Intracellular localization of Rickettsia tsutsugamushi in polymorphonuclear leukocytes.

Rickettsia tsutsugamushi (Gilliam strain) was serially propagated in BHK-21 cell cultures and incubated with guinea pig peritoneal polymorphonuclear leukocytes to study the ultrastructural features of rickettsial uptake and entry into the leukocytes. Significant numbers of rickettsiae were phagocytized selectively by these leukocytes within 30 min. About one-half of these rickettsiae remained sequestered in phagosomes but the other one-half were free from the phagosome and localized directly in the polymorphonuclear leukocyte cytoplasm. Various stages of rickettsial release from the phagosomes were observed. Once free within the polymorphonuclear leukocyte cytoplasm, the rickettsiae were preferentially localized in the glycogen-packed areas which are devoid of lysosomes and other cytoplasmic organelles. This study indicates that rickettsiae phagocytized by polymorphonuclear leukocytes can escape from the phagosome into the cytoplasm.

Clinical and biological aspects of infection caused by Ehrlichia chaffeensis.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects the monocyte-macrophage. E. chaffeensis, which is transmitted to humans by ticks primarily from infected deer, causes human monocytic ehrlichiosis, an acute febrile systemic illness. This paper reviews current knowledge of clinical and biological aspects of infections caused by E. chaffeensis.

Gossypolone suppresses progesterone synthesis in bovine luteal cells.

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc) activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 microM) induced morphological changes in cellular organelles.

Ehrlichia sennetsu groE operon and antigenic properties of the GroEL homolog.

A clone expressing an immunoreactive 55-kilodalton (kDa) protein of Ehrlichia sennetsu, the causative agent of human Sennetsu ehrlichiosis, was isolated from a gene library of this organism. Sequence analysis of the DNA insert revealed two open reading frames, encoding proteins of 10,620 and 58,225 kDa, respectively. These deduced amino acid sequences were homologous to those of the GroES and GroEL heat shock proteins (HSP) of other bacteria, respectively. Phylogenetic trees based on GroES and GroEL homologs of several bacteria including E. sennetsu showed a relationship similar to that based on 16S rRNA gene sequences. The recombinant and native 55-kDa proteins of E. sennetsu, GroEL homolog, reacted with a monoclonal antibody (SPA807) which recognizes a homologous sequence between human and mycobacterial HSP60 and a polyclonal antibody (SPA804) to cyanobacteria HSP60, but not with antibodies to HSP60 of several other organisms used. Furthermore, anti-recombinant E. sennetsu 55-kDa protein antibody prepared in a rabbit was reactive to HSP60 antigens of other Ehrlichia and Rickettsia species, but not GroEL of E. coli. The recombinant 55-kDa protein would be a useful tool for studying the role of this antigen in the immune response to E. sennetsu infection.

Effects of age and luteinizing hormone antiserum on human chorionic gonadotropin-stimulated testosterone secretion in young male rats.

The steroidogenic capacity of young male rats of different ages was studied. Two days prior to sacrifice at 5, 10, 15, 20, 25 and 30 days of age, the rats in treatment groups were given intramuscularly either human chorionic gonadotropin (HCG) at 20 I.U. twice daily/rat or luteinizing hormone (LH) antiserum (AS) at 0.25 ml twice daily/rat. Either saline or normal sheep serum (NSS) was given to control rats. The serum and testicular testosterone concentrations in the control rats averaged 0.85 +/- 0.03 ng/ml and 1.35 +/- 0.06 ng/mg testicular protein, respectively. At day-15 the serum and testicular testosterone concentrations in the HCG-treated rats had significantly increased to 9.30 +/- 0.85 ng/ml and 11.92 ng/mg of testicular protein, respectively. At the same age, the HCG-induced higher levels of serum and testicular testosterone concentrations were significantly reduced to 2.80 +/- 0.70 ng/ml and 6.02 +/- 1.00 ng/mg protein by concomitant administration of LH/AS and HCG. Our results suggest that the testosterone production in response to HCG stimulation is age-related. It was also determined that neutralization of circulating gonadotropin in LH/AS-treated rats decreased the sensitivity of Leydig cells to gonadotropin stimulation. This in vivo model should provide an excellent opportunity for the investigation of the testicular function in developing young males.

Inhibitory effect of gossypol on human chorionic gonadotropin (hCG)-induced progesterone secretion in cultured bovine luteal cells.

