Risk factors of gastrointestinal nematode parasite infections in small ruminants kept in smallholder mixed farms in Kenya

Background

We have evaluated a sensitive screening assay for Mycobacterium tuberculosis (MTB) complex organisms and a specific assay for detecting Mycobacterium bovis DNA in lymph nodes taken from cattle with evidence of bovine tuberculosis. Underlying these series of experiments was the need for a versatile DNA extraction protocol which could handle tissue samples and with the potential for automation. The target for the screening assay was the multi-copy insertion element IS1081, present in 6 copies in the MTB complex. For confirmation of M. bovis we used primers flanking a specific deletion in the genome of M. bovis known as region of difference 4 (RD4). The sensitivity and specificity of these PCRs has been tested on genomic DNA from MTB complex reference strains, mycobacteria other than tuberculosis (MOTT), spiked samples and on clinical material.

Results

The minimum detection limits of the IS1081 method was < I genome copy and for the RD4 PCR was 5 genome copies. Both methods can be readily adapted for quantitative PCR with the use of SYBR Green intercalating dye on the RotorGene 3000 platform (Corbett Research). Initial testing of field samples of bovine lymph nodes with visible lesions (VL, n = 109) highlighted two shortfalls of the molecular approach. Firstly, comparison of IS1081 PCR with the "gold standard" of culture showed a sensitivity of approximately 70%. The sensitivity of the RD4 PCR method was 50%. Secondly, the success rate of spoligotyping applied directly to clinical material was 51% compared with cultures. A series of further experiments indicated that the discrepancy between sensitivity of detection found with purified mycobacterial DNA and direct testing of field samples was due to limited mycobacterial DNA recovery from tissue homogenates rather than PCR inhibition. The resilient mycobacterial cell wall, the presence of tissue debris and the paucibacillary nature of some cattle VL tissue may all contribute to this observation. Any of these factors may restrict application of other more discriminant typing methods. A simple means of increasing the efficiency of mycobacterial DNA recovery was assessed using a further pool of 95 cattle VL. Following modification of the extraction protocol, detection rate with the IS1081 and RD4 methods increased to 91% and 59% respectively.

Conclusion

The IS1081 PCR is a realistic screening method for rapid identification of positive cases but the sensitivity of single copy methods, like RD4 and also of spoligotyping will need to be improved to make these applicable for direct testing of tissue extracts.     

Improved Detection of Mycobacterium bovis Infection in Bovine Lymph Node Tissue Using Immunomagnetic Separation (IMS)-Based Methods

Immunomagnetic separation (IMS) can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR) or culture (IMS-MGIT). This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL), 74 non-visibly lesioned (NVL)) collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1%) lymph node samples tested positive by culture, 162 (57.8%) by IMS-PCR (targeting IS6110), and 191 (68.2%) by IMS-MGIT culture. Twelve (6.9%) of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 79 M. bovis positive lymph node samples (27 (13.1%) VL and 52 (70.3%) NVL) were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle) decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant) which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 70.3% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.     

Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.

Methodology/Principal Findings

We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.

Conclusions

M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10⁵ cells g⁻¹ using Griffiths and 4.25×10⁶ cells g⁻¹ using FastDNA® Spin kit.     

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory

Background

The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis) for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only.

Results

During 1998–2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern.

Conclusions

In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days). However, culture is needed after all to assess the antibiotic susceptibility of the strains.

     

Comparative analysis of mycobacterial infections in wild striped bass Morone saxatilis from Chesapeake Bay

During an ongoing epizootic of mycobacteriosis, wild striped bass Morone saxatilis from Chesapeake Bay were analyzed using 3 methods for detection of either mycobacterial infection or associated granulomatous pathology. The specific detection techniques, which utilized aseptically collected splenic tissue, were histology, quantitative culture and nested PCR. Based on analysis of 118 samples, detection of infection differed significantly between the 3 methods (chi-square, p = 0.0007). Quantitative culture and nested PCR detected similar, higher rates of infection (69 and 75%, respectively) than the histological method (52%). Although primary PCR assays for a 924 to 940 bp segment of the mycobacterial 16S rRNA gene were positive for genomic DNA from mycobacterial cultures, a secondary, nested PCR reaction for an internal 300 bp gene segment was required in order to detect mycobacteria within splenic tissue. A similar rate of mycobacterial infection was present in fish collected from all sites tested. Although all detection methods found that striped bass age 4.0 to 4.9 yr had the highest positive incidence, nested PCR detected a higher frequency of mycobacterial infection in fish > or = 6.0 yr of age than the other 2 methods. Quantitative bacteriology was a more sensitive detection technique when the fish tissue contained < or = 10(3) mycobacteria g(-1).     

