Resistance of experimental antibiotic resistant variants of Yersinia pestis EV76 (NIIEG line) to modern marker antibiotics]

Lower susceptibility of previously designed experimental polyresistant variants of Yersinia pestis EV76 (NIIEG line) with inserted R plasmid or transposons to some present antibiotics efficient in the treatment of plague, i. e. doxycycline, amikacin and ceftriaxone, was shown. Clones, more resistant to them vs. the initial variants, were selected. They accustomed in vivo in laboratory animals per se and after administration of antibiotics. The data on the protective activity of the new variants in the treatment of experimental plague after combined vaccination and antibiotic prophylaxis are presented.     

Doxycycline in the prevention of experimental plague induced by plague microbe variants]

A comparative study was performed on the efficacy of doxycycline in experimental plague infection induced in albino mice by strain 231 of the plague microbe and its variant 231 Fra- deprived of the ability to produce the fraction I antigen. It was shown that the LD50 for strain 231 during animal treatment with doxycycline was significantly higher than that for variant 231 Fra-. Prophylaxis of the plague infection caused by the Fra- forms of the plague microbe required significantly higher doses of doxycycline (ED50) than that of the infection caused by the Fra+ forms. The use of the daily maximum permissible doses of doxycycline (50 to 100 mg/kg a day) for 10 days in treatment of albino mice infected with the strain Fra- did not provide animal survival at the level higher than 60 to 70 per cent while the survival rate in the animals infected with the strain Fra+ of the plague microbe and treated according to the same scheme amounted to 90-100 per cent. The lower therapeutic efficacy of doxycycline in the treatment of the infection caused by the fractionless variant of the plague microbe should be considered in development of rational schemes for prophylaxis and treatment of plague.     

Lack of levofloxacin and moxyfloxacin efficacy in experimental plague of albino mice infected with nalidixic acid resistant pathogen (Nal(r))

The efficacy of levofloxacin and moxyfloxacin vs. the previously tested fluoroquinolones was studied on albino mice with experimental plague due to the Nal(r) mutants of Yersinia pestis 231 and 231 FI-. The plague microbe mutants resistant to nalidixic acid (Nal(r)) generated at a frequency of 10(-10)-10(-9). The resistance to nalidixic acid was not accompanied by the strains loss of the virulence. The Nal(r) mutants were cross resistant to fluoroquinolones (ciprofloxacin, moxyfloxacin). The LD50 for the nontreated animals did not differ from that for the mice treated with nalidixic acid and the fluoroquinolones (when the animals were infected with Nal(r) mutants). The results showed that the criteria of the plague microbe susceptibility/resistance to fluoroquinolones should be revised.     

Transmission of crayfish plague

Two possible means of transmission of crayfish plague were investigated: via fish (as vectors), and via crayfish (as hosts or vectors when dead). The crayfish transmission experiments focussed on both the viability of the fungus in dead crayfish when kept in simulated field conditions, and on the treatments which kill viable forms of Aphanomyces astaci within the recently dead host (cadaver). It was found that A. astaci remains viable for 5 d, and possibly longer in crayfish kept in water at 21 degrees C after dying of crayfish plague. Heating (boiling for 1 min) was the quickest way of decontaminating crayfish cadavers. Freezing took considerably longer: after 48 h at -20 degrees C, viable stages were still present. It was also found that A. astaci is unlikely to survive passage of the gastrointestinal tract of either mammals or birds as no viable stages were found after 12 h at 37 degrees C. Two basic modes of transmission of crayfish plague via fish were investigated: (1) after passage of initially viable forms of A. astaci through the digestive tract of fish and (2) via fish skin. If A. astaci was fed to fish as infected abdominal cuticle, it was still viable after passage through the gastrointestinal tract. When pure mycelium or spores were fed to fish there was no indication of viable forms of A. astaci after passage through the gastrointestinal tract. Transmission via fish skin was not observed under the experimental conditions applied. The results on fish as vectors have practical importance for fish transport and stocking because the present study shows that there is a risk of transmission of crayfish plague via fish faeces. The investigation of crayfish as vectors delivers methods that could be used for treatment of crayfish imported for human consumption into crayfish-plague-free areas. This application is particularly important for the importation of American crayfish, which are in general suspected to carry A. astaci in their cuticle.     