Inhibitory effects of gossypol on the female reproductive system have been recently reported. This study investigated a possible site of gossypol action on progesterone synthesis. Bovine luteal cells were cultured with hCG and forskolin in the presence or absence of gossypol. At 10 micrograms/ml, gossypol significantly inhibited hCG- and forskolin-stimulated progesterone secretion and intracellular cAMP formation; at 20 micrograms/ml, gossypol completely abolished the stimulative effect of hCG and forskolin. The results suggest that adenylate cyclase may be a site of gossypol action on steroidogenesis of bovine luteal cells.

Antifertility effect of gossypol in male Japanese quail.

Conventionally housed 130-160 g adult male Japanese quail were given gossypol acetic acid (gossypol) im at 25 mg/kg in 0.5 ml of 10% EtOH for 12 and 24 days (Groups 1 and 2), respectively. One day after treatment was terminated they were allowed to mate with laying females individually for 20 days. Fertility was 0% from mating of the Group 1 birds on days 1-2 and increased to 25, 35, 55 and 65% on days 3-6 after cessation of gossypol treatment. At day 11, fertility was 80 vs 84% in controls, whereas hatchability was 70% for both. By comparison, eggs from Group 2 mated quail were infertile for up to 20 days after the termination of gossypol treatment. In a parallel experiment, the percent testes to body weight ratio in control and 7, 14, 21, and 28-day gossypol-treated quail was 2.5, 2.2, 1.8, 0.5, and 0.2%, respectively. In 12 vs 24-day treated birds, 7, 14, 21, and 28 days after gossypol treatment, the ratios were 1.0 vs 0.5%, 2.0 vs 0.8% and 2.8 vs 1.9%, respectively. The decreased fertility and hatachability, and testicular atrophy resulting from gossypol given to male quail was dose-time related. Furthermore, the androgen-dependent cloacal gland was drastically reduced in size by the treatment with gossypol. The mode of action of gossypol in male quail is different than it is in mammals in that the testicular size of mammals remains unchanged with long-term gossypol treatment. It is concluded that quail may be a useful avian animal model for investigating the antifertility effects of gossypol.

Gossypol in female fertility control: ovum implantation and early pregnancy inhibited in rats.

Intramuscular administration of gossypol to normally cycling female rats induced an irregularity of the cyclic pattern for as long as the treatment was continued. Furthermore, administration of gossypol from days 0 (day of sperm-positive vaginal smear) to 8 of pregnancy prevented the normal maintenance of pregnancy. Serum values of progesterone and estradiol 17 beta in gossypol-treated normally cycling and pregnant rats were significantly lower than the control levels. The supplement of a combination of exogenous progesterone and estradiol 17 beta eliminated the inhibitory effects of gossypol on ovum implantation and the maintenance of pregnancy. Our results indicate that gossypol may have some usefulness in female fertility control.

Binding of 3H-gossypol in organelles of cultured bovine luteal cells.

Gossypol is an antifertility agent which inhibits steroidogenesis in both sexes. The present study investigated the binding of gossypol in organelles of cultured bovine luteal cell to elucidate its inhibitory site of action in steroid biosynthesis. Cultured bovine luteal cells were incubated with 3H-gossypol (4.3 or 2.15 microM) for 3 hours. At the end of treatment, cultured bovine luteal cells were harvested, homogenized and centrifuged for organelle preparation. The radioactivity of gossypol was measured in each subcellular fraction. The cell membrane fraction has the highest binding capacity for gossypol, and the majority of gossypol was located in the particulate fractions. Results of the present study provide information in understanding the regulatory mechanism of gossypol on antisteroidogenic and/or toxic effects in cultured bovine luteal cells.

Inhibition of in vitro fertilization and early embryonic development in hamsters by gossypol.

We have previously reported an inhibitory effect of gossypol and its metabolite on bovine and mouse early embryonic development. In the present study, eggs were collected from oviducts of superovulated hamsters. Epididymal sperm were used for in vitro fertilization (IVF). Gossypol at 5, 10, and 30 micrograms/ml significantly inhibited the formation of 2 pronuclei by 45, 65 and 95%, respectively. On the first day of pregnancy, hamsters were given an intrauterine treatment of 200 micrograms of gossypol in 100 microliters of corn oil per uterine horn. On day 3, embryos from controls were in morula (65%) and early morula (17%) stages, while less than 2% of embryos from the gossypol-treated hamsters were in the morula stage. The numbers of embryo implantation sites on day 8 and pups in controls (14 +/- 2.0 and 12 +/- 1.5, respectively) were significantly higher than those in the gossypol-treated hamsters (8.5 +/- 2.0 and 4.0 +/- 1.5, respectively). Our results suggest that gossypol is able to affect fertilization, embryonic development, embryo implantation, and the number of pups in hamsters through a not-yet-defined mechanism.

