Anatomia do sistema reprodutor feminino de Alouatta belzebul (Linnaeus, 1766) / [Anatomy of the female reproductive system of the Alouatta belzebul (Linnaeus, 1766)]

O conhecimento da anatomia de qualquer animal silvestre é de fundamental importância para sua preservação e proteção. Neste contexto, o presente estudo objetivou descrever a morfologia do sistema reprodutor feminino de Alouatta belzebul. Foram utilizados seis espécimes de A. belzebul, fêmeas, adultas, e livres de lesões. Observou-se macroscopicamente que os ovários têm características morfológicas em formato ovoides, com superfície lisa, e, na análise histológica na região de córtex, evidenciou-se folículos ovarianos em diferentes estágios de desenvolvimento. As tubas uterinas anatomicamente são finas e curvilíneas, apresentando uma camada mucosa, uma muscular e outra serosa. O útero possui formato simples, com fundo globoso, com um miométrio altamente vascularizado, sendo organizado em feixes de fibras musculares lisas. A estrutura anatômica da vagina apresentou-se como um tubo muscular longo de paredes finas, onde, na região vestibular, o óstio externo da uretra é marcado por uma papila uretral bilobada e, na região de vulva, em sua porção caudal, contatou-se um clitóris bem desenvolvido. No que concerne à análise histológica da vagina, verificou-se, em região de mucosa vaginal, um extrato basal composto por epitélio estratificado pavimentoso não queratinizado atrófico. As descrições morfológicas fornecem, de forma inédita, informações importantes relativas à anatomia macroscópica e microscópica do sistema reprodutor feminino dessa espécie.(AU)

Knowledge of the anatomy of any wild animal is of fundamental importance for its preservation and protection. In this context the present study aimed to describe the morphology of the female reproductive system of A. belzebul. We used 6 specimens of A. belzebul, female, adult and free of lesions. It was macroscopically observed that the ovaries are ovoid with smooth surface and the histological analysis in cortical region showed ovarian follicles in different stages of development. The fallopian tubes are anatomically thin and curvilinear, with one mucous layer, one muscular and one serous layer. The uterus was presented in a simple format with a globular fundus, with a highly vascularized myometrium, being organized in bundles of smooth muscle fibers. The anatomical structure of the vagina presented itself as a long thin-walled muscular tube where in the vestibular region the external orifice of the urethra is marked by a bilobed urethral papilla and in the caudal portion in its caudal portion a well-developed clitoris. Regarding the histological analysis of the vagina, a basal extract composed of atrophic non-keratinized stratified squamous epithelium was found in the vaginal mucosa region. The morphological descriptions provide important information regarding the macroscopic and microscopic anatomy of the female reproductive system of this species in an unprecedented way.(AU)

Heat-treated (in single aliquot or batch) colostrum outperforms non-heat-treated colostrum in terms of quality and transfer of immunoglobulin G in neonatal Jersey calves.

The objective of this randomized clinical trial was to describe the effect on colostrum characteristics and passive transfer of IgG in neonatal calves when using the Perfect Udder colostrum management system (single-aliquot treatment; Dairy Tech Inc., Greeley, CO) compared with a negative control (fresh refrigerated or fresh frozen colostrum) and a positive control (batch heat-treated colostrum). First-milking Jersey colostrum was pooled to achieve 31 unique batches with a minimum of 22.8 L per batch. The batch was then divided into 4 with 3.8 L allocated to each treatment group: (1) heat-treated in Perfect Udder bag at 60°C for 60 min and then stored at -20°C (PU); (2) heat-treated in a batch pasteurizer (Dairy Tech Inc.) at 60°C for 60 min and then stored at -20°C in Perfect Udder bag (DTB; positive control); (3) fresh frozen colostrum stored at -20°C in Perfect Udder bag (FF; negative control); and (4) fresh refrigerated colostrum stored at 4°C in Perfect Udder bag (FR; negative control). Colostrum from all treatments was sampled for analysis of IgG concentration and bacterial culture immediately after batch assembly, after processing, and before feeding. Newborn Jersey calves were randomly assigned to be fed 3.8 L of colostrum from 1 of the 4 treatment groups. A prefeeding, 0-h blood sample was collected, calves were fed by esophageal tube within 2 h of birth, and then a 24-h postfeeding blood sample was collected. Paired serum samples from 0- and 24-h blood samples were analyzed for IgG concentration (mg/mL) using radial immunodiffusion analysis. The overall mean IgG concentration in colostrum was 77.9 g/L and was not affected by treatment. Prefeeding total plate counts (log10 cfu/mL) were significantly different for all 4 treatments and were lower for heat-treated colostrum (PU=4.23, DTB=3.63) compared with fresh colostrum (FF=5.68, FR=6.53). Total coliform counts (log10 cfu/mL) were also significantly different for all 4 treatments and were lower for heat-treated colostrum (PU=0.45, DTB=1.08) compared with fresh colostrum (FF=3.82, FR=4.80). Mean 24-h serum IgG concentrations were significantly higher for calves in the PU (41.0 mg/mL) and DTB (40.6 mg/mL) groups compared with FF (35.1 mg/mL) and FR (35.5 mg/mL) groups. Mean apparent efficiency of absorption of IgG was significantly higher for the PU (37%) and DTB (37%) groups compared with the FF (32%) and FR (32%) groups. Calves fed heat-treated colostrum (PU or DTB) experienced significantly improved AEA and serum IgG concentrations.