Experimental evaluation of prospects for the use of beta-lactams in plague infection caused by pathogens with plasmid resistance to penicillins]

High therapeutic efficacies of ceftriaxone, ceftazidime, cefotaxime and azthreonam in the treatment of experimental plague induced by beta-lactamase-producing strains of the plague microbe containing R plasmids RP-1, R57b and R40a were shown to correlate with their in vitro antibacterial activities. The therapeutic efficacy of sulbactam/ampicillin was recorded in the treatment of plague induced by the strain containing R plasmids R57b and R40a (the treatment course of 7 days). However, it was lower when the infection was due to the strain containing plasmid RP-1 (beta-lactamase TEM-2). Cefoperazone was not active in the treatment of experimental plague induced by the strains containing plasmids RP-1 and R57b (beta-lactamases TEM-2 and OXA-3). Ceftriaxone versus the antibiotics tested was considered to be the drug of choice for the etiotropic therapy of plague induced not only by the type strains of the plague microbe but also by its variants with the plasmid pattern resistance to penicillins.     

Antibiotics of the aminoglycoside group (gentamicin, sisomicin and amikacin) in the prevention and treatment of experimental plague]

Activity of aminoglycosides such as gentamicin, sisomicin and amikacin against plague microbe strains of natural origin was studied in vitro. It was also studied in prophylaxis and treatment of experimental plague infection in albino mice. The MAC of gentamicin and sisomicin for 50 strains of the plague microbe was 0.2-1.6 micrograms/ml. For the majority of the strains it was 0.4 micrograms/ml. The amikacin MICs were 0.4-3.2 and 0.8 micrograms/ml, respectively. High efficacy of gentamicin, sisomicin and amikacin was shown in prophylaxis and treatment of experimental plague infection in albino mice. The optimal doses of the antibiotics were determined. Under definite conditions such as the use of short-term regimens and higher intervals, advantages of sisomicin over gentamicin and amikacin in prophylaxis of experimental plague infection were observed.     

Isepamycin in prophylaxis and treatment of experimental plague due to FI+ and FI- variants of plague microbe]

The efficacy of isepamycin vs. other aminoglycosides was studied in vitro and on albino mice with experimental plague due to natural antigen valuable strains of the plague microbe and the pathogen variants deprived of the ability to produce the capsular antigen fraction I (FI- phenotype). The MICs of isepamycin for the strains of the plague microbe (20 FI+ and 20FI-) were 1.0-4.0 mg\l, that did not differ from those of streptomycin, kanamycin, amikacin and tobramycin. The ED50 of isepamycin in the prophylaxis and treatment of the experimental plague of the mice had no statistically significant differences from the ED50 of the other aminoglycosides. The efficacy index of isepamycin was > 10(4), that did not differ from that of streptomycin, amikacin and gentamicin, irrespective of the strain phenotype (Y. pestis 231 FI+ or Y. pestis 231 FI-). The same as the other aminoglycosides, isepamycin in doses equivalent to the human average daily doses, protected 80-100% of the albino mice from death when used in the prophylaxis and therapy of plague irrespective of the strain phenotype. The results of the study made it possible to consider isepamycin as an agent promising for the prophylaxis and treatment of plague.     

The prostate response to prolactin modulation in adult castrated rats subjected to testosterone replacement