Distribution of Environmental Mycobacteria in Karonga District, Northern Malawi

The genus Mycobacterium includes many species that are commonly found in the environment (in soil and water or associated with plants and animals), as well as species that are responsible for two major human diseases, tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The distribution of environmental mycobacteria was investigated in the context of a long-term study of leprosy, tuberculosis, Mycobacterium bovis BCG vaccination, and the responses of individuals to various mycobacterial antigens in Karonga District, northern Malawi, where epidemiological studies had indicated previously that people may be exposed to different mycobacterial species in the northern and southern parts of the district. A total of 148 soil samples and 24 water samples were collected from various locations and examined to determine the presence of mycobacteria. The detection method involved semiselective culturing and acid-fast staining, following decontamination of samples to enrich mycobacteria and reduce the numbers of other microorganisms, or PCR with primers specific for the mycobacterial 16S rRNA gene, using DNA extracted directly from soil and water samples. Mycobacteria were detected in the majority of the samples, and subsequent sequence analysis of PCR products amplified directly from soil DNA indicated that most of the products were related to known environmental mycobacteria. For both methods the rates of recovery were consistently higher for dry season samples than for wet season samples. All isolates cultured from soil appeared to be strains of Mycobacterium fortuitum. This study revealed a complex pattern for the environmental mycobacterial flora but identified no clear differences between the northern and southern parts of Karonga District.     

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

Aim: To develop a multiplex real‐time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real‐time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross‐reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real‐time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real‐time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real‐time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.     

Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10⁴ times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.     

Homogenisation of cystic fibrosis sputum by sonication--an essential step for Aspergillus PCR

The importance of Aspergillus as a lung pathogen in cystic fibrosis (CF) is becoming increasingly recognised. However, fungal culture of CF sputum is unreliable and there is no consensus for identifying phenotypes beyond ABPA that may benefit from antifungal therapy. There are no published studies using real-time PCR to detect Aspergillus in CF sputum. The major barrier to sensitive detection of Aspergillus using PCR is sputum homogenisation. This study aimed to optimise sputum homogenisation utilising sonication to improve Aspergillus DNA extraction. Sonication amplitude and duration that enabled sputum homogenisation but ensured preservation of DNA integrity were first determined. 160 sputum samples were collected from CF patients. 49 of the sputum samples were split, one half was used for standard culture and the other half was homogenised with NALC-NaOH before undergoing DNA extraction. The subsequent 111 samples were homogenised with dithiothreitol plus sonication prior to culture and DNA extraction. Real-time PCR targeting a portion of the 18S rDNA of Aspergillus was performed on all DNA extractions. In the 49 samples with no sonication 8 (16%) were culture positive but only 4 of these were PCR positive. However, PCR was positive in 11 culture negative samples. PCR after sonication showed a significant improvement in sensitivity: 33 (30%) were culture and PCR positive, 48 (43%) were culture negative, but PCR positive (p < 0.0001) and 30 (27%) were culture and PCR negative. The combination of dithiothreitol and sonication to homogenise sputum increases PCR yield, with PCR being substantially more sensitive than culture.     

Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

Background

Nucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1) tissue disruption procedures; 2) DNA extraction protocols; and 3) inhibition of bacterial PCR by host DNA.

Principal Findings

Regarding DNA extraction, we found that 1) grinding was not necessary if bead-beating is done, 2) the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3) lysozyme digestion of 1 hour was sufficient, and 4) repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was < 3 µg in a final volume of 25 µl and the quality was high (260/280 nm ratio = 1.89±0.08). Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M. tuberculosis, M. avium subsp. paratuberculosis).

Conclusions

Our findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA.     

Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the <Emphasis Type="Italic">Mycoplasma bovis</Emphasis>

Background

Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics.

Results

Here, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30?min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR.

Conclusions

To the author’s knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings.

     

Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10³ to 10⁴ oocysts, and the nested PCR method was able to detect 10⁰ to 10² oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.     