Susceptibility testing of Mycobacterium tuberculosis: comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method.

Tuberculosis is a leading cause of morbidity and mortality worldwide. Susceptibility testing of the causative agent, Mycobacterium tuberculosis, is critical for control of the disease. This study compared the flow cytometric susceptibility assay with the proportion method and the BACTEC TB-460 system. There was agreement between the flow cytometric and proportion methods for 73 (94%) of 78 isoniazid tests, and complete agreement for 26 ethambutol and rifampicin tests. In contrast, the proportion and BACTEC methods failed to agree for 22%, 15% and 8% of isoniazid, ethambutol and rifampicin tests, respectively. These findings indicated that susceptibility testing by the flow cytometric assay is accurate, with results available within 24 h of initiation of the testing procedure.

Interleukin-6 enhances production of anti-OspC immunoglobulin G2b borreliacidal antibody.

Protection against infection with Borrelia burgdorferi is dependent primarily on induction of complement-dependent antibody that can kill the spirochete. Measuring the production of sustained high levels of borreliacidal antibody is thus paramount for determining potential vaccine efficacy. We investigated the borreliacidal antibody response in sera and the amount of antibody produced by cultured lymph node cells of C3H/HeJ mice vaccinated with outer surface protein C (OspC). We showed that recombinant OspC was a weak stimulant of borreliacidal antibody production compared to whole cells of OspC-expressing B. burgdorferi. Mice vaccinated with B. burgdorferi in adjuvant produced a high level (titer, 5,120) of anti-OspC borreliacidal antibody, which waned rapidly. Similarly, borreliacidal antibody production by cultured lymph node cells from vaccinated mice peaked soon after vaccination and then decreased. Treatment of lymph node cells with interleukin-6 (IL-6) augmented borreliacidal antibody production, particularly immunoglobulin G2b, whereas treatment with anti-IL-6 inhibited the borreliacidal response. These findings demonstrate a previously unrecognized role for IL-6 in borreliacidal antibody production that may have important implications for vaccine development.

Detection of borreliacidal antibodies in dogs after challenge with Borrelia burgdorferi-infected ixodes scapularis ticks.

Detection of borreliacidal antibodies is an accurate serodiagnostic test for confirmation of Lyme disease in humans. In this study, 13 pathogen-free beagles, 12 to 26 weeks old, were infected with Borrelia burgdorferi by tick challenge. Dogs were monitored for clinical signs and symptoms of Lyme disease along with borreliacidal antibody production against B. burgdorferi sensu stricto isolates 297 and 50772. Ten (77%) dogs developed lameness in one or more legs within 210 days after attachment of Ixodes scapularis ticks. Eight (80%) of the lame animals had concurrent fever of > or =38 degrees C. Spirochetes were also recovered from the skin and joints of 12 (92%) dogs, but rarely from other organs. Borreliacidal antibodies against B. burgdorferi isolate 297 were detected in only four (31%) dogs, and the levels of killing antibodies remained low for the duration of the infection. In contrast, borreliacidal antibodies against B. burgdorferi isolate 50772 were detected in 13 (100%) dogs within 21 days of infection. Furthermore, the borreliacidal antibody levels correlated with the severity of B. burgdorferi infection. Detection of borreliacidal antibodies, especially against B. burgdorferi isolate 50772, is also a reliable serodiagnostic test for detection of Lyme disease in dogs.

Production of borreliacidal antibody to outer surface protein A in vitro and modulation by interleukin-4.

Borreliacidal antibody production is one of several parameters for establishing the effectiveness of Borrelia burgdorferi vaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages and B. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum hydroxide, but not recombinant OspA. Anti-OspA borreliacidal antibody was detected in supernatants of cultures of lymph node cells obtained on day 7 after vaccination, peaked on day 17, and rapidly declined. The borreliacidal activity was attributable to immunoglobulin G1 (IgG1), IgG2a, and IgG2b antibodies. When lymph node cells were treated with interleukin-4 (IL-4), production of borreliacidal antibody was inhibited but was unaffected by treatment with anti-IL-4 antibodies. These results suggest that other cytokines, but not IL-4, are mainly responsible for production of the secondary borreliacidal antibody response.

