IS900 PCR Research Articles

Evaluation of the association of SLC11A1 gene polymorphism with incidence of paratuberculosis in goats.

Paratuberculosis is one of the chronic granulomatous enteritis that predominantly affects ruminants world wide, caused by Mycobacterium avium ssp. paratuberculosis (MAP). In ruminants, microsatellite polymorphisms of the 3' untranslated region (3'UTR) of the solute carrier family 11 member A1 (SLC11A1) gene were associated with resistance to intracellular pathogen infections. This research was carried out to detect the polymorphisms in A and B regions of the 3'UTR of SLC11A1 gene and to evaluate the potential association between these polymorphisms and MAP infection in goats. MAP-specific antibodies were detected by ELISA and MAP infection was confirmed by IS900 PCR in 150 adult goats from different regions of Kerala, India. The polymorphism of microsatellite regions A and B at 3'UTR of the SLC11A1 gene was analysed in goats by an automated technique, fragment analysis, using fluorescent-tagged forward primers. Eight alleles with sizes ranging from 221 to 239 bp were found in region A. Region B revealed two alleles, 117 bp (B₇) and 119 bp (B₈). Animals with B₈ alleles were found to have higher incidence of paratuberculosis than animals with B₇ alleles (P < 0.01). There was no statistically significant association found between region A genotypes and paratuberculosis incidence. These results suggest that caprine SLC11A1 gene has significant role in paratuberculosis resistance in goats and further studies might help in development of a PCR-based genotyping test for paratuberculosis resistance and selection of superior animals for future goat breeding programmes.

Detection of mycobacterial infection in non-human primates using the Xpert MTB/RIF molecular assay

Tuberculosis is a major public health concern, and diagnostic strategies applied to animal populations are scarce. As part of ongoing efforts to control tuberculosis dissemination at our animal facility, two non-human primates (NHP, Saimiri sciureus) presenting cutaneous lesions were examined for mycobacterial infection. Both animals tested positive for acid-fast bacilli and Mycobacterium tuberculosis using a molecular assay (IS6110 PCR). Animals were euthanized and several samples were tested for M. tuberculosis using the Xpert MTB/RIF assay. Many samples were positive for M. tuberculosis and rifampicin resistance, and some produced mycobacterial growth. Oral swabs from cage mates were then tested with Xpert MTB/RIF, and the majority tested positive for M. tuberculosis and rifampicin resistance, and produced growth in culture. To our knowledge, this is the first report of multidrug-resistant mycobacterial infection in NHP. Additionally, our data shows that the Xpert MTB/RIF assay can be useful as a screening tool for tuberculosis infection in NHP.

Short communication: Passive shedding of Mycobacterium avium ssp. paratuberculosis in commercial dairy goats in Brazil

Goat farming is a low-cost alternative to dairy production in developing countries. In Brazil, goat production has increased in recent years due in part to the implementation of programs encouraging this activity. Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a disease that causes chronic granulomatous enteritis in ruminants, but MAP transmission dynamics are still poorly understood in goats. In a previously published study of our research group, 10 dairy goat farms (467 animals) from Minas Gerais state were analyzed for MAP detection; 2 fecal cultures and 11 milk samples tested positive for MAP by conventional PCR and were confirmed by sequencing. Because no clinical signs were observed over 1 yr of monitoring, we hypothesized that these MAP-positive goats could be passive shedders. Thus, in the present study, 4 positive goats (4/13) from the previous study were purchased and feces and milk samples were collected for evaluation (twice, with an interval of 3 mo between tests) by culture of MAP, IS900 PCR, or both. All analyses were negative for MAP. At the last time point, blood samples were collected for ELISA, the animals were killed, and tissues collected for tissue culture and histopathology. At necropsy, no macroscopic lesions related to paratuberculosis were observed. Similarly, no histological changes were observed and MAP in samples stained by Ziehl-Neelsen was not detected. These animals were characterized as potential passive shedders with upward contamination of the teat canal by MAP. This is the first report of the passive shedding phenomenon in goats in Brazil and it highlights the importance of identifying these animals for control programs and to ensure the quality of dairy products.