Identification of Malassezia Species in Patients with Seborrheic Dermatitis in China

Background

The causes of seborrheic dermatitis (SD) are complex and incompletely understood. Among the factors, Malassezia yeasts have been reported to play a major etiological role in SD. Many previous studies adopted conventional culture methods that were disadvantaged to detect Malassezia microflora in SD patients, resulting in a low detection rate for each species and high variance in types of microflora observed.

Objective

This study analyzed Malassezia microflora in SD patients by applying a transparent dressing to the lesional skin and using direct detection of fungal DNA using nested PCR.

Methods

We collected samples from the lesional skin of 146 SD patients in China and extracted fungal DNA directly from the lesional samples without culture. Specific primers for each Malassezia species were designed to amplify existing yeasts in each sample. Some samples were randomly selected to culture and identified by morphological and physiologic criteria.

Results

M. globosa and M. restricta were found in 87.0 and 81.5 % of seborrheic dermatitis patients, respectively, which together accounted for more than 50 % of Malassezia spp. recovered in these Chinese patients. The majority of SD patients (82.9 %) showed co-colonization of two or more Malassezia species.

Conclusion

M. globosa and M. restricta predominated in Malassezia colonization in Chinese SD patients. Compared with conventional culture, non-culture-based methods may more accurately reflect Malassezia microflora constitution.     

First-Time Detection of Mycobacterium bovis in Livestock Tissues and Milk in the West Bank,Palestinian Territories

Background

Bovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area.

Methodology/principal findings

A total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample.

Significance

Detection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel.     

Detection of Mycobacterium bovis and Mycobacterium tuberculosis from Cattle: Possible Public Health Relevance

Mycobacterium bovis and Mycobacterium tuberculosis infect both animals and humans. The disease epidemiology by these agents differs in developed and developing countries due to the differences in the implementation of the prevention and control strategies. The present study describes the detection of M. bovis and M. tuberculosis from specimens of lungs and pulmonary lymph nodes of four cattle died in an organized herd of 183 cattle in the state of Himachal Pradesh, India, with inconclusive skin test results. Identification and distinction of these closely related mycobacterial species was done by PCR-RFLP targeting hsp65 gene followed by spacer oligonucleotide typing. Mixed infection of M. bovis and M. tuberculosis was detected in one cattle.     

Enhanced Detection of Tuberculous Mycobacteria in Animal Tissues Using a Semi-Nested Probe-Based Real-Time PCR

Bovine tuberculosis has been tackled for decades by costly eradication programs in most developed countries, involving the laboratory testing of tissue samples from allegedly infected animals for detection of Mycobacterium tuberculosis complex (MTC) members, namely Mycobacterium bovis. Definitive diagnosis is usually achieved by bacteriological culture, which may take up to 6–12 weeks, during which the suspect animal carcass and herd are under sanitary arrest. In this work, a user-friendly DNA extraction protocol adapted for tissues was coupled with an IS6110-targeted semi-nested duplex real-time PCR assay to enhance the direct detection of MTC bacteria in animal specimens, reducing the time to achieve a diagnosis and, thus, potentially limiting the herd restriction period. The duplex use of a novel β-actin gene targeted probe, with complementary targets in most mammals, allowed the assessment of amplification inhibitors in the tissue samples. The assay was evaluated with a group of 128 fresh tissue specimens collected from bovines, wild boars, deer and foxes. Mycobacterium bovis was cultured from 57 of these samples. Overall, the full test performance corresponds to a diagnostic sensitivity and specificity of 98.2% (CI_P95% 89.4–99.9%) and 88.7% (CI_P95% 78.5–94.7%), respectively. An observed kappa coefficient was estimated in 0.859 (CI_P95% 0.771–0.948) for the overall agreement between the semi-nested PCR assay and the bacteriological culture. Considering only bovine samples (n = 69), the diagnostic sensitivity and specificity were estimated in 100% (CI_P95% 84.0–100%) and 97.7% (CI_P95% 86.2–99.9%), respectively. Eight negative culture samples exhibiting TB-like lesions were detected by the semi-nested real-time PCR, thus emphasizing the increased potential of this molecular approach to detect MTC-infected animal tissues. This novel IS6110-targeted assay allows the fast detection of tuberculous mycobacteria in animal specimens with very high sensitivity and specificity, being amenable and cost effective for use in the routine veterinary diagnostic laboratory with further automation possibilities.     

Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.