Footpad Research Articles

152 Leveraging Rfid Technology to Investigate the Genetic Associations Between Feeding Behavior and Leg Health in Floor-Raised Broilers

Abstract Leg disorders (LD) pose serious welfare issues in broiler chickens. The objective of this study was to investigate the suitability of using feeding behavior (FB) traits as potential leg health indicators in floor-raised broilers. The data used were recorded from 95,711 pure-line chickens of both sexes. The individual FB was monitored continuously on a feeder visit basis with a radio-frequency antenna system. The visit-log data were clustered into daily observations for different FB traits and averaged for the 1st week period after the trial started. The FB traits were daily feed intake (DFI1), number of visits (NV1), time spent at the feeder (TSF1), visiting activity interval (VAI1), number of visited feeders (NVF1), and walked distance visiting feeders (WD1). The LD traits were tibial dyschondroplasia (TD), hock-burn (HB), footpad (FP), rotated legs (RO), bowed-in (BI), and bowed-out (BO) leg structure deformities. Multi-trait linear-threshold models were fitted using Bayesian inference for estimating (co)variance components. The heritability (h2) posterior averages and standard deviations (within parentheses) ranged between 0.09 (0.01) and 0.52 (0.03) for LD traits, and between 0.09 (0.01) and 0.47 (0.02) for FB traits. Genetic correlations (rg) between FB and LD traits ranged from null to weak. TD presented negative rg with NV1 (-0.13), TSF1 (-0.14), NVF1 (-0.11), and WD1 (-0.14), indicating that some alleles involved in broiler activity are favorably associated with a potential reduction in the risk of TD-caused bone lesions. Conversely, FP presented unfavorable rg with the same traits (ranging from 0.21 to 0.32), except for TSF1 (-0.08). When compared with the remaining FB traits, DFI1 generally presented the opposite relationships with TD and FP, with positive (0.16) and negative (-0.18) genetic correlations observed for the first and the latter, respectively. Angular and rotational deformities (RO, BI, and BO) did not present significant correlations with FB traits, apart from the weak correlation found between BO and TSF1 (0.11). General activity indicators such as NV1, NVF1, and WD1 could represent earlier selection criteria to reduce TD incidence, although the potential undesired selection response for FP must be further investigated.

Effects of negative air ions (NAIs) on Leishmania major: A novel tool for treatment of zoonotic cutaneous leishmaniasis (ZCL).

BackgroundCutaneous leishmaniasis (CL) is a Neglected Tropical Disease (NTD) that causes high morbidity in the tropics and sub-tropics. Despite the remarkable advancements in the treatment of CL, the available therapeutics are far from ideal and also cause serious adverse side effects. Negative air ions (NAIs) generators are widely available for domestic and industrial uses. Several studies have reported on positive effects of NAIs therapy on human health as a non-pharmaceutical treatment for respiratory disease, allergy, or stress-related health conditions, including infectious diseases. To our knowledge, no studies have examined the effectiveness of the NAIs therapy against Leishmania parasites. The aims of this study were to investigate the effect of NAIs therapy on Leishmania major (L. major) the causative agent of CL in in vitro and in a murine model.Methodology/Principal findingsIn vitro anti-leishmanial effects of NAIs therapy were measured by parasitological methods. NAIs therapy was assessed in vivo in L. major infected BALB/c mice by measuring the footpad (FP) lesion size and parasite load using metric caliper tool and qPCR, respectively. Immune responses in treated and non-treated mice were assessed by measuring the levels of IFN-γ, IL-4, NO and arginase activity. In vitro NAIs therapy significantly decreased the viability of Leishmania promastigotes and of amastigotes cultured in macrophages, but did not affect the host cells. NAIs therapy of L. major infected BALB/c mice resulted in reduced FP lesion size, diminished parasite burden, and importantly decreased induction of IL-4 and arginase activity in the presence of NAIs. In contrast IFN-γ and NO levels were significantly enhanced. NAIs therapy significantly diminished the progression of disease compared to the control group, but was less effective than amphotericin B treatment.ConclusionsOur study shows that NAIs treatment was effective in vitro and in Leishmania-infected mice, elicited a T-helper 1 (Th1) response and increased efficient cellular immunity, resulting in a diminished parasite load. Therefore, NAIs therapy can be considered as a useful and safe tool that can contribute to clearing L. major infections without inducing toxicity in host cells. The applications and mechanisms of NAIs therapy warrant further investigation especially in humans suffering from CL.