Despite the androgenic dependence, other hormones, growth factors, and cytokines are necessary to support prostatic growth and maintain the glandular structure; among them, prolactin is a non-steroidal hormone secreted mainly by the pituitary gland. However, extra-pituitary expression of prolactin, such as in the prostate, has also been demonstrated, highlighting the paracrine and autocrine actions of prolactin within the prostate. Here, we investigated whether prolactin modulation alters ventral prostate (VP) morphophysiology in adult castrated rats. Sprague Dawley rats were castrated and after 21 days, divided into ten experimental groups (n?=?6/group): castrated control: castrated animals that did not receive treatment; castrated+testosterone: castrated animals that received T (4 mg/kg/day); castrated+PRL (PRL): castrated animals receiving prolactin (0.3 mg/kg/day); castrated+T+PRL: castrated animals that received a combination of testosterone and prolactin; and castrated+bromocriptine (BR): castrated animals that received bromocriptine (0.4 mg/kg/day). The control group included intact animals. The animals were treated for 3 or 10 consecutive days. At the end of experimental period, the animals were euthanized, and the blood and VP lobes were collected and analyzed by different methods. The main findings were that the administration of prolactin to castrated rats did not exert anabolic effects on the VP. Although we observed activation of downstream prolactin signaling after prolactin administration, this was not enough to overcome the prostatic androgen deficiency. Likewise, there was no additional glandular involution in the castrated group treated with bromocriptine. We concluded that despite stimulating the downstream signaling pathway, exogenous prolactin does not act on VP in the absence or presence of high levels of testosterone.     

Further study of the effectiveness of oral immunization against plague in experiments on animals]

It was shown in comparative experiments on guinea pigs that the oral method of immunization with plague vaccine was much less effective than the inhalation and subcutaneous. Guinea pigs were found to be a poor model for studying the efficacy of oral immunization with dry live plague vaccine rehydrated before use: with the use of this preparation the animals were 46 times less resistant to aerosol plague infection than in application of strain EB cultured in agar.     

Chemotherapeutic effectiveness of a new derivative of 5-alkyl-3N-furanones in experimental staphylococcal infection

High chemotherapeutic efficacy of the compound 1929, a new derivative of 5-alkyl-3H-furanones was shown in albino mice with experimental staphylococcal infection caused by intraperitoneal administration to the animals. The efficacy was found to be higher than that of furagin used for comparison. The average therapeutic dose (AD50) of the compound for intraperitoneal administration amounted to 40 mg/kg while the average lethal dose (LD50) was 3000 mg/kg.     

Use of monoclonal antibodies to plague microbe antigens at the early stage of infection in white mice for enhancement of the late streptomycin and doxycycline treatment]

It was demonstrated that use for prophylaxy (after 5 h of infection) or for treatment (after 24 h after infection) of the monoclonal antibodies mixture to specific epitops of capsule antigen (fraction 1), lipopolysacharide, murine toxine can prevent development of plague pathogen at 100 of mice infected by approximately 1000 LD50 Yersinia pestis 231. 5-day course of prophylaxy by monoclonal antibodies provided survival of 50 per cent animals. Subsequent use of fraction 1 antigen for 5 days followed by treatment with streptomycin or doxycycline at 6-7-8-9-10 days after infection with Y. pestis 231 prevented infection manifestation at 80 per cent of animals, etiotropic therapy started at the same period was ineffective. When white mice were infected with Y. pestis 231 Fra-, with deleted ability to produce capsule antigen (fraction 1) 80% level of efficacy can be provided by subsequent administration of antibodies to fraction 1 combinated with lipopolysacharide, murine toxine and streptomycin. Use of monoclonal antibodies followed by doxycycline was ineffective.     

Allogeneic hepatic cell suspension in the treatment of liver insufficiency

A study was made of a possibility of using isolated hepatocytes for the treatment of diseases of the liver in animals. The optimal dose of the cell suspension in intravascular, intraperitoneal, intrapleural and subcutaneous administration was determined; a reaction of the experimental animals to this biological substrate was studied. The efficacy of the isolated hepatocytes in the treatment of hepatic insufficiency is demonstrated; also a comparative assessment of the mentioned methods of the cell suspension administration is given.     

Histopathological observation of immunized rhesus macaques with plague vaccines after subcutaneous infection of Yersinia pestis

In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 μg F1 and 10 μg rV270) and SV2 (200 μg F1 and 100 μg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×10(6) CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague.     

Effectiveness of phosphomycin and its combinations with amikacin and cefotaxime in experimental plague infection]

When administered intramuscularly in doses of 8 and 16 mg/mouse, phosphomycin was highly active in the treatment of albino mice with experimental plague infection (80-100-percent protection of the animals from death). Combinations of phosphomycin with cefotaxime in inefficient or not sufficiently efficient doses had a synergistic effect. When the albino mice were treated with combinations of phosphomycin and amikacin, the percentage of the survived animals significantly increased in comparison to that after the use of the antibiotics alone.     

