Simple and rapid liquid chromatographic and electrophoretic methods for phenol quantification and its stability in tuberculin purified protein derivative preparations.

Phenol is commonly used as an antimicrobial agent with an initial concentration of 0.35% (w/v) in injectable diluted tuberculin purified protein derivative (TPPD) solution. The anti-microbial action of phenol in TPPD is directly concentration dependent. Furthermore, high phenol content (>0.5%) may have a negative effect on the stability and clinical effectiveness of TPPD solution. Therefore, simple, rapid and reliable reversed phase liquid chromatographic (RPLC) and capillary zone electrophoretic (CZE) methods were firstly developed and validated for phenol quantification in Connaught tuberculin (CT68) PPD diluted preparations at 5 TU per test dose of 0.1 mL. In RPLC, the elution was carried out by 80% (v/v) ACN mixed with 20% (v/v) phosphate buffer containing 0.05% (v/v) triflouroacetic acid (pH 3.2) at 0.2 mL min-1 flow rate and 20.0 °C column temperature. In addition, phenol was separated from tuberculin (CT68) protein with a resolution of (R = 2.81) and was quantified within 3 min. In CZE, the migration of phenol was performed by 50 mmol L-1 borate buffer (pH 9.8) at -20 kV applied voltage and 25.0 °C capillary temperature. Furthermore, excellent linearity was achieved within 0.17-0.53% (w/v) for the phenol content with coefficients of determination (r2) higher than 0.9995. Moreover, the detection and quantification limits were found to be 0.046 & 0.153% and 0.051 & 0.171% (w/v) with RPLC and CZE respectively. Additionally, the intraday precision (RSD%, n = 9) was ranged between 0.18 and 0.39 and 0.33-54 with RPLC and CZE respectively. Moreover, the interday precision (RSD%, n = 27) was varied between 2.06 and 2.99 and 2.25-3.40 by RPLC and CZE, respectively. Furthermore, the obtained mean recoveries were ranged between 91.32 and 107.51% with RPLC and 90.71-108.92% with CZE. In addition, the effect of different storage temperatures at 4, 25 and 37 °C over storage periods of 2, 7, 14, 21 and 30 days was also studied on the TPPD product. The obtained results have revealed that the phenol content was effectively decreased about 37% of its original content after 30 days at storage temperatures of 25 and 37 °C. However, the phenol content did not change and was stable up to 21 days at storage temperature of 4 °C. Therefore, the simple and rapid proposed analytical methods could be used for a rapid expiry investigation of TPPD products based on phenol quantification, as a marker.

Small-Volume Injections: Evaluation of Volume Administration Deviation From Intended Injection Volumes.

BACKGROUND: In the perioperative period, anesthesiologists and postanesthesia care unit (PACU) nurses routinely prepare and administer small-volume IV injections, yet the accuracy of delivered medication volumes in this setting has not been described. In this ex vivo study, we sought to characterize the degree to which small-volume injections (≤0.5 mL) deviated from the intended injection volumes among a group of pediatric anesthesiologists and pediatric postanesthesia care unit (PACU) nurses. We hypothesized that as the intended injection volumes decreased, the deviation from those intended injection volumes would increase. METHODS: Ten attending pediatric anesthesiologists and 10 pediatric PACU nurses each performed a series of 10 injections into a simulated patient IV setup. Practitioners used separate 1-mL tuberculin syringes with removable 18-gauge needles (Becton-Dickinson & Company, Franklin Lakes, NJ) to aspirate 5 different volumes (0.025, 0.05, 0.1, 0.25, and 0.5 mL) of 0.25 mM Lucifer Yellow (LY) fluorescent dye constituted in saline (Sigma Aldrich, St. Louis, MO) from a rubber-stoppered vial. Each participant then injected the specified volume of LY fluorescent dye via a 3-way stopcock into IV tubing with free-flowing 0.9% sodium chloride (10 mL/min). The injected volume of LY fluorescent dye and 0.9% sodium chloride then drained into a collection vial for laboratory analysis. Microplate fluorescence wavelength detection (Infinite M1000; Tecan, Mannedorf, Switzerland) was used to measure the fluorescence of the collected fluid. Administered injection volumes were calculated based on the fluorescence of the collected fluid using a calibration curve of known LY volumes and associated fluorescence.To determine whether deviation of the administered volumes from the intended injection volumes increased at lower injection volumes, we compared the proportional injection volume error (loge [administered volume/intended volume]) for each of the 5 injection volumes using a linear regression model. Analysis of variance was used to determine whether the absolute log proportional error differed by the intended injection volume. Interindividual and intraindividual deviation from the intended injection volume was also characterized. RESULTS: As the intended injection volumes decreased, the absolute log proportional injection volume error increased (analysis of variance, P < .0018). The exploratory analysis revealed no significant difference in the standard deviations of the log proportional errors for injection volumes between physicians and pediatric PACU nurses; however, the difference in absolute bias was significantly higher for nurses with a 2-sided significance of P = .03. CONCLUSIONS: Clinically significant dose variation occurs when injecting volumes ≤0.5 mL. Administering small volumes of medications may result in unintended medication administration errors.