Comparative evaluation of FAT, IS900 PCR and microscopy vis-a-vis histopathology for the detection of Mycobacterium avium subsp paratuberculosis infection in tissues of goats naturally died in herds endemic for Johne’s disease

Study investigated infection of Mycobacterium avium subspecies paratuberculosis (MAP) in paired tissues viz. ileum and mesenteric lymph nodes (MLN) from 74 goats using histopathology, acid fast staining of tissue sections, fluorescent antibody test (FAT), IS900 tissue PCR and culture. Histopathology of H&amp;E stained tissue sections revealed variable grades of lesions of Johne's disease (JD) in 37 (50.0%) ileae and 21 (28.4%) MLN. In AFB staining, 16 (21.6%) intestine and 10 (13.5%) MLN revealed presence of acid fast bacilli indistinguishable to MAP. Thirteen (17.5%) intestine and 9 (12.1%) MLN were positive in FAT. Eleven (14.8%) intestine and 7 (9.4%) MLN were positive by IS900 tissue PCR, whereas 7 (9.5%) intestine and 8 (10.8%) MLN were positive in culture. The study showed that sensitivity of histopathology (H&amp;E staining) was highest as compared to other routine tests for the diagnosis of MAP infection in tissues of goats died (irrespective of cause of deaths) during study period from goat herds endemic for Johne's disease. Lower sensitivity of FAT than histology was due to the strong cell mediated immunity exhibited by MAP. Though difference was not significant between sensitivities of FAT and PCR, FAT and AFB staining, and agreement was very good; however, results of the study suggested, that FAT may be a useful test to improve the diagnostic efficacy of caprine Johne's disease and more effective in the detection of grade I lesions as compared to culture and PCR. Study also showed that it was essential to collect both tissues (intestines and MLNs) for the diagnosis of MAP infection at necropsy.

Serological, culture and molecular survey of Mycobacterium avium paratuberculosis in a goat flock in Tuscany

Mycobacterium avium paratuberculosis (Map) is a pathogen which causes a chronic progressive granulomatous enteritis known as paratuberculosis or Johne's disease and it primarily affects wild and domestic ruminants. The aim of this research was to examine a flock which consisted of 294 goats and was located in Garfagnana district (Tuscany, Italy) performing ELISA tests, culture and IS900 PCR assay; direct diagnostic methods were carried out not only on bulk tank milk and cheese samples but also on individual milk and tissue specimens collected from nine subjects positive to ELISA tests. Out of 294 animals, 20 goats (6.8%) were positive to ELISA surveys. Bulk tank milk samples were negative to culture and to PCR assay carried out on the DNA extracted directly from them, while, with respect to cheese, Map was detected by culture in 2/12 (16.66%) cheeses ripened for 3-7days, and by PCR in 2/12 (16.66%) cheeses ripened for 3-7days and in 3/12 (25%) cheeses ripened for 45days. Regarding individual milk samples, Map was detected by culture in 2/9 (22.22%) specimens and by PCR in 5/9 (55.55%) samples. Furthermore, Map was isolated from the intestine in 9/9 (100%) animals, from the mesenteric lymph nodes in 8/9 (88.88%) subjects, from the liver in 4/9 (44.44%) goats, from the spleen in 5/9 (55.55%) animals, while Map DNA was found in all the tissue samples analyzed.The results demonstrated the presence of paratuberculosis in a goat flock located in Garfagnana district (Tuscany, Italy).

Diagnosis of tuberculous meningitis: Current scenario from a Tertiary Neurocare Centre in India

BackgroundTuberculous meningitis (TBM) is a condition that is caused by Mycobacterium tuberculosis complex and is difficult to diagnose due to the nonspecificity of the presentations. The study analyzed the different modes of diagnosis available in a developing country set up over a period of five years to understand the diagnostic values of the current conventional and automated methods of diagnosis of TBM among the patients suspected with chronic meningitis. MethodsA total of 10,281 cerebrospinal fluid samples (CSF) were collected from suspected chronic meningitis patients, of which 790 samples were from individuals who had clinically suspected TBM. The samples were subjected to CSF cytology and staining, culturing, immunological tests, molecular techniques, and methods for detection of drug resistance. ResultsThe TBM patients were predominantly male, with a mean age of 21–40 years. CSF pleocytosis and lymphocytic predominance were noted. Culture had 40.13% positivity among clinically suspected TBM patients. The multidrug-resistant M. tuberculosis (MDR-TB) constituted 3.14% of the total clinical isolates. With IS6110 PCR, a specificity of 92.86% and sensitivity of 100% are seen with an assay threshold of 30pg/ml. Line probe assay (LPA) using culture isolates had a sensitivity of 97.67% and a specificity of 100%. Direct CSF LPA showed a sensitivity of 96.15% and a specificity of 100%. ConclusionsA combination of techniques that involved culture, cytology, and DNA amplification methods was found to be promising in specific, accurate, and rapid detection of M. tuberculosis in the CSF samples from patients.