The usual suspects: Co-occurrence of integument injuries in turkey flocks

The present study investigated the prevalence and co-occurrence of integument injuries in Canadian turkeys. Participating farmers scored 30 birds in their flock for integument injuries to the head/neck (HN), back/tail (BT), and footpad (FP) using a simplified scoring system (0: no sign of injury, 1: mild injury, 2: severe injury). Information from 62 flocks was used to calculate the prevalence of any (score ≥1) and severe (score 2) injuries on a flock- and individual-level. Chi-square analyses were performed to determine the likelihood of integument injury co-occurrence. The prevalence of each type of injury varied between flocks. While the majority of flocks reported injuries, the within-flock prevalence was relatively low and largely comprised of mild cases (score 1). Given their higher prevalence, the data indicate that FP injuries are overall more widespread and more severe among Canadian turkey flocks than HN and BT injuries. Co-occurrence of different integument injuries was observed in 7% of birds and 58.1% of flocks reported at least one bird with co-occurring injury types. Despite the low prevalence of multiple injury types, birds with one type of injury were more likely to present with other injury types. Indeed, birds with HN injuries were 4 times more likely to have BT injuries, and birds with FP injuries were 1.5 times more likely to have BT injuries compared to birds that do not have these respective injuries. The data increase our understanding of the co-occurrence of these common integument injuries which can help inform a holistic management approach to rear turkeys with healthy skin and feather cover.

Potential therapeutic effects of curcumin coated silver nanoparticle in the treatment of cutaneous leishmaniasis due to Leishmania major in-vitro and in a murine model

BackgroundLeishmaniasis is a destructive protozoan parasitic and vector-borne infection characterized as a neglected tropical disease (NTD) that is currently endemic in more than 100 countries. Herbal compounds such as curcumin derived from turmeric with complex and extensive structures have a wide range of applications in the treatment of parasitic infections; however, its usage has a major challenge due to low water solubility and bioavailability. Besides, metal nanoparticles such as silver have been applied for parasitic infection treatment. Synthesis of curcumin and silver nanoparticles conjugate facilitates in enhancing curcumin solubility and bioavailability. Here curcumin-coated silver nanoparticles (Cur@AgNPs) were synthesized, characterized, and then their cell cytotoxicity and antileishmanial activities were investigated both in-vitro and in mouse models. Materials and methodsCurcumin coated silver nanoparticles (Cur@AgNPs) synthesized by a simple one-pot green chemistry technique. After confirming their stability, in vitro cell cytotoxicity and leishmanicidal activity against promastigotes and amastigotes of the protozoan parasite, Leishmania major (L. major) was evaluated by using colourimetric yellow methyl thiazole tetrazolium (MTT) assay. Furthermore, antiparasitic properties of Cur@AgNPs in the mouse model were studied through quantifying footpads (FPs) lesion size and determining parasite burden in lymph nodes (LNs) and FPs of infected BALAB/c mice with L. major. ResultsCur@AgNPs represent a plasmonic peak of silver nanoparticles at 420 nm in UV–Vis spectra. The size of synthesized NPs was 29.1 ± 5.6 nm based on the analyzing TEM micrographs. The hydrodynamic diameter of the synthesized particles is 32.4 ± 2.3 nm. The zeta potential of washed Cur@AgNPs (4 rounds of washing) was −19.8 ± 1.5 mV. The stability of Cur@AgNPs at different time intervals (up to two months) in PBS buffer media has been investigated thoroughly. We confirmed that Cur@AgNPs decreased significantly in both forms of promastigotes and amastigotes of Leishmania parasite in a single treatment. The nanoparticle elucidated potent antileishmanial activity with IC50 of 58.99 μg/ml for promastigotes and EC50 58.99 μg/ml for amastigotes at 48 h post-infection with no harmful negative toxicity on the mice macrophages. In the treated infected BALB/c mice using Cur@AgNPs, the cutaneous leishmania (CL) lesion size in the FPs and Leishmania burden in both LNs and FPs were decreased significantly. ConclusionThe results of this study revealed that Cur@AgNPs could be, novel, safe, and effective promising candidates as an antileishmanial agent in the treatment of CL infection.