Preliminary investigation of the transmission of tuberculosis between farmers and their cattle in smallholder farms in northwestern Ethiopia: a cross-sectional study.

BACKGROUND: The feeding habits and close physical contact between Ethiopian farmers and their cattle promote the transmission of tuberculosis (TB) between the farmers and their cattle. This study aimed to investigate the transmission of TB between farmers and their cattle in smallholder farms in northwestern Ethiopia. RESULTS: A total of 70 human TB lymphadenitis (TBLN) cases visiting the Felegehiwot Comprehensive Specialized Hospital in Bahir Dar City and 660 cattle were investigated. Half of the cattle were owned by households with TB cases, and the remaining half by TB free households. Among the 70 human TBLN patients interviewed, 65.7% (46 out of 70) of the respondents were not aware of zoonotic TB, and 67.1% (47/70) of them consumed raw milk. Positive cultures of TB were obtained in 40 of the 70 cases where TBLN tests were positive with fine needle aspiration cytology. Spoligotyping resulted in 31 different patterns, of which 25 isolates were Mycobacterium (M.) tuberculosis, and the remaining were M. africanum (4 isolates) and M. bovis (2 isolates). None of the animals showed positive test results for bovine TB by comparative intradermal tuberculin test. CONCLUSIONS: Based on the identification of M. bovis from two patients diagnosed with TBLN, we obtained preliminary evidence of zoonotic transmission of TB in northwestern Ethiopia. We did not identify a direct route of transmission between cattle and its owners. This is the objective of further investigations.

A Dynamic Model of Human and Livestock Tuberculosis Spread and Control in Urumqi, Xinjiang, China.

We establish a dynamical model for tuberculosis of humans and cows. For the model, we firstly give the basic reproduction number R 0. Furthermore, we discuss the dynamical behaviors of the model. By epidemiological investigation of tuberculosis among humans and livestock from 2007 to 2014 in Urumqi, Xinjiang, China, we estimate the parameters of the model and study the transmission trend of the disease in Urumqi, Xinjiang, China. The reproduction number in Urumqi for the model is estimated to be 0.1811 (95% confidence interval: 0.123-0.281). Finally, we perform some sensitivity analysis of several model parameters and give some useful comments on controlling the transmission of tuberculosis.

Biomarkers for risk of developing active tuberculosis in contacts of TB patients: a prospective cohort study.

Identifying those Mycobacterium tuberculosis latent-infected individuals most at risk of developing active tuberculosis (TB) using routine clinical and laboratory tests remains a huge challenge in TB control efforts. We conducted a prospective longitudinal study of clinical and laboratory markers associated with the risk of developing active TB in contacts with latent M. tuberculosis infection.HIV-negative household contacts (n=296) of pulmonary TB patients underwent monitoring of clinical features, full blood cell counts, tuberculin skin text (TST) and chest radiography performed regularly during 18 months of follow-up. Paired statistical tests, a Kaplan-Meier analysis and Cox proportional hazard modelling were performed on variables between contacts progressing or not progressing to active TB.The appearance of TB disease symptoms in contacts was significantly associated with an elevated peripheral percentage of blood monocytes (adjusted hazard ratio (aHR) 6.25, 95% CI 1.63-23.95; p<0.01), a ≥14 mm TST response (aHR 5.72, 95% CI 1.22-26.80; p=0.03) and an increased monocyte:lymphocyte ratio (aHR 4.97, 95% CI 1.3-18.99; p=0.03). Among contacts having TST ≥14 mm, a strong association with risk of progression to TB was found with an elevated blood monocyte percentage (aHR 8.46, 95% CI 1.74-41.22; p<0.01).Elevated percentage of peripheral blood monocytes plus an elevated TST response are potential biomarkers for identifying contacts of TB patients at highest risk of developing active TB.

Chitin microneedles for an easy-to-use tuberculosis skin test.

An easy-to-use tuberculosis skin test is developed with chitin microneedles that deliver purified protein derivative at the correct skin depth and result in a positive test in BCG-immunized guinea pigs.

National shortage of purified-protein derivative tuberculin products.

Tubersol, a product of Sanofi Pasteur Limited, is in short supply nationwide until at least the end of May 2013. Tubersol is one of two purified-protein derivative (PPD) tuberculin products licensed by the Food and Drug Administration (FDA). The manufacturer has notified CDC that 50-dose vials of Tubersol will remain unavailable until the end of May 2013 and that supplies of 10-dose vials are still being reestablished: the product is available only by contacting Sanofi directly (at https://www.vaccineshoppe.com/index.cfm? or telephone, 800-822-2463). JHP Pharmaceuticals, LLC, the manufacturer of Aplisol, the other PPD tuberculin product licensed by FDA, has notified FDA that the product is available in restricted quantity. Acute local shortages of Aplisol also have been reported to CDC by TB control officials, as health-care providers switch from Tubersol to Aplisol. The shortages of Aplisol probably will diminish as Tubersol supplies are restored to their preshortage availability in the normal distribution networks. This report advises public health officials, clinicians, and workers in occupational health and infection control about how to adapt to the shortage.

Mesoporous silica-calcium phosphate-tuberculin purified protein derivative composites as an effective adjuvant for cancer immunotherapy.

The synthesis of mesoporous silica/calcium phosphate composite loaded with the immunopotentiator tuberculin purified protein derivative (PPD-MS/CaP) as an effective adjuvant for cancer immunotherapy is reported here. The PPD-MS/CaP adjuvant is prepared by immersing mesoporous silica in a supersaturated calcium phosphate solution supplemented with the immunopotentiator PPD for 24 h. PPD is coprecipitated with calcium phosphate inside and on the surface of mesoporous silica. By loading the immunopotentiator PPD in the PPD-MS/CaP adjuvant, an enhanced activation of antigen-presenting cells, such as GM-CSF secretion by THP-1 differentiated macrophages, is obtained probably due to sustained PPD release and an efficient cellular uptake of PPD. The PPD-MS/CaP adjuvant mixed with liquid-N2 -treated tumor tissue effectively triggers anti-tumor immune response and markedly inhibits in vivo tumor growth. The PPD-MS/CaP adjuvant is a promising alternative for cancer immune therapy.

[Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis]. / Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of yIFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis / Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP)

Los derivados proteicos purificados (PPD) son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb) y PPD aviar (PPDa) son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control), no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of ?IFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis / Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP)

Los derivados proteicos purificados (PPD) son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb) y PPD aviar (PPDa) son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control), no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.(AU)

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ÒInterferon-release assay. The stimulation of ÒInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of ?IFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.(AU)

Purified protein derivatives of tuberculin--past, present, and future.

The tuberculin skin test, which involves monitoring the immune reaction to an injection of purified protein derivative (PPD), has been the most widely used method for detecting infection with Mycobacterium tuberculosis since its development in 1930s. Until recently, the molecular composition of PPD was unknown. This thwarted the discovery of improved skin testing reagents and drastically hindered efforts to define the mechanism of action. Proteomic evaluation of PPD combined with a detailed analysis in the guinea pig model of tuberculosis led to further definition of the molecular composition of PPD. This communication reviews the history and current status of PPD, in addition to describing candidate next-generation PPD reagents, based on the use of an individual protein or protein cocktails.

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis.

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.

The stability of human, bovine and avian tuberculin purified protein derivative (PPD).

INTRODUCTION: Guidelines recommend storing tuberculin purified protein derivative (PPD) refrigerated. However, especially in developing countries, maintaining the product refrigerated under field conditions can be difficult, limiting its use. Here we determine the effect of prolonged exposure to high temperatures on the potency of human, bovine and avian tuberculin PPD. METHODOLOGY: Human, bovine and avian tuberculin PPD were stored for several weeks exposed to temperatures ranging from 37º to 100ºC. The potency was evaluated in vivo, in sensitized or naturally infected animals. RESULTS: Most test situations didn't affect the biological activity of the tuberculin PPDs and only very long and extreme incubations (several days at 100 °C) compromised the potency. CONCLUSIONS: Tuberculin PPD is very stable and can be stored or transported for long periods without refrigeration. 

The comparative performance of the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations from different manufacturers.