Mycobacterium avium subsp. paratuberculosis and associated risk factors for inflammatory bowel disease in Iranian patients

BackgroundInflammatory bowel disease (IBD) is described as a relapsing condition with high morbidity and uncertain complex pathogenesis. The association of Mycobacterium avium ssp. paratuberculosis (MAP) with Crohn’s disease (CD) in human has been debated for decades, however there is no confirmed data to verify such relations in Iran. The aim of this study was to investigate risk factors and a possible role of MAP in Iranian patients with CD.MethodsAnti-MAP antibodies were detected in serum of IBD patients and subjects without IBD (nIBD) through ELISA; MAP DNA and viable MAP cells were identified in patients’ biopsies through nested PCR and direct culture methods, respectively. Principal component analysis (PCA) was used to investigate the risk factors in relation to IBD and MAP infection.ResultsPositivity for IS900 PCR was detected in 64% (n = 18) of CD, 33% (n = 10) of ulcerative colitis (UC) and 9.7% (n = 6) of nIBD samples. Live MAP cells were isolated from biopsies of 2 CD patients only. Among 28 patients with CD, 46% (n = 13) and 39% (n = 11) were positive for antibodies against MAP3865c133–141 and MAP3865c125–133 peptides, respectively, whereas much lower seroreactivity was detected in UC subjects accounting for 3% (n = 1) for MAP3865c133–141 and 16.7% (n = 5) for MAP3865c125–133. A high immune reactivity to MAP epitopes among CD patients was positively correlated with consumption of fast food meals and IBD familiarity. For both CD and UC, breastfeeding period and consumption of fruit/vegetables presented negative correlation with the presence of anti-MAP antibodies.ConclusionsThis study provided evidences that high prevalence of MAP DNA and anti-MAP antibodies in CD patients is significantly associated with the development of CD. Despite the role of several factors contributing to IBD, the presence of MAP DNA and anti-MAP antibodies in Iranian CD patients highlights a possible transmission of MAP from animal-derived products to humans.

Molecular identification of Mycobacterium tuberculosis in cattle

Bovine tuberculosis continued to be a re-emerging problem in some countries especially in endemic areas due to the fact that human and animal health surveillance system is not adopted to diagnose the infection. This crisis can be attributed due to sharing of the same habitat especially in rural areas. In the present study, a total of 148 samples were collected from cattle for isolation over a period of 3 years from cattle with and without lesions, of which 67 isolates were obtained by culture. Fifty one isolates were identified as Mycobacterium tuberculosis complex (MTBC) by IS6110 PCR of which 43 (84.3%) were identified as M. tuberculosis and 08 (15.6%) were identified as M. bovis by using 12.7kb fragment multiplex PCR. Among this, 31 isolates which were positive for IS6110 PCR were subjected to spoligotyping and revealed 28 isolates belonging to MANU1 strain of M. tuberculosis. This study clearly indicates that high prevalence of M. tuberculosis than M. bovis in bovine was identified by means of culture and by molecular methods M. tuberculosis can affect cattle producing lesion in contradiction to the earlier thoughts. This study speculates that M. tuberculosis MANU1 strain infection in cattle could be due to spill over from human or other non specific hosts in tuberculosis endemic areas. Though bovine tuberculosis due to M. tuberculosis in cattle is not considered a serious threat worldwide, in countries where human TB is endemic, M. tuberculosis infection of cattle needs to be considered.