Licking behaviour induces partial anthelmintic efficacy of ivermectin pour-on formulation in untreated cattle

Licking behaviour in cattle has been reported to account for the disposition of topically administered macrocyclic lactones. However, its impact on anthelmintic efficacy remains to be established. Therefore, we evaluated the impact of ivermectin exchange between cattle on the reduction in the faecal egg count (FEC) after pour-on administration in a group of 10 heifers experimentally infected with Ostertagia ostertagi and Cooperia oncophora. Four treated (500 μg/kg, pour-on) and six untreated animals were put together after treatment and plasma and faecal exposure to ivermectin as well as the FECs were evaluated before and over 40 days after treatment. Ivermectin was detected in plasma and faeces of the six untreated heifers, with maximal exposures two- to three-fold lower than the minimal exposures in treated animals. The interindividual variability of exposure was very high in untreated animals, with a ten-fold difference between the upper and lower limits compared with treated heifers, where there was only a two-fold difference. Anthelmintic efficacy, expressed as an average reduction of the FECs over the experimental period, was maximal in the treated group. In untreated heifers, anthelmintic efficacies ranged from zero to maximal efficacy, with intermediary values between 30% and 80%. The use of a classical pharmacodynamic model demonstrated a clear relationship between exposure and efficacy and enabled us to define the critical plasma or faecal ivermectin concentrations delimiting an exposure window associated with partial anthelmintic efficacy. This range of ivermectin plasma concentrations (0.1-1 ng/mL) could be considered as a potential selection window for anthelmintic resistance. Finally, our results show that macrocyclic lactone exchange between cattle after pour-on administration, resulting from natural grooming behaviour, can significantly impact on anthelmintic efficacy. This raises several issues such as the design of comparative clinical trials and the occurrence of partial efficacy which is considered a risk factor for the development of anthelmintic resistance.     

Efficacy of beta-cyclodextrin against experimental cryptosporidiosis in neonatal lambs

The efficacy of beta-cyclodextrin against experimental Cryptosporidium parvum infection was evaluated in neonatal lambs. The animals were treated by oral administration of the drug at 1 g/kg of body weight during 3 consecutive days. Preventive treatment was started within 1 day of birth, and therapeutic treatment was initiated at the onset of diarrhea following confirmation of infection. Disease development and drug efficacy were evaluated by monitoring the presence or absence of diarrhea and oocyst shedding from birth until 30 days of age. Weight gains at 15 and 30 days of age were also recorded. Beta-cyclodextrin was highly effective as a prophylactic treatment; 1 animal did not acquire the infection, diarrhea was prevented in infected animals, and there was a considerable decrease in oocyst shedding. The therapeutic treatment was effective in decreasing the severity of diarrhea and the duration of oocyst shedding. The animals tolerated the drug well, and there was a significant increase in their body weights.     

Detection of micronucleated cells and gene expression changes in glandular stomach of mice treated with stomach-targeted carcinogens

To assess the genotoxicity of chemicals on the stomach, we developed in vivo assays that can detect micronucleus induction and gene expression changes in epithelial cells of the glandular stomach in mice. Male BALB/c mice were orally given a single dose (100 mg/kg) of N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as stomach-targeted carcinogens. The glandular stomach was excised at 4h, 3 and 4 days after administration, and a single cell suspension of epithelial cells was prepared from the everted glandular stomach by EDTA treatment. For determination of micronucleus induction, gastric epithelial cells on days 3 and 4 after administration were fixed with 10% neutral-buffered formalin, stained with a combination of AO-DAPI, and analyzed under fluorescence microscopy. We also examined the induction of micronuclei in peripheral blood of these mice on days 2 and 3 after administration. Moreover, total RNA was extracted from gastric epithelial cells at 4h after administration, and p21 and plk2 expression was analyzed using a quantitative RT-PCR technique. 1) A significant increase of micronucleated cells was observed in the glandular stomach in mice treated with N-nitroso-N-methylurea (MNU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to mice treated with vehicle. 2) In peripheral blood, induction of micronuclei was observed in mice treated with MNU but not with MNNG. 3) p21 and plk2, which related to cell cycle arrest, were up-regulated in the glandular stomach in mice treated with MNU or MNNG compared to mice treated with vehicle. The present study showed that these assays using glandular stomach may help to evaluate the genotoxicity of chemicals after oral administration.     