Protection against murine potomac horse fever by an inactivated Ehrlichia risticii vaccine.

Ehrlichia risticii propagated in a murine macrophage cell line were freed from the host cell by hypotonic lysis of the infected cells. The cell-free ehrlichiae were inactivated with beta-propiolactone and combined or not combined with polymyxin-B. The vaccines were administered to mice with Quil-A (saponin) as an adjuvant twice at 2 to 3 week intervals and the mice were challenged with live E. risticii 2 to 3 weeks after the last vaccination. With or without the addition of polymyxin-B, the vaccine preparations protected mice from developing clinical signs and gross pathologic changes such as thymic atrophy, splenomegaly, and increase in whole intestinal weight. Mice vaccinated with or without polymyxin-B developed high titer IgG antibody against E. risticii before and after the challenge with live E. risticii. Spleen lymphocyte proliferative response assay at 11 days post challenge revealed that with polymyxin-B a higher lymphocyte proliferation occurred as compared with that of the mice which received polymyxin-B-free vaccine. Spleen lymphocytes of the placebo (polymyxin-B and Quil-A) pretreated/challenged mice showed no proliferative activity. Western blot analysis revealed that vaccinated mice reacted mainly with 110, 57 and 33 kDa antigen bands before and after challenge. The placebo (polymyxin-B and Quil-A)/challenged mice showed a very weak response to ehrlichial antigens at day 10 to 11 post challenge. Comparison with inactivated Renografin-purified E. risticii or 0.25% SDS-insoluble fraction of E. risticii with the inactivated host cell-free vaccine revealed no increased protection. These results indicate that inactivated host cell-free E. risticii can protect mice from murine Potomac horse fever. The presence of polymyxin-B appeared to be not harmful but rather beneficial for lymphocyte proliferation response upon challenge with live E. risticii.

Application of the polymerase chain reaction for the detection of Ehrlichia canis in tissues of dogs.

Doxycycline treatment resulted in a carrier status in 3 dogs and complete clearance of Ehrlichia canis in 2 dogs (Iqbal and Rikihisa, 1994). Using specimens obtained during that study applicability of polymerase chain reactions (PCRs) in detecting E. canis DNA in tissue specimens and correlation of PCR results with our previous cell culture isolation results were evaluated. PCRs using a pair of primers specific to E. canis 16SrRNA gene sequence were used to detect DNA of E. canis in tissues of 5 experimentally-infected dogs 2 months after doxycycline treatment. An approximately 600 bp product defined by the specific primers was amplified in blood, kidney, lymph nodes, liver, and/or spleen of 3 dogs from which E. canis was reisolated in cell culture. In contrast, E. canis DNA was not detected in tissue or blood specimens of the 2 dogs from which E. canis was not reisolated after doxycycline treatment or in 2 control uninfected dogs. The findings indicate PCR is effective in detecting E. canis in tissues.

Effect of antibiotics on clinical, pathologic and immunologic responses in murine Potomac horse fever: protective effects of doxycycline.

Effects of three antibiotics on clinical, pathologic and immunologic responses in murine Potomac horse fever caused by Ehrlichia risticii infection were examined. When antibiotics were given after the development of clinical signs, antibiotics ranked in the order of reducing clinical signs and in preventing body weight loss and an intestinal enlargement were doxycycline, demeclocycline and rifampin. Infected mice treated with doxycycline and demeclocycline developed greater splenomegaly than rifampin-treated or untreated infected mice. All antibiotics used prevented thymic atrophy due to E. risticii infection. Indirect fluorescent antibody titers were highest with doxycycline treatment. Mice treated with demeclocycline and rifampin produced higher antibody titer than those without treatment. Ehrlichia risticii was reisolated from the spleens of both untreated and rifampin-treated infected mice. The effects of administering single doses of doxycycline at different times after infection were examined. Body weight loss was prevented by the drug given at every treatment day examined, i.e. Days 3, 5 and 7 post-infection (PI). Thymic atrophy was minimum in mice treated at Day 5 PI, while splenomegaly was found on every treatment day. Splenocyte proliferative response to concanavalin A and lipopolysaccharide, and specific antibody development against E. risticii was best in mice treated at Day 5 PI followed by those treated at Day 3 and Day 7 PI.