Post-exposure prophylaxis (PEP) efficacy of rifampin, rifapentine, moxifloxacin, minocycline, and clarithromycin in a susceptible-subclinical model of leprosy.

BackgroundSubclinical infection with Mycobacterium leprae is one potential source of leprosy transmission, and post-exposure prophylaxis (PEP) regimens have been proposed to control this source. Because PEP trials require considerable investment, we applied a sensitive variation of the kinetic mouse footpad (MFP) screening assay to aid in the choice of drugs and regimens for clinical trials.Methodology/Principal findingsAthymic nude mice were inoculated in the footpad (FP) with 6 x 103 viable M. leprae and treated by gastric gavage with a single dose of Rifampin (SDR), Rifampin + Ofloxacin + Minocycline (SD-ROM), or Rifapentine + Minocycline + Moxifloxacin (SD-PMM) or with the proposed PEP++ regimen of three once-monthly doses of Rifampin + Moxifloxacin (RM), Rifampin + Clarithromycin (RC), Rifapentine + Moxifloxacin (PM), or Rifapentine + Clarithromycin (PC). At various times post-treatment, DNA was purified from the FP, and M. leprae were enumerated by RLEP quantitative PCR. A regression analysis was calculated to determine the expected RLEP value if 99.9% of the bacilli were killed after the administration of each regimen. SDR and SD-ROM induced little growth delay in this highly susceptible murine model of subclinical infection. In contrast, SD-PMM delayed measurable M. leprae growth above the inoculum by 8 months. The four multi-dose regimens delayed bacterial growth for >9months post-treatment cessation.Conclusions/SignificanceThe delay in discernable M. leprae growth post-treatment was an excellent indicator of drug efficacy for both early (3–4 months) and late (8–9 months) drug efficacy. Our data indicates that multi-dose PEP may be required to control infection in highly susceptible individuals with subclinical leprosy to prevent disease and decrease transmission.

Characterization of immune response in mice vaccinated with highly efficacious vaccine 0ΔNLS against HSV-1

Abstract Using an experimental mouse model we compared the host immune response of a mutant HSV-1, termed 0ΔNLS (deficient in the nuclear location signal of ICP0), a highly efficacious prophylactic vaccine against ocular HSV-1 infection, to that of the parental HSV-1 virus, termed GFP105 (KOS background). C57BL/6 mice were infected in the footpad (FP) with 1×105 plaque forming units of virus and at times post infection (PI), organized lymphoid tissues were analyzed for changes in the immune profile. 0ΔNLS infected animals were more efficient in clearance of virus from the FP and generated a more robust chemokine responses and myeloid cell infiltration in FP at early time points (day 1–3 PI) compared to GFP105 infected mice. By day 7 PI, there were significantly more GC B cells and isotype switched B cells in the PLN of mice infected with 0ΔNLS in comparison to GFP105 infected animals. In addition, an increased number of follicular helper CD4 T cells along with enhanced IL-21 cytokine production and an increased number of antigen-specific CD8 T cells against major and sub-major HSV-1 epitopes was noted. In contrast, by day 21 PI, no difference in T cell and B cell subset was found in the PLN comparing the two groups of infected mice. Furthermore, mice which were re-exposed to 0ΔNLS 21 days PI showed an increased number of metabolically active plasma cells from the bone marrow and secretion of IgG by memory B cells after pre-activation compared to those re-infected with GFP105. In addition, an increased number of IFN-ɤ secreting, HSV-1-specific CD8 T cells from the re-exposed 0ΔNLS mice was observed as well. Collectively, these results warrant further studies to fully understand the efficacy of the 0ΔNLS virus as an effective vaccine against ocular HSV-1 infection.