Spatio-Temporal Distribution of Mycobacterium tuberculosis Complex Strains in Ghana

BackgroundThere is a perception that genomic differences in the species/lineages of the nine species making the Mycobacterium tuberculosis complex (MTBC) may affect the efficacy of distinct control tools in certain geographical areas. We therefore analyzed the prevalence and spatial distribution of MTBC species and lineages among isolates from pulmonary TB cases over an 8-year period, 2007–2014.MethodologyMycobacterial species isolated by culture from consecutively recruited pulmonary tuberculosis patients presenting at selected district/sub-district health facilities were confirmed as MTBC by IS6110 and rpoß PCR and further assigned lineages and sub lineages by spoligotyping and large sequence polymorphism PCR (RDs 4, 9, 12, 702, 711) assays. Patient characteristics, residency, and risks were obtained with a structured questionnaire. We used SaTScan and ArcMap analyses to identify significantly clustered MTBC lineages within selected districts and spatial display, respectively.ResultsAmong 2,551 isolates, 2,019 (79.1%), 516 (20.2%) and 16 (0.6%) were identified as M. tuberculosis sensu stricto (MTBss), M. africanum (Maf), 15 M. bovis and 1 M. caprae, respectively. The proportions of MTBss and Maf were fairly constant within the study period. Maf spoligotypes were dominated by Spoligotype International Type (SIT) 331 (25.42%), SIT 326 (15.25%) and SIT 181 (14.12%). We found M. bovis to be significantly higher in Northern Ghana (1.9% of 212) than Southern Ghana (0.5% of 2339) (p = 0.020). Using the purely spatial and space-time analysis, seven significant MTBC lineage clusters (p< 0.05) were identified. Notable among the clusters were Ghana and Cameroon sub-lineages found to be associated with north and south, respectively.ConclusionThis study demonstrated that overall, 79.1% of TB in Ghana is caused by MTBss and 20% by M. africanum. Unlike some West African Countries, we did not observe a decline of Maf prevalence in Ghana.

Loop-mediated isothermal amplification (LAMP) assay for speedy diagnosis of tubercular lymphadenitis: The multi-targeted 60-minute approach

Tuberculous lymphadenitis (TBLA), the most common presentation of tuberculosis, poses a significant diagnostic challenge in the developing countries. Timely, accurate and cost-effective diagnosis can decrease the high morbidity associated with TBLA especially in resource-poor high-endemic regions. The loop-mediated isothermal amplification assay (LAMP), using two targets, was evaluated for the diagnosis of TBLA. LAMP assay using 3 sets of primers (each for IS6110 and MPB64) was performed on 170 fine needle aspiration samples (85 confirmed, 35 suspected, 50 control cases of TBLA). Results were compared against IS6110 PCR, cytology, culture and smear. The overall sensitivity and specificity of LAMP assay, using multi-targeted approach, was 90% and 100% respectively in diagnosing TBLA. The sensitivity of multi-targeted LAMP, only MPB64 LAMP, only IS6110 LAMP and IS6110 PCR was 91.7%, 89.4%, 84.7% and 75.2%, respectively among confirmed cases and 85.7%, 77.1%, 68.5% and 60%, respectively among suspected cases of TBLA. Additional 12/120 (10%) cases were detected using multi-targeted method. The multi-targeted LAMP, with its speedy and reliable results, is a potential diagnostic test for TBLA in low-resource countries.

Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop-mediated isothermal amplification assay.

Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug-resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing-specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time-consuming and technically demanding. In the present study, a loop-mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain-identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c-targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207-PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c-multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.

Persistence of bacterial indicators and zoonotic pathogens in contaminated cattle wastes.