Development of neutralizing antibody in horses infected with Ehrlichia risticii.

The role of the humoral immune response in ehrlichial infection is unknown. Development of neutralizing antibodies during a course of Ehrlichia risticii infection in a pony was examined in vitro by determining the inhibition of E. risticii infection of P388D1 cells in the presence of the sera. The pony experimentally infected with E. risticii developed significant neutralizing activity in the sera by 15 days postinfection when parasitemia started to decline. Neutralizing activity continued to rise after recovery from the disease up to 34 days postinfection at which time the experiment was terminated. In vitro neutralizing activities in the sera from 3 additional ponies infected with E. risticii were lower at 2 weeks than at 4 weeks postinfection. The sera from vaccinated/challenged ponies had comparable neutralizing activity to those of the recovered ponies at approximately 3 to 4 weeks postchallenge. Equine sera from infected or vaccinated/challenged ponies were also effective in protecting mice from E. risticii infection. These studies demonstrated the significant development of neutralizing activity in the sera of recovered or vaccinated/challenged ponies.

Effect of gossypol on thyroid hormones in young female rats.

The study was designed to examine the effect of gossypol on thyroid function in young female rats. Forty young Sprague-Dawley female rats were randomly assigned to four treatment groups. The dosages of gossypol acetic acid (gossypol) used for this experiment were 1, 5 and 10 mg/kg body weight/day in 0.5 ml potassium phosphate buffer subcutaneously started from day 34 of age for 15 consecutive days. The control group was treated with vehicle alone. Animals were sacrificed 15 days after gossypol treatment. Blood samples and sera were collected and prepared for measurement of thyroid hormones by radioimmunoassay. The levels of free thyroxine (T4), 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (reverse T3, rT3) in control rats at age 49 days averaged 55.90 +/- 7.7, 670 +/- 90, and 71.7 +/- 5.7 pg/ml of serum, respectively. Fifteen days after gossypol treatment at 5 and 10 mg/kg/day, the free T4 levels significantly decreased to 21.96 +/- 2.1 and 11.12 +/- 1.5 pg/ml of serum, the T3 levels significantly decreased to 430 +/- 89, 359 +/- 90 pg/ml of serum and the rT3 levels were significantly decreased to 37.20 +/- 7.3, and 24.20 +/- 6.0 pg/ml of serum, respectively. However, the serum levels of free T4, T3 and rT3 in the 1 mg/kg/day of gossypol-treated animals were not significantly different from the controls. Five to 15 days after initiation of gossypol treatment, both body weight gain and food intake appeared to be significantly reduced at 5 and 10 mg/kg/day of gossypol treatment. This trend continued to the end of the 15-day gossypol treatment. The results provide new evidence that gossypol might have exerted its antithyroid function by an unknown mechanism that triggered an interference in body metabolism, thus causing the loss of food intake and body weight gain in young female animals.

Effect of gossypol on spermatozoal lactate dehydrogenase-X (LDH-X) in male rats.

The studies in this report evaluate the inhibitory effects of long-term gossypol acetic acid (gossypol) administration on spermatozoal lactate dehydrogenase (LDH-X) activity, fertility and reversibility. Gossypol at 20 mg/kg/day or vehicle only was administered orally to adult male rats for 5 consecutive weeks. Groups of control and gossypol-treated rats were sacrificed at 2-, 4-, and 5-week intervals when LDH-X analysis of spermatozoa were prepared from the tail of the epididymis. An additional group of control and gossypol-treated rats were allowed to recover for up to 8 weeks after withdrawal of the treatment. In the gossypol-treated rats, the spermatozoal LDH-X activity was depressed by 80% of control level after a 5-week treatment period. There was no significant difference in spermatozoal LDH-X activity between the control and treated animals at 2 and 4 weeks. However, the number of ejaculated sperm estimated from the vaginal smear preparations after each mating was significantly less than the control values after 2, 4 and 5 weeks of treatment. At 5 weeks after the cessation of gossypol treatment, the spermatozoal LDH-X activity had only partially recovered but the fertility (number of ejaculated sperm, embryo implantation sites and pups born) had recovered to a level comparable to the control values 5 weeks after the cessation of gossypol treatment. The inconsistencies in the response of spermatozoal LDH-X activity, sperm number and fertility to gossypol may suggest that the antifertility action of gossypol and spermatozoal LDH-X activity in adult male rats may not be directly related as suggested by the results generated from the in vitro studies.