Murine experimental leprosy: Evaluation of immune response by analysis of peritoneal lavage cells and footpad histopathology.

This study evaluated the immune response of nude and BALB/c mice inoculated in the footpads (FP) with Mycobacterium leprae after 3, 5 and 8months. At each timepoint peritoneal cells, peripheral blood, FP and popliteal lymph nodes (PLN) were collected. Peritoneal cell cultures were performed to measure the H2 O2 , O2- , NO, IL-2, IL-4, IL-10, IL-12, IFN-γ and TNF levels. Serum levels of anti-PGL-I antibodies were also quantified. The results showed that the infection was progressive in nude mice with bacterial multiplication, development of macroscopic lesions in the FP and presence of bacilli in the PLN at 8months. In BALB/c mice, the infection reached a plateau of bacillary multiplication at 5months and regressed at 8months. Histopathological analysis of FP revealed a mononuclear inflammatory infiltrate with a large number of neutrophils at 5months, with a higher number in nude mice. At 8months, the number of neutrophils decreased and the infiltrate was predominantly mononuclear in both mouse strains. There was no H2 O2, O2- , IL-2, IL-4, IL-10 and IFN-γ production in the course of infection in nude mice; however, in BALB/c, O2- and IL-12 production was higher at 5months and NO, IFN-γ and TNF production was higher at 8months when there was a decrease in the number of bacilli. The level of anti-PGL-I antibodies was higher in BALB/c mice. Thus, nude and BALB/c mice can be used as experimental models for the study of various aspects of leprosy.

Foot Pad Health as Part of On-Farm-Monitoring in Turkey Flocks.

Currently, there is no consistent approach to on-farm and post-mortem foot pad (FP) assessment in turkey husbandry in sampling of both feet, sample sizes of birds and scoring schemes during the production period. Therefore, in a field study, 11,400 turkeys, i.e., 22,800 feet, were macroscopically scored at 4-week intervals, 60 birds per flock per date, in accordance with the scale system of Hocking et al. (1). Spearman's rho was calculated between the foot pad dermatitis (FPD) score of both feet of an individual turkey. Sample size for FPD monitoring was calculated for several flock sizes, considering expected FPD prevalence and the error and confidence level (α = 0.01, 0.05, 0.1). To compare macroscopic to histological findings, ten excised FPs were histopathologically investigated by hematoxylin & eosin staining. To align manual macroscopic FPD evaluations with a technical system, 20 photographic images of FPD were measured using the ImageJ program. The scores of both feet of an individual turkey correlated between r = 0.252 and r = 1.000. Thus, both feet of a bird should be monitored, while the worse foot should be evaluated. As an exemplary sample size for on-farm FPD assessment, 77 turkey poults were calculated in a flock of 4,000 birds with an expected FPD prevalence of 40% and α = 0.1. The sample size of monitored birds within a flock should differ and depend on flock size and expected FPD prevalence. Histopathological findings showed normal and non-affected structures of a macroscopic Score 0 and a moderate ulcer of the macroscopic Score 1 and Score 2. The applied assessment scheme should distinguish first alterations and scar tissue as separate scores to differentiate the need for management intervention vs. the success of management measures that were already implemented. FPD affected areas were given lower Scores and assessed to be healthier when evaluated by an image system, compared to a manual assessment. Furthermore, with regard to an increase in camera-based assessments, the boundary of the metatarsal pad needs to be clarified. In conclusion, a new scoring system is required, as the size of the FP cannot be clearly defined and different tissue textures, as well as valid sample sizes are not currently sufficiently considered.