Ireland currently obtains its avian and bovine tuberculin purified protein derivatives (PPDs) from a single source. Because problems of supply or quality cannot be discounted, it is prudent that Ireland identify alternative supplier(s) as part of a broad risk management strategy. Therefore, the aim of this study was to compare the performance of a number of different tuberculin combinations (that is, pairings of bovine and avian PPD; with different manufacturers) in the single intradermal comparative tuberculin test (SICTT), as currently performed in Ireland. The study was randomised, controlled and double-blinded. A total of 2172 cattle were used in the study. Each animal was tested using two SICTTs, the first based on the tuberculin combination in current use, and the second using one of six trial tuberculin combinations. Analyses were conducted to compare both reactor-status and skin increase. For each control/trial tuberculin combination, there was good agreement between the control and trial reactor-status. Differences in skin increases were mainly confined to animals categorised as either negative or severe inconclusive. However, the measured differences were minor, and unlikely to have a significant impact on the actual test outcome, either for individual animals or for herds. In conclusion, while further studies determining sensitivity and specificity in Ireland would have to be done in the event of a change in tuberculin PPD there should be minimal disruption of the national programme if alternative tuberculin PPDs meeting WHO, OIE and EU regulations were used. In this study, the precision of the guinea pig bio-assay to assess tuberculin potency was low and therefore Ireland should maintain its practice of periodically assessing potency in naturally infected cattle, even though this is not currently required under WHO, OIE or EU Regulations.

Factors influencing the performance of an interferon-γ assay for the diagnosis of tuberculosis in goats.

The interferon-gamma (IFN-γ) assay is an effective tool for the diagnosis of tuberculosis (Tb) in goats. The objectives of this study were to evaluate factors that might affect assay performance: (1) the phenol concentration of the purified protein derivative (PPD, tuberculin) used; (2) dialysis of PPD; and (3) delaying antigenic stimulation of blood samples for 8, 16 and 24h after collection. The assay was performed in duplicate with two cut-off points. Dialysis of PPD reduced test sensitivity, whereas the concentration of phenol did not significantly affect test outcome. Delaying antigenic stimulation of samples >8h resulted in a reduction in test sensitivity, compromising the capacity of the assay to detect infected animals. Performing the assay in duplicate was unnecessary, which has implications for reducing assay costs. These findings will facilitate the effective application of the IFN-γ assay as an ancillary test in Tb eradication programmes in goats.

Antibody response to a sterile filtered PPD tuberculin in M. bovis infected and M. bovis sensitized cattle.

BACKGROUND: Bovine tuberculosis, caused by Mycobacterium bovis, afflicts approximately 50 million cattle worldwide and is detected by the tuberculin skin test (TST). While it has long been recognized that purified protein derivative (PPD) tuberculin is composed of a mixture of M. bovis derived protein components, little is known about the quality, relative quantity and identity of the proteins that make up PPD tuberculin. We manufactured a sterile filtered PPD tuberculin (SF-PPD) from a nine-week-old M. bovis culture supernatant in order to characterise the culture filtrate proteins (CFP) which make up M. bovis PPD tuberculin and to compare the antibody response of M. bovis infected versus M. bovis sensitized cattle. RESULTS: SF-PPD resolved into approximately 200 discrete spots using two-dimensional polyacrylamide gel electrophoresis (2-DE) while fewer than 65 spots could be discerned from 2-DE gels of tuberculin derived from autoclaved culture supernatant. Two dimensional Western blot analyses indicated that sera from M. bovis sensitized cattle recognized additional SF-PPD antigens as compared to M. bovis infected cattle at seven weeks post infection/sensitization. However, application of a comparative tuberculin skin test resulted in an antibody boosting response to the same set of M. bovis CFPs in both the M. bovis infected and M. bovis sensitized cattle. CONCLUSIONS: We concluded that it is the heat sterilization of the M. bovis CFPs that causes severe structural changes to the M. bovis proteins. This work suggests that M. bovis infected cattle and cattle artificially sensitized to M. bovis with an injection of heat killed cells exhibit similar antibody responses to M. bovis antigens.

Immunological and cytotoxicological characterization of tuberculin purified protein derivative (PPD) conjugated to single-walled carbon nanotubes.

Tuberculosis (TB) represents one of the leading killers among all infectious disease. Protection against TB depends on the activation of T-helper type I (Th1) immune response. Carbon nanotubes (CNTs) have attracted considerable attention because of their potential applications as new nanovehicle. In the current study, tuberculin purified protein derivative (PPD) was conjugated to carboxylated single-walled carbon nanotubes (SWCNTs). Cytotoxicity of the carboxylated SWCNT and SWCNT-PPD conjugate was analyzed with MTT assay and by reactive oxygen species (ROS) and nitric oxide (NO) generation. Male BALB/c mice were immunized with BCG, PPD, SWCNT-PPD conjugate and PPD in complete Freund's adjuvant (CFA). Induction of cellular immune response was analyzed by measuring the levels of Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-10 and IL-5). Immunization with non-conjugated PPD or PPD in Freund's adjuvant induced a Th2 cytokine response while immunization with BCG resulted to a mixed Th1/Th2 cytokine response. In contrast, PPD in conjugation with SWCNT generated preferentially a Th1-type cytokine response in the absence of potential cytotoxic effects.