Role of antibodies and effect of BCG vaccination in experimental candidiasis in mice

The role of humoral antibodies and the effect of BCG vaccination were studied in the experimental candidiasis in mice. The antibody suppressed, B-cell deficient animals were prepared by repeated administration of rabbit anti-mouse--antiserum to the new born mice from birth onwards. Such immunodeficient animals along with controls were infected intravenously with Candida albicans, to study the course of candidal infection. It was observed that B-cell-deficient animals were found to be more susceptible to candidal infection than the controls, as indicated by their steady loss of body weight, longer mean time to death and higher viable counts of candidal cells in different organs. The anti-candidal antibodies were absent in all B-cell-deficient animals but present in the controls. These results suggest that antibodies make a contribution in protection against candidal infection in mice. The BCG vaccinated animals were prepared by repeated intravenous administration of BCG to mice and these vaccinated animals along with unvaccinated controls were challenged intravenously with C. albicans, to study the course of candidal infection. It was observed that BCG vaccination prolonged meantime to death and reduced the number of candidal cells in their kidneys.     

Treatment with beta-cyclodextrin of natural Cryptosporidium parvum infections in lambs under field conditions.

Following the unexpected activity of the excipient beta-cyclodextrin against experimental infection by Cryptosporidium parvum in suckling mice, its efficacy in the prevention and treatment of natural infections in lambs was evaluated under field conditions. Fifty-three crossbred neonatal lambs were randomly selected for the study. Treatment consisted of oral administration of an aqueous suspension of beta-cyclodextrin at a dose of 500 mg/kg of body weight. To test prophylactic efficacy, the suspension was administered at 1, 2 and 3 days of age. To evaluate therapeutic efficacy, the suspension was administered on each of the 3 days following onset of diarrhoea. Infection was monitored by daily examination of faecal samples, from birth to 30 days. The criteria studied in evaluating efficacy were: oocyst shedding, the presence of diarrhoea, and weight gain at 15 and 30 days. In the group that received prophylactic treatment with beta-cyclodextrin, there were no mortalities and, compared with control lambs, there was a decrease in the number of animals infected, a longer prepatent period and notable reduction in the patent period and the duration of diarrhoea. Therapeutic treatment also reduced the patent period and the severity of diarrhoea. beta-cyclodextrin was well tolerated by all of the treated animals.     

Long-term isoprenaline administration produces an increase in capillarity in the soleus muscle of the rat

The effects of isoprenaline administration (300 micrograms/kg for 5 weeks) on rat soleus muscle capillarity and glycolytic and oxidative capacities were evaluated. The treatment resulted in ventricular hypertrophy. The activities of lactic dehydrogenase, pyruvate kinase, citrate synthase, and cytochrome c oxidase in soleus muscle homogenates were not different between control and isoprenaline-injected animals. Capillaries were visualized in muscle cross sections treated to demonstrate ATPase activity after acid preincubation. Capillary density was higher in the experimental (873 +/- 38 capillaries/mm2) than in the control (713 +/- 33 capillaries/mm2) animals. Capillary to fiber ratio was also higher in the experimental (2.47 +/- 0.10) than in control (2.09 +/- 0.08) animals, but fiber cross-sectional area was not changed by the treatment (2836 +/- 87 microns2 in controls and 2951 +/- 136 microns2 in experimental). A plot of capillary to fiber ratio vs. fiber cross-sectional area showed that at a given fiber cross-sectional area the value of capillary to fiber ratio of the treated animals was higher than that of the controls. This indicates that treatment resulted in the proliferation of microvessels. The results suggest that prolonged beta-adrenergic stimulation results in the development of new capillaries but that this is not accompanied by increases in the oxidative capacity of the soleus muscle of the rat.