Evaluation of whole-cell and OspC enzyme-linked immunosorbent assays for discrimination of early lyme borreliosis from OspA vaccination.

A recombinant Lyme borreliosis vaccine consisting of outer surface protein A (OspA) is commercially available for vaccination of humans against infection with Borrelia burgdorferi. Vaccination with OspA induces an antibody response that makes serologic interpretation of infection with B. burgdorferi difficult, especially by screening tests based on whole-cell preparations of B. burgdorferi. We show that an enzyme-linked immunosorbent assay with B. burgdorferi sensu stricto 50772, which lacks the plasmid encoding OspA and OspB, or a full-length recombinant OspC protein can identify patients infected with B. burgdorferi. We found that 69 and 65% of serum samples from patients with case-defined early Lyme borreliosis had anti-B. burgdorferi sensu stricto 50772 and anti-OspC reactivities, respectively. In addition, little or no reactivity was detected with sera obtained from individuals vaccinated with OspA. Unfortunately, 51 and 33% of sera from healthy patients and sera from patients with other illnesses were also reactive against B. burgdorferi sensu stricto 50772 and OspC, respectively. Although these assays can discriminate B. burgdorferi infection from vaccination with OspA, their lack of specificity highlights the necessity for confirming equivocal or positive reactivities with more specific serodiagnostic tests.

Occurrence of severe destructive lyme arthritis in hamsters vaccinated with outer surface protein A and challenged with Borrelia burgdorferi.

Arthritis is a frequent and major complication of infection with Borrelia burgdorferi sensu stricto. The antigens responsible for the induction of arthritis are unknown. Here we provide direct evidence that a major surface protein, outer surface protein A (OspA), can induce arthritis. Hamsters were vaccinated with 30, 60, or 120 microg of recombinant OspA (rOspA) in aluminum hydroxide and challenged with B. burgdorferi sensu stricto isolate 297 or C-1-11. Swelling of the hind paws was detected in 100, 100, and 50% of hamsters vaccinated with 30, 60, or 120 microg of rOspA, respectively. In addition, arthritis developed in 57% of hamsters vaccinated with a canine rOspA vaccine after infection with B. burgdorferi sensu stricto. When the canine rOspA vaccine was combined with aluminum hydroxide, all vaccinated hamsters developed arthritis after challenge with B. burgdorferi sensu stricto. Histopathologic examination confirmed the development of severe destructive arthritis in rOspA-vaccinated hamsters challenged with B. burgdorferi sensu stricto. These findings suggest that rOspA vaccines should be modified to eliminate epitopes of OspA responsible for the induction of arthritis. Our results are important because an rOspA vaccine in aluminum hydroxide was approved by the Food and Drug Administration for use in humans.

Flow cytometric testing of susceptibilities of Mycobacterium avium to amikacin, ciprofloxacin, clarithromycin and rifabutin in 24 hours.

OBJECTIVE: To develop a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing Mycobacterium avium organisms in drug-free and antimycobacterial agent-containing medium. METHODS: Prior to analysis by flow cytometry, all M. avium susceptibility test samples were inactivated by exposure to paraformaldehyde. The susceptibilities of 20 clinical isolates of M. avium to amikacin, ciprofloxacin, clarithromycin, and rifabutin were tested by the flow cytometric and BACTEC methods. RESULTS: Agreement was 97% between the results of the two methods. The results of flow cytometric susceptibility tests were available 24 h after inoculation of drug-containing medium, while the BACTEC method required 4-8 days to complete. CONCLUSIONS: The flow cytometric assay is safe, simple and reproducible.

Detection of borreliacidal antibodies in Lyme borreliosis patient sera containing antimicrobial agents.

The borreliacidal-antibody test has been used for the serological detection and confirmation of Lyme borreliosis. However, the presence of antimicrobial agents in serum can confound the accurate detection of borreliacidal antibodies. In this study, we developed a Bacillus subtilis agar diffusion bioassay to detect small concentrations of antimicrobial agents in serum. We also used XAD-16, a nonionic polymeric resin, to adsorb and remove high concentrations of amoxicillin, cefotaxime, ceftriaxone, cefuroxime, doxycycline, and erythromycin without significantly affecting even small concentrations of immunoglobulin M (IgM) or IgG borreliacidal antibodies. High concentrations of penicillin could also be removed by adding 1 U of penicillinase without significantly influencing the levels of borreliacidal antibodies. These simple procedures greatly enhance the clinical utility of the borreliacidal-antibody test.

Macrophages interact with enriched populations of distinct T lymphocyte subsets for the induction of severe destructive Lyme arthritis.

