. This result shows that BgAMA-1 is a new member of the AMA-1 family and that its immune response is characteristic of canine B. gibsoni infection.