Suppression of Ia antigen expression on gamma interferon treated macrophages infected with Ehrlichia risticii.

Ehrlichia risticii is an obligate intracellular bacterium of monocytes/macrophages. In this report, using immunofluorescence staining, flow cytometry, and Kolmogorov-Smirnov analysis of histograms, the response of P338D1 and peritoneal macrophages stimulated with recombinant murine interferon-gamma (rIFN-gamma) was examined for the expression of major histocompatibility complex Class II gene product (Ia) and effect of E. risticii infection on induction of Ia surface expression. Maximal expression of Ia by sham-infected P388D1 cells was observed 2 days post rIFN-gamma addition followed by a progressive decline. These stimulatory effects of rIFN-gamma were dose dependent. Relative to sham-infected P388D1 cells, the induction of Ia by rIFN-gamma (200 U ml-1) on E. risticii-infected P388D1 cells was significantly suppressed at each time point tested through Day 5 with maximal suppression of 88% occurring on Day 2. Similarly, the induction of Ia by rIFN-gamma on E. risticii-infected peritoneal macrophages was significantly suppressed by 77% (fluorescent microscopy) when compared to sham-infected peritoneal macrophages. The higher dose of rIFN-gamma (2000 U ml-1) failed to restore Ia surface expression by E. risticii-infected P388D1 cells. The suppression of Ia on P388D1 cells in response to RIFN-gamma was not related to the degree of infection of these cells by E. risticii. A soluble inhibitor substance was not demonstrable in the supernatant from E. risticii-infected cells, nor were inhibitor levels of prostaglandin E2 levels found in the supernatant. Suppression of surface Ia expression on the macrophage suggests a mechanism whereby I. risticii may evade T-lymphocyte recognition, hinder antigen-specific T-lymphocyte activation, and promote their own survival.

Porcine lymphoblastoid cell lines of B-cell origin.

Two lymphoblastoid cell lines were isolated from different pigs and were maintained in culture for over 100 passages or 20 months. These cell lines were characterized by their cell surface antigens, ability to stimulate a mixed lymphocyte reaction and production of immunoglobulin. When tested against a panel of monoclonal anti-cell surface antigen antibodies, only those monoclonal antibodies which detect porcine class I or II molecules reacted against the lymphoblastoid cell lines in a microcytotoxicity assay. The two pig cell lines could stimulate peripheral blood mononuclear cells in a mixed lymphocyte reaction. P-SC(1) and P-16(2) also demonstrated a dependency upon the presence of 2-mercaptoethanol for cell division. The secretion of pig immunoglobulin by P-SC(1) or P-16(2) was first demonstrated by ELISA using a polyclonal anti-swine IgG (heavy and light chain) serum. By the use of monoclonal anti-IgA, IgG or IgM antibodies in an enzyme-linked assay on Western blots of P-SC(1) or P-16(2) lysate/supernatant, the two cell lines were demonstrated to be producing a whole monomeric IgA molecule and a mu chain.

Preparation of anti-canine serum amyloid A (SAA) serum and purification of SAA from canine high-density lipoprotein.

Antiserum to canine serum amyloid A (SAA) was prepared in rabbits by immunization with crude SAA which was prepared from high-density lipoprotein 3 (HDL3) obtained from canine acute-phase serum. The antiserum was absorbed for contaminating antibodies by affinity chromatography using Sepharose 4B coupled with normal canine serum proteins. The rabbit anti-canine SAA serum reacted with a protein and formed a single precipitin line at the position of the alpha 1-region of the immunoelectrophoresis of canine acute-phase serum but did not react with the normal canine serum on immunoelectrophoresis. The antibody to canine SAA was also confirmed by Western blotting analysis. Canine SAA was purified as a low molecular weight protein component from crude SAA by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after gel filtration chromatography. Purified canine SAA had a molecular weight of 15,000 as estimated by SDS-PAGE. This SAA level was found by enzyme-linked immunosorbent assay (ELISA) to increase 1 day after inoculation with Bordetella bronchiseptica to 9.0-20.1 times the preinoculation value.