Severe destructive Lyme arthritis was detected in the hind paws of hamsters infused with enriched populations of either CD4+ or CD4- T lymphocytes along with macrophages exposed in vitro to formalin-inactivated Borrelia burgdorferi and then infected with the Lyme spirochete. Swelling was detected 4 days after infection, increased rapidly, peaked on day 8 of infection, and gradually decreased. Similarly, severe destructive arthritis was induced in hamsters infused with enriched populations of unfractionated T lymphocytes and macrophages exposed to spirochetes after infection with B. burgdorferi. Histopathological examination affirmed that hamsters infused with CD4+, CD4-, or unfractionated T lymphocytes and macrophages exposed to B. burgdorferi-induced arthritis. In addition, macrophages exposed in vitro to B. burgdorferi demonstrated both conventional and coiling phagocytosis, suggesting a mechanism by which CD4+ and CD4- T lymphocytes induce arthritis, respectively. These findings demonstrate that both CD4+ and CD4- subpopulations of T lymphocytes are capable of interacting with macrophages for the induction of severe destructive Lyme arthritis.

Safe determination of susceptibility of Mycobacterium tuberculosis to antimycobacterial agents by flow cytometry.

We showed previously that susceptibility testing for Mycobacterium tuberculosis labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing M. tuberculosis organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of M. tuberculosis to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.

Performance of the Pollen Tube Growth Test in the COLIPA Validation Study on Alternatives to the Rabbit Eye Irritation Test.

In the present paper, we describe and analyse the performance and the results of the pollen tube growth (PTG) test applied to the COLIPA international validation study of in vitro alternatives to the Draize eye irritation test. The PTG test, based on photometric quantification of in vitro pollen tube mass production, was used by three independent laboratories to estimate the acute eye irritation potentials of 23 ingredients and 32 cosmetic formulations. Basing on historical Draize test data and on IC(50) values of previously tested cosmetic formulations, a mathematical formula was generated to predict rabbit eye irritation potentials from PTG test results. Statistical evaluation of the calculated modified maximal average scores (MMAS) revealed a high prediction capability of the PTG test in regard to the finished formulations but a relatively low one for alcohols, higher concentrated cationic surfactants, and acidic and alkaline materials. Furthermore, our results indicated that the PTG test was able to produce precise IC(50) values without any limitations from all of the 55 test substances with good intra- and interlaboratory reproducibility. From these findings we suggest that the PTG test is not a validated test at present but is considered to be a potent candidate for further validation processes. For this purpose an additional prediction model for ingredient classes as mentioned above must be generated.

Borreliacidal antibody production against outer surface protein C of Borrelia burgdorferi.

Early Lyme borreliosis sera with significant titers of anti-outer surface protein C (OspC) borreliacidal antibodies were identified. Human anti-OspC borreliacidal antibodies could be either IgM or IgG. Significant concentrations of borreliacidal activity were detected after vaccination of mice with OspC. Detection of anti-OspC borreliacidal activity was dependent on surface expression of OspC by Borrelia burgdorferi isolate 50772. The ability of OspC to induce borreliacidal antibodies in vivo and after vaccination offers another possible explanation for the ability of vaccination with OspC to protect against infection with B. burgdorferi. Furthermore, detection of anti-OspC borreliacidal antibodies, especially IgM antibodies, in early Lyme borreliosis sera provides additional evidence that borreliacidal antibody detection may be useful for the serodiagnosis of early Lyme borreliosis.

Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours.

Susceptibility testing of Mycobacterium tuberculosis is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.

Inhibition of the production of anti-OspA borreliacidal antibody with T cells from hamsters vaccinated against Borrelia burgdorferi.

The serious morbidity associated with Lyme borreliosis has focused considerable effort on the development of a comprehensive vaccine for protection against infection with Borrelia burgdorferi. Induction of borreliacidal antibody by vaccination or infection has been shown to correlate with protection of humans and animals against infection with the Lyme spirochete. In this report, we showed that high levels of borreliacidal antibody (titer of 1,280) were produced in vitro when T and B cells from hamsters 14 days after vaccination were incubated with macrophages and B. burgdorferi. By contrast, T and B cells from hamsters 7 or 21 days after vaccination failed to initiate production of borreliacidal activity. Furthermore, the T cells from hamsters 7 or 21 days after vaccination inhibited the in vitro production of borreliacidal antibody when cocultured with T and B cells obtained from hamsters 14 days after vaccination. When cell-free supernatants from the suspensions of T and B cells from hamsters 14 days after vaccination were absorbed with recombinant OspA, they lost nearly all borreliacidal activity. The removal of anti-OspA antibody resulted in a decrease in borreliacidal titer from 1,280 to less than 4. These results demonstrate that T cells from vaccinated animals can prevent a sustained production of protective borreliacidal antibody.