BackgroundManure can provide a favourable environment for pathogens’ survival. De-contamination may be conducted by extended storage, until field conditions are suitable for application to land as source of agricultural nutrients.ResultsThe hygienic evaluation of manure and slurry coming from a plant that collects cattle livestock wastes from a big slaughterhouse was carried out. Samples were even collected from spillages in the area around the plant. Microbial analyses highlighted the massive presence of faecal indicators in all samples: mean counts of Escherichia coli and enterococci were always above EU limits for marketable processed manure products.Cultures referable to the genus Brucella spp. were recorded in two samples of fresh manure but not in the aged ones. Conventional isolation techniques failed to detect members of the Mycobacterium genus, while by means of IS900 and F57 PCR real-time system on DNA directly extracted from environmental samples, the pathogen was detected in all cases.ConclusionsThoughtful design of manure storage infrastructure is critical to prevent spills and over-topping of an open structure. The documented overload situation seems to lay the basis for an ongoing environmental contamination by enteric organisms and opportunistic pathogens circuit faecal-oral route. Moreover, the type of wastes analysed during this study, namely a mixture of fresh cattle manure, bedding and rumen content, needs a longer storage period or, alternatively, of specific chemical, biological or thermal treatments for stabilization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0705-8) contains supplementary material, which is available to authorized users.

Comparative evaluation of in-house Real time IS 6110, nested MPT 64 PCR and Roche AMPLICOR 16s rRNA PCR for diagnosing tuberculous meningitis

Background: Early diagnosis of tuberculous meningitis (TBM) is still a diagnostic challenge due to paucibacillary nature of the disease and conventional methods being quite insensitive and time consuming. We evaluated performance of three real time PCR assays targeting MPT64, IS6110 and 16s rRNA gene sequences for early diagnosis of TBM Methods & Materials: A total of 100 consecutive probable TBM patients and 50 non TBM patients were enrolled from an ongoing prospective study on tuberculous meningitis (July 2012 to Dec 2014). CSF specimens were subjected to microscopy and automated BACTEC MGIT culture. In-house Real time nested MPT64 and IS6110 PCR were standardized using H37Rv spiked CSF and evaluated for diagnostic utility along with commercially available Roche Amplicor Real time PCR assay. Results: Out of 100 clinical specimens processed, the sensitivity of smear microscopy and culture was 4% and 42% respectively. The sensitivity, specificity of Real time IS6110 PCR, Nested MPT 64 assay and Roche Amplicor Real time PCR assay was 71%, 98%; 69%, 98% and 25%, 100% respectively against probable TBM as reference standard. The Real time IS6110 PCR, Nested MPT 64 assay and Roche Amplicor Real time PCR assay could detect M. tuberculosis in only 84%, 77% and 30% of culture positive patient's respectively Conclusion: Real time IS6110 PCR proved to be a simple and rapid method to diagnose TBM with sensitivity, specificity of 71%, 98% only. Nested MPT 64 assay though had comparable sensitivity and specificity was much more difficult due to nested design and higher risks of contamination. None of the PCR was 100% sensitive for detecting all culture positive samples suggesting that for TBM diagnosis there is no single rule out test and all the tests are contingent upon their ability to pick the target in tested volume of CSF.

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation.

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.

Use of Bactec MGIT 960 ParaTB system for the diagnosis of bovine paratuberculosis

In the present study, Bactec MGIT 960 ParaTB system, a liquid culture system has been used to culture the Mycobacterium avium paratuberculosis (Map). Faecal, milk and serum samples were collected in separate sterile containers from 20 milking crossbred cows, aged above 4 years with a history of chronic diarrhea, weak body condition and weight loss. Five (25.0%, 5/20) animals reacted positively to single intra-dermal Johnin test. Four (20.0%, 4/20) samples each of serum and milk were positive by ELISA. The positive milk and serum samples belonged to the same individuals. Direct PCR could not detect Map DNA in any of the faeces/milk samples. However, 1 out of 20 faecal samples was tagged positive in system on day 19 post culture. Z-N staining, conventional IS900 PCR and real-time PCR confirmed these to be Map.

Short communication: Herd-level prevalence of Mycobacterium avium ssp. paratuberculosis is not associated with participation in a voluntary Alberta Johne’s disease control program