Effects of light intensity in different stocking densities on tibial measurements and incidence of lesions in broilers

The possibility for use of high light intensity in broiler production management to improve the health of broilers? legs was examined in a study aiming to determine the effect of two levels of light intensity in different stocking densities of broiler chickens. The study was carried out on 1200 Ross 308 broilers according to a random block system with 6 treatments (2 x 3 factorial design) and 4 replicates per treatment. The examined light intensity levels of 150 lx (high light intensity-HLI) and 20 lx (low light intensity-LLI) were applied to 3 stocking densities of broilers: 10, 13 and 15 birds/m2. Scoring of foot pad (FP) and hock burns (HB) was carried out on the 42nd day, on a sample of 240 broilers. Osteometric and biomechanical measurements on tibia were performed after slaughtering and primary processing, on a sample of 72 broilers. The high level intensity of light exerted no significant effect on the development and firmness of the tibia of broilers grown in the densities of 10, 13 and 15 birds/m2. There are indications that in the highest studied stocking density the increased light intensity can improve the individual properties of tibia. The differences in the average assessment of hock burns were concluded to be the result of the stocking density that was observed only in conditions of the increased intensity of light. The average score of foot pad lesions was under the significant main effect of both investigated factors. The differences in the average FP estimation between the light intensity treatments were confirmed in the stocking density of 13 birds/m2.

Effect of Avian Reovirus Infection on Antibody Response, Footpad Thickness and Body Weight in SPF Chicken

Avian reoviruses (ARVs) are designated to be non-pathogenic to virulent viruses and cause asymptomatic to severe infections in poultry. Particularly in broiler breeder flocks, infection results in huge economic losses to farmers due to virulent ARV strain. Hence, this study was designed to isolate the reovirus from field outbreaks and to study the pathogenicity of ARV isolate in specific pathogen-free (SPF) chickens. The isolated ARV strain was inoculated by footpad (FP) route at the dose rate of 0.1 ml (104 TCID/0.1 ml) per bird into 30 specific pathogen-free (SPF) birds (inoculated group). Thirty (30) SPF birds were used as control group (uninoculated group). The antibody response to ARV infection from six birds per group and FP thickness in all the birds was measured on Days 7, 14, 21 and 28. Body weights were recorded on Days 1 and 28 of the study. The study indicated that the orthotropic ARV virus infection showed decreased body weights in SPF birds. A significant increase in FP thickness at virus inoculation site and progressive increase in percent positive birds for ARV antibodies with mean titre was observed in ARV infected birds.

Study of murine experimental Jorge Lobo's disease by analysis of peritoneal lavage cells and footpad histopathology: early versus chronic lesions.

The murine model of Jorge Lobo's disease is characterized by histological alterations similar to those seen in human disease, including a large number of viable fungi. This study evaluated the immune response of mice with early and late macroscopic lesions (5 and 13 months post-inoculation [p.i.], respectively) by the analysis of peritoneal lavage cells and footpad (FP) histology. The FP of mice were inoculated with 1 × 10(6) fungi (viability index of 41%). At 5 and 13 months p.i., the granuloma mainly consisted of macrophages and multinucleated giant cells, but a larger number of neutrophils was observed at 5 months and lymphocytes at 13 months. The number of fungi in the FP and fungal viability were 1.8 ± 1.1 × 10(6) fungi/ml and 38.5% at 5 months p.i. and 30.8 ± 11.7 × 10(6) fungi/ml and 9% at 13 months (P < .05). Higher production of H₂O₂, O₂(-), IL-10, and TNF-α were observed at 13 months (P < .05), but there was no significant difference in the production of NO, IL-2, IL-4, IL-12 and IFN-γ. The results showed significant differences between early and late lesions and support the use of BALB/c mice for evaluation of the different phases of infection.

IL-10 and NOS2 modulate antigen-specific reactivity and nerve infiltration by T cells in experimental leprosy.

BackgroundAlthough immunopathology dictates clinical outcome in leprosy, the dynamics of early and chronic infection are poorly defined. In the tuberculoid region of the spectrum, Mycobacterium leprae growth is restricted yet a severe granulomatous lesion can occur. The evolution and maintenance of chronic inflammatory processes like those observed in the leprosy granuloma involve an ongoing network of communications via cytokines. IL-10 has immunosuppressive properties and IL-10 genetic variants have been associated with leprosy development and reactions.Methodology/Principal FindingsThe role of IL-10 in resistance and inflammation in leprosy was investigated using Mycobacterium leprae infection of mice deficient in IL-10 (IL-10−/−), as well as mice deficient in both inducible nitric oxide synthase (NOS2−/−) and IL-10 (10NOS2−/−). Although a lack of IL-10 did not affect M. leprae multiplication in the footpads (FP), inflammation increased from C57Bl/6 (B6)<IL-10−/−<NOS2−/−<10NOS2−/−. While IL-10−/− mice exhibited modest FP induration compared to B6, NOS2−/− and 10NOS2−/− mice developed markedly enlarged FP marking distinct phases: early (1 month), peak (3–4 months), and chronic (8 months). IFN-γ-producing CD4+CD44+ cells responding to M. leprae cell wall, membrane, and cytosol antigens and ML2028 (Ag85B) were significantly increased in the evolved granuloma in NOS2−/− FP compared to B6 and IL-10−/− during early and peak phases. In 10NOS2−/− FP, CD4+CD44+ and especially CD8+CD44+ responses were augmented even further to these antigens as well as to ML0380 (GroES), ML2038 (bacterioferritin), and ML1877 (EF-Tu). Moreover, fragmented nerves containing CD4+ cells were present in 10NOS2−/− FP.Conclusions/SignificanceThe 10NOS2−/− strain offers insight on the regulation of granuloma formation and maintenance by immune modulators in the resistant forms of leprosy and presents a new model for investigating the pathogenesis of neurological involvement.

Semi-automated protocol for purification of Mycobacterium leprae from tissues using the gentleMACS™ Octo Dissociator

Mycobacterium leprae, etiologic agent of leprosy, is propagated in athymic nude mouse footpads (FPs). The current purification protocol is tedious and physically demanding. A simpler, semi-automated protocol was developed using gentleMACS™ Octo Dissociator. The gentleMACS protocol provided a very effective means for purification of highly viable M. leprae from tissue.

Rift valley Fever virus encephalitis is associated with an ineffective systemic immune response and activated T cell infiltration into the CNS in an immunocompetent mouse model.

BackgroundRift Valley fever virus (RVFV) causes outbreaks of severe disease in livestock and humans throughout Africa and the Arabian Peninsula. In people, RVFV generally causes a self-limiting febrile illness but in a subset of individuals, it progresses to more serious disease. One manifestation is a delayed-onset encephalitis that can be fatal or leave the afflicted with long-term neurologic sequelae. In order to design targeted interventions, the basic pathogenesis of RVFV encephalitis must be better understood.Methodology/Principal FindingsTo characterize the host immune responses and viral kinetics associated with fatal and nonfatal infections, mice were infected with an attenuated RVFV lacking NSs (ΔNSs) that causes lethal disease only when administered intranasally (IN). Following IN infection, C57BL/6 mice developed severe neurologic disease and succumbed 7–9 days post-infection. In contrast, inoculation of ΔNSs virus subcutaneously in the footpad (FP) resulted in a subclinical infection characterized by a robust immune response with rapid antibody production and strong T cell responses. IN-inoculated mice had delayed antibody responses and failed to clear virus from the periphery. Severe neurological signs and obtundation characterized end stage-disease in IN-inoculated mice, and within the CNS, the development of peak virus RNA loads coincided with strong proinflammatory responses and infiltration of activated T cells. Interestingly, depletion of T cells did not significantly alter survival, suggesting that neurologic disease is not a by-product of an aberrant immune response.Conclusions/SignificanceComparison of fatal (IN-inoculated) and nonfatal (FP-inoculated) ΔNSs RVFV infections in the mouse model highlighted the role of the host immune response in controlling viral replication and therefore determining clinical outcome. There was no evidence to suggest that neurologic disease is immune-mediated in RVFV infection. These results provide important insights for the future design of vaccines and therapeutic options.

Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice

BackgroundThe inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues.Methodology/Principle FindingsTwo M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations.Conclusions hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

Migration of dendritic cells from murine skeletal muscle

To better understand the role of dendritic cells (DCs) in skeletal muscle, we investigated the migration of DCs from murine skeletal muscle and compared that to previously studied footpad (FP) DC trafficking. We adoptively transferred carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled mature DCs to syngeneic mice and followed them in various lymphatic tissues at different time points. Injection of DCs into the tibialis anterior muscle resulted in the peak number of CFSE + DCs recovered in spleen at 12 h, not at 24 h, when the largest number of these cells appeared in the draining lymph nodes. Interestingly, this result for adoptive transfer of DCs to skeletal muscle differs with what is previously reported for adoptive transfer to the FP, a result that we also confirmed in parallel studies. These findings could have a significant impact on (1) understanding muscle diseases with immunological complications such as muscular dystrophies and (2) the immunologic effects of treatments for muscle diseases.

Lymphotoxin-α and TNF Have Essential but Independent Roles in the Evolution of the Granulomatous Response in Experimental Leprosy

Recent studies identified an association between genetic variants in the lymphotoxin-alpha (LTalpha) gene and leprosy. To study the influence of LTalpha on the control of experimental leprosy, both low- and high-dose Mycobacterium leprae foot pad (FP) infections were evaluated in LTalpha-deficient chimeric (cLTalpha(-/-)) and control chimeric (cB6) mice. Cellular responses to low-dose infection in cLTalpha(-/-) mice were dramatically different, with reduced accumulation of CD4(+) and CD8(+) lymphocytes and macrophages and failure to form granulomas. Growth of M. leprae was contained for 6 months, but augmented late in infection. In contrast, tumor necrosis factor knockout and tumor necrosis factor receptor 1 knockout FPs exhibited extensive inflammatory infiltration with an increase in M. leprae growth throughout infection. Following high-dose infection, cB6 FP induration peaked at 4 weeks and was maintained for 12 weeks. Induration was not sustained in cLTalpha(-/-) FPs that contained few lymphocytes and no granulomas. There was a reduction in the expression levels of inflammatory cytokines, chemokines, and chemokine receptors, including nitric oxide synthase 2, vascular cell adhesion molecule, and intercellular cell adhesion molecule. Furthermore, cLTalpha(-/-) popliteal lymph nodes contained a higher proportion of naïve CD44(lo)CD62L(hi) T cells than cB6 mice, suggestive of reduced T cell activation. Therefore, both LTalpha and tumor necrosis factor are essential for the regulation of the granuloma, but they have distinctive roles in the recruitment of lymphocytes and maintenance of the granulomatous response during chronic M. leprae infection.

Emergence of an effective adaptive cell mediated immune response toMycobacterium lepraeis not impaired in reactive oxygen intermediate-deficient mice

Cytokine-activated macrophages (MPhi) employ reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to combat pathogens. The requirement for ROI for an effective host response to experimental leprosy using mice which have a disruption in the 91-kD subunit of the NAPDH oxidase cytochrome b (phox91-/-) was examined. Mycobacterium leprae multiplication in phox91-/- foot pads (FP) was elevated early in infection but subsequently arrested similarly to control mice within a noninvasive granuloma. Using a modified lepromin test model, a similar cellular composition in the M. leprae-induced FP granuloma in both strains with lymphocyte infiltration consisting primarily of CD4+CD44(hi)CD62L(lo) effector cells was found. Of great interest was the disparity in the T cell population between the granuloma and the draining lymph node which contained predominantly naïve CD4+CD44(lo)CD62L(hi) cells and was, therefore, not representative of the infection site. TH1 cytokines, chemokines and inducible nitric oxide synthase were comparably expressed in the FP of both strains. When infected in vitro, normal MPhi from B6 and phox91-/- mice supported bacterial viability, whereas IFNgamma-activated MPhi killed M. leprae in a RNI-dependent manner, emphasizing that ROI was dispensable. These data show that phox91-/- mice generate a strong adaptive immune response and control long-term infection with M. leprae.

Effects of Age and Diet on Rat Skin Histology

To document age-related histologic morphometric changes of rat skin and the effects of calorie restriction on such changes. Fischer 344 rats of three age groups (young, 4 mo; adult, 1 year; old, 24+ months) were procured from ad libitum (AL) diet and calorie-restricted (CR) colonies of the National Institute of Aging and were used for histologic study. Each study group consisted of six animals. Skin samples from the dorsum (DS) and footpad (FP) of these animals were excised and processed for histology with staining techniques for general morphology (hematoxylin-eosin-phloxine) and for differentiation of collagen bundles and elastic fibers (Verhoeff-van Gieson technique). Light microscopic morphometric and stereologic point counting procedures were applied manually to tissue sections to obtain quantitative data on the depth of the epidermis, dermis, and stratum corneum, epidermal nuclear number, and percentage fraction of collagen, elastic fibers, capillaries, and pilosebaceous units. Data were analyzed with two-way of analysis of variance (ANOVA) to determine significant effects of age, diet, and age-diet interaction on these parameters in AL rats and their age-matched cohorts. Significant effects of age, diet, or age-diet interaction were observed in respect of the thickness of epidermis, dermis, stratum corneum of FP, epidermal nuclear number, collagen percentage fraction, and area fraction of capillaries. DS epidermis showed increasing thickness in AL group, but this was reduced in CR rats. A similar trend in DS dermal depth was observed. Fewer capillaries were present in aging CR rats. The DS epidermal nuclear profiles and collagen area fraction also showed effects of diet and age-diet interaction. Aging changes, especially the effect of CR, was more evident in the measured parameters of dorsal skin. No alterations were observed in the distribution of pilosebaceous units and elastic fiber profiles of the skin. The Fischer 344 rat shows many age-related changes in the skin, some of which are different from data reported in literature. The pattern of aging changes in skin parameters was different in the two groups, suggesting an in influence of CR. CR appears to modify the aging rate of some skin components, and this may be caused by metabolic changes imposed by diet.

“PERFEXT”: a direct method for quantitative assessment of cytokine production in vivo at the local level

A method termed "PERFEXT", based on sequential perfusion and detergent extraction of lymphoid and non-lymphoid organs, has been developed for the quantitative measurement of cytokines produced at a local level in a given tissue. In vivo treatment of mice with Staphylococcus enterotoxin B (SEB) or lipopolysaccharide (LPS) served as the model systems. Interleukin-2 (IL2) and interferon-gamma (IFN gamma) levels were monitored by ELISA analysis of extracted samples. After local footpad (FP) injection with SEB, spleen and serum IL2 levels peaked at 2-4 h, while IL2 levels peaked at around 4-8 h in both FP and popliteal lymph nodes. SEB injection resulted in increased IFN gamma levels both in the FP and the draining lymph node. The detection of cytokines in the intestine allows for the application of the method at mucosal sites as well, provided enzyme inhibitors are present during the extraction procedure. After FP injection with LPS, IFN gamma production was significantly increased in the draining lymph node and was detectable in the FP, whereas IL2 was undetectable in any organ examined. IL2 and IFN gamma could also be detected at the site of elicitation of a delayed-type hypersensitivity reaction following local FP challenge. Local cytokine production correlated with the swelling response, whereas cytokine production in the spleen did not. IL2 peaked early, followed by a late increase in IFN gamma production, corresponding to the maximum swelling. This simple method should prove useful for analysing the production of other cytokines in vivo in distinct anatomical compartments.