Johne’s disease (JD) control programs for dairy farms have the general objective of reducing both cow- and herd-level prevalence of Mycobacterium avium ssp. paratuberculosis (MAP). An important aspect of many programs is herd testing for MAP to determine the infection status of participating farms. However, it is uncertain whether MAP herd-level prevalence on farms voluntarily participating in a JD control program is different from that on nonparticipating farms. Therefore, the aim was to compare MAP infection status of participants and nonparticipants in the Alberta Johne’s Disease Initiative (AJDI), a voluntary JD control program initiated in 2010 in Alberta, Canada. Between September 2012 and August 2013, environmental fecal samples were collected from 93 randomly selected farms not enrolled in the AJDI. Additionally, 81 farms that initially enrolled in the AJDI during the same time interval were also sampled. Samples were collected from 6 defined locations on each farm and cultured for MAP. Results were confirmed using conventional IS900 PCR and F 0285 quantitative PCR. Overall, 51% of participating and 51% of nonparticipating farms were identified as being MAP-infected. Furthermore, based on multivariable logistic regression, the number of MAP-positive samples was not associated with AJDI participation (taking herd size into account as a potentially modifying or confounding variable). In conclusion, there was no indication that voluntary participation in the AJDI was associated with herd-level MAP prevalence.

Noninvasive Tuberculosis Screening in Free-Living Primate Populations in Gombe National Park, Tanzania.

Recent advances in noninvasive detection methods for mycobacterial infection in primates create new opportunities for exploring the epidemiology of tuberculosis in free-living species. Chimpanzees (Pan troglodytes schweinfurthii) and baboons (Papio anubis) in Gombe National Park, Tanzania, were screened for infection with pathogens of the Mycobacterium tuberculosis Complex using Fecal IS6110 PCR; none was positive. This study demonstrates the feasibility of large-scale mycobacterial screening in wild primates.

Microcalcificaciones cerebrales en un recién nacido con tuberculosis congénita

Tuberculosis is a serious public health problem worldwide. In 2012, the World Health Organization estimated 8.6 million new cases and 1.3 million deaths due to the disease. In 2011, the incidence in Colombia was 24 cases per 100,000 inhabitants. There is little information about tuberculosis in pregnant women, and congenital infection is considered a rare disease that is difficult to diagnose, leads to high mortality, and may be confused with tuberculosis acquired after birth. In addition, it has been associated with HIV infection in mothers and infants. Moreover, there is increasing incidence of congenital syphilis in the world. In Colombia, the prevalence is 2.5 cases per 1,000 births and its frequency in the Instituto Materno Infantil-Hospital La Victoria is one case per 57 births. We report the case of a newborn under treatment for congenital syphilis and in whom microcalcifications were found in a transfontanelar ultrasound. This finding warned about the existence of another infectious agent. PCR was negative for cytomegalovirus, and IgM titers for toxoplasma, rubella and herpes I and II were also negative.After learning about a history of incomplete treatment for tuberculosis in the mother, we suspected the presence of an infection by the tubercle bacillus in the newborn. No acid-fast bacilli were demonstrated in three gastric juice samples. The IS6110 PCR assay was found positive in cerebrospinal fluid and urine, but not in blood. The newborn was treated with crystalline penicillin for 10 days along with isoniazid, rifampicin, pyrazinamide and streptomycin. The patient is currently under clinical monitoring.

Pathology and diagnosis of Mycobacterium bovis in naturally infected dromedary camels (Camelus dromedarius) in India.

The present study investigated the pathological features of tuberculosis (TB) caused by Mycobacterium bovis and its diagnosis in naturally infected dromedary camels from an organized farm in India. During the period of the 5-year study, a total of 18 (19.56%) camels out of 92 examined showed gross lesions compatible with TB at post-mortem. The clinical signs and pathological lesions in these camels were studied, and the efficacy of different diagnostic tests was also assessed. On the basis of occurrence and distribution of gross TB lesions, the infected camels revealed two different lesional patterns as pulmonary (n = 15) and disseminated (n = 3) form. The histopathology of affected organs revealed typical granulomatous lesions wherein the giant cells and acid-fast bacilli were occasionally observed in pulmonary form whereas they frequently observed in disseminated form. The single intradermal tuberculin test (SIDT) detected TB in 10 (55.55%) whereas the Ziehl-Neelsen (ZN) stain and IS6110 PCR from tissue lesions detected 13 (72.22%) and 18 (100%) of the infected camels, respectively. The study suggests that pulmonary form of the TB is more common in camels indicating respiratory route as the major source of exposure in camel herds. Moreover, very low sensitivity of SIDT was observed which highlights the difficulty for confirmation of TB in live camels.

Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis

The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections.