Peptide aMptD-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Bulk Milk Samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 × 10² CFU ml⁻¹ for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 × 10² CFU ml⁻¹) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 × 10³ and an association constant of 1.33 × 10⁹. The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

Evaluation of two recovery methods for detection of Mycobacterium avium subsp. paratuberculosis by PCR: direct-dilution--centrifugation and C(18)-carboxypropylbetaine processing

A duplex polymerase chain reaction (PCR)-hybridization assay based on Mycobacterium avium subsp. paratuberculosis (MAP)-specific IS900 integration sites was used to evaluate two mycobacterial recovery methods from bovine feces: a direct-dilution-centrifugation method and a C(18)-carboxypropylbetaine (CB-18)-based method. All MAP PCR results were confirmed for absence of inhibitors using a novel PCR system based on the rpoB gene of plant chloroplasts as an internal control. The detection limits of both MAP recovery methods when coupled with PCR were determined to be between 100 and 1000 organisms. Using culture as a 'gold standard' PCR following the direct-dilution-centrifugation protocol was 92.6% sensitive and 83.7% specific, whereas PCR following the CB-18 method was 100% sensitive and 53.5% specific. Both methods were 100% specific when 60 'true' negatives from two uninfected herds were tested. Both the CB-18 and direct processing methods coupled with a target-specific amplification technique may provide greater sensitivity to diagnose subclinical animals as they were able to detect more positives, on samples derived from infected herds, than conventional culture methods; however, more extensive investigation and follow-up of suspect animals will be required to fully validate the MAP recovery and molecular detection protocols described.     

Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg²⁺ concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 × 10⁶ to 3 × 10¹ copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.     

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.     

Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log₁₀ CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5′-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log₁₀ were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log₁₀ CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 10³ spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Changes in Sodium,Calcium, and Magnesium Ion Concentrations That Inhibit Geobacillus Biofilms Have No Effect on Anoxybacillus flavithermus Biofilms

This study investigated the effects of varied sodium, calcium, and magnesium concentrations in specialty milk formulations on biofilm formation by Geobacillus spp. and Anoxybacillus flavithermus. The numbers of attached viable cells (log CFU per square centimeter) after 6 to 18 h of biofilm formation by three dairy-derived strains of Geobacillus and three dairy-derived strains of A. flavithermus were compared in two commercial milk formulations. Milk formulation B had relatively high sodium and low calcium and magnesium concentrations compared with those of milk formulation A, but the two formulations had comparable fat, protein, and lactose concentrations. Biofilm formation by the three Geobacillus isolates was up to 4 log CFU cm⁻² lower in milk formulation B than in milk formulation A after 6 to 18 h, and the difference was often significant (P ≤ 0.05). However, no significant differences (P ≤ 0.05) were found when biofilm formations by the three A. flavithermus isolates were compared in milk formulations A and B. Supplementation of milk formulation A with 100 mM NaCl significantly decreased (P ≤ 0.05) Geobacillus biofilm formation after 6 to 10 h. Furthermore, supplementation of milk formulation B with 2 mM CaCl₂ or 2 mM MgCl₂ significantly increased (P ≤ 0.05) Geobacillus biofilm formation after 10 to 18 h. It was concluded that relatively high free Na⁺ and low free Ca²⁺ and Mg²⁺ concentrations in milk formulations are collectively required to inhibit biofilm formation by Geobacillus spp., whereas biofilm formation by A. flavithermus is not impacted by typical cation concentration differences of milk formulations.     

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 × 10² CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.     

Dynamics of Escherichia coli Virulence Factors in Dairy Herds and Farm Environments in a Longitudinal Study in the United States

Pathogenic Escherichia coli or its associated virulence factors have been frequently detected in dairy cow manure, milk, and dairy farm environments. However, it is unclear what the long-term dynamics of E. coli virulence factors are and which farm compartments act as reservoirs. This study assessed the occurrence and dynamics of four E. coli virulence factors (eae, stx₁, stx₂, and the gamma allele of the tir gene [γ-tir]) on three U.S. dairy farms. Fecal, manure, water, feed, milk, and milk filter samples were collected from 2004 to 2012. Virulence factors were measured by postenrichment quantitative PCR (qPCR). All factors were detected in most compartments on all farms. Fecal and manure samples showed the highest prevalence, up to 53% for stx and 21% for γ-tir in fecal samples and up to 84% for stx and 44% for γ-tir in manure. Prevalence was low in milk (up to 1.9% for stx and 0.7% for γ-tir). However, 35% of milk filters were positive for stx and 20% were positive for γ-tir. All factors were detected in feed and water. Factor prevalence and levels, expressed as qPCR cycle threshold categories, fluctuated significantly over time, with no clear seasonal signal independent from year-to-year variability. Levels were correlated between fecal and manure samples, and in some cases autocorrelated, but not between manure and milk filters. Shiga toxins were nearly ubiquitous, and 10 to 18% of the lactating cows were potential shedders of E. coli O157 at least once during their time in the herds. E. coli virulence factors appear to persist in many areas of the farms and therefore contribute to transmission dynamics.     

Practical Method for Extraction of PCR-Quality DNA from Environmental Soil Samples

Methods for the extraction of PCR-quality DNA from environmental soil samples by using pairs of commercially available kits were evaluated. Coxiella burnetii DNA was detected in spiked soil samples at <1,000 genome equivalents per gram of soil and in 12 (16.4%) of 73 environmental soil samples.The detection of pathogenic organisms in the environment often relies on PCR analysis of DNA purified from environmental soil (6). For effective detection, a reliable method to obtain PCR-quality DNA from soil is necessary. Although a variety of complex techniques have been effective for specific soil samples (1-3, 7, 8), it is not clear which methods would be the best for the wide variety of samples encountered in a large-scale environmental sampling study. In addition, many published techniques would be difficult to use on a large number of samples (1-3, 7, 8).This study evaluates the abilities of commercially available DNA extraction kits to provide DNA from environmental soil samples that are suitable for PCR detection of Coxiella burnetii. C. burnetii is an obligate intracellular, Gram-negative, zoonotic pathogen and the causative agent of Q fever (5). It is classified as a category B agent of bioterrorism by the CDC.Three commercially available DNA purification kits were evaluated. Twenty different soil samples obtained from diverse locations in the southeastern United States were used for testing. These samples consisted of light sandy soil and were all initially processed through one of three DNA purification kits, the UltraClean soil DNA isolation kit (MoBio Laboratories, Carlsbad CA), the QIAamp DNA minikit (Qiagen, Valencia, CA), or the QIAamp DNA stool minikit (Qiagen), or through a combination of two of the kits used sequentially. Thus, all 20 samples were each processed through nine extraction protocols. To process soil samples, five grams of soil was mixed with 10 to 30 ml of phosphate-buffered saline (PBS) to create a homogenized slurry. Samples were mixed for 1 h at room temperature and then centrifuged for 5 min at 123 × g. The supernatant was removed and centrifuged at 20,000 × g for 15 min. The supernatant was then carefully discarded and the pellet resuspended in 1 ml of PBS.For the UltraClean soil kit, 700 μl of the resuspended soil extraction pellet was processed by the manufacturer''s alternative protocol (for maximum yields). For preps done using the QIAamp DNA minikit (tissue protocol) and the QIAamp stool kit (stool protocol), 700 μl (high volume) of the soil extract was processed according to the instructions for the particular kit. For 17 of the samples the tissue protocol and stool protocol were applied using only 200 μl of the soil extract (low volume). For all of the kits, the final elutions were performed with 55 μl of water.To further purify the products of the commercial DNA isolation kits, eluates were passed through a second round of extraction. When the MoBio UltraClean kit was used for the second round of extraction, eluates were added to the bead-containing tubes and mixed with 60 μl of solution 1 and 200 μl of the MoBio inhibitor removal solution (IRS). The manufacturer''s protocol was then followed. When the QIAamp tissue protocol was utilized for the second round of extraction, eluates were diluted to 200 μl with water and then mixed with 200 μl of buffer ATL plus 200 μl of buffer AL and then incubated at 70°C for 10 min. Following this step, the manufacturer''s protocol was followed. When the QIAamp stool protocol was used for the second round of extraction, eluates were mixed with 1.2 ml of the ASL buffer, followed by addition of the InhibitEX tablet. The manufacturer''s protocol was then followed.PCR inhibition in all of the DNA samples was then evaluated by running a quantitative PCR that detects the IS1111 gene from C. burnetii (4). PCRs were run on 200 genome equivalents of C. burnetii (strain Nine Mile Phase 1) DNA. Reaction mixtures spiked with 1-μl aliquots of the environmental DNA samples were compared to reaction mixtures spiked with 1 μl of water. Inhibition was considered present if the DNA sample caused an increase of 1 in the threshold cycle value.Use of the MoBio UltraClean procedure by itself resulted in removal of inhibitors from 35% of the samples, whereas after use of the Qiagen tissue protocol (high volume) only 4% of the samples were free of inhibition (Fig. ​(Fig.1).1). The Qiagen stool kit (high volume) resulted in 96% of the samples showing lack of inhibition with a low volume of soil eluate and 62.5% of the samples when the high volume was used. The DNA extracted from these three kits was then used as starting material for a subsequent DNA extraction step using the same set of three commercial kits. The MoBio UltraClean kit followed by the Qiagen stool kit eliminated inhibition in all samples, as did these two kits when used in the reverse order, even if the Qiagen stool kit was loaded with 700 μl of material (high volume). When a low volume of starting material was used, combinations of the two Qiagen kits also removed inhibitors from 100% of the samples when either the Qiagen tissue protocol was used first or the Qiagen stool protocol was used first (Fig. ​(Fig.1).1). The raw data for all of the inhibition assays are included as supplemental data (see Table S1 in the supplemental material).Open in a separate windowFIG. 1.Twenty environmental soil samples were used for the isolation of DNA with the indicated protocols. The samples were then tested for the ability to inhibit an IS1111 PCR with C. burnetii Nine Mile DNA as template. The percentages of samples that did not show any inhibition are indicated.To determine the yield of DNA obtained by the various protocols, nine aliquots (5 g each) of a single rich organic soil sample were each mixed with 5 ml PBS, spiked with 1 × 10⁶ Nine Mile Phase 2 C. burnetii organisms, and then processed by the nine (high-volume) extraction protocols described above. An additional 1 × 10⁶ Nine Mile Phase 2 C. burnetii organisms were used directly in the Qiagen tissue protocol to prepare DNA for the purpose of determining the exact amount of C. burnetii input into the assays. The quantitative IS1111 PCR assay (4) was used to determine the yield of C. burnetii DNA by using the various methods for processing soil. The yield was calculated by dividing the number of genome equivalents of C. burnetii DNA obtained from the spiked soil samples by the number of genome equivalents obtained when C. burnetii was included directly in the Qiagen tissue protocol. A common feature of all of the protocols was that they all produced a low yield of C. burnetii DNA when purified from a complex soil mixture (Fig. ​(Fig.2).2). The yields ranged from 0.02% to 4.3% and were variable. Although the 4.3% yield obtained when the stool kit was used alone was the highest on average, the high variability observed with these extractions suggests that most of these protocols provide similar yields. The stool kit followed by the MoBio kit clearly resulted in the lowest yield.Open in a separate windowFIG. 2.Five-gram aliquots of a single soil sample were all spiked with approximately 1 × 10⁶ C. burnetii Phase 2 Nine Mile strain cells. The samples were then subjected to the indicated extraction protocol(s). The resulting DNA was tested for inhibition, and then the genome equivalents of C. burnetii DNA were determined by quantitative IS1111 PCR. The exact input amount of C. burnetii was determined by running an aliquot directly through the QIAamp tissue protocol followed by IS1111 PCR. Yield was calculated as genome equivalents obtained from the spiked soil samples divided by the genome equivalents obtained from the direct extraction through the QIAamp tissue protocol. Values represent the mean ± standard deviation of five experiments. Statistically significant differences (Student''s t test) were found between stool versus MoBio plus stool kits (P = 0.05), stool plus tissue versus MoBio plus stool kits (P = 0.01), and stool plus tissue versus tissue plus MoBio kits (P = 0.03). For the protocol using the stool kit followed by the MoBio kit the yield was significantly different from stool, stool plus tissue, MoBio plus tissue, and MoBio protocols (P < 0.05).Although these yields are low, the IS1111 PCR assay used to detect C. burnetii DNA amplifies a multicopy gene, and the assay can detect a single genome equivalent (4). This suggests that these protocols are adequate for the detection of C. burnetii in soil samples with 500 to 2,000 organisms per gram of soil. To test this, a 5-g sample of organic soil was spiked with 800 C. burnetii organisms per gram, and the DNA was extracted using the MoBio UltraClean kit followed by the QIAamp stool protocol. C. burnetii DNA was detected after 38 cycles using the IS1111 PCR assay.While these results are focused on soil samples, the procedures described also work well on vacuum samples and sponge wipe samples (data not shown). Based on removal of inhibitors and yield, our data suggest that the QIAamp tissue protocol (high volume) followed by the QIAamp stool protocol and the MoBio UltraClean kit followed by the QIAamp stool protocol are both suitable for extraction of DNA from environmental soil samples. To test the application of the latter method to a larger number of samples, 73 bulk soil samples from the southeastern United States were processed according to this method. Inhibition was removed from all 73 samples, and 12 of the samples were positive in the C. burnetii IS1111 PCR assay. This suggests that this practical method for extraction of PCR-quality DNA can be successfully used to detect DNA from C. burnetii and other pathogens in large numbers of environmental samples.      

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Effect of Commercial-Scale High-Temperature, Short-Time Pasteurization on the Viability of Mycobacterium paratuberculosis in Naturally Infected Cows' Milk

Raw cows' milk naturally infected with Mycobacterium paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73°C for 15 s or 25 s with and without prior homogenization (2,500 lb/in² in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73°C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73°C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. paratuberculosis, but the extended 25-s holding time at 73°C was found to be no more effective at killing M. paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.     

Diagnostic Performance of Schistosoma Real-Time PCR in Urine Samples from Kenyan Children Infected with Schistosoma haematobium: Day-to-day Variation and Follow-up after Praziquantel Treatment

Background

In an effort to enhance accuracy of diagnosis of Schistosoma haematobium, this study explores day-to-day variability and diagnostic performance of real-time PCR for detection and quantification of Schistosoma DNA compared to other diagnostic tools in an endemic area before and after treatment.

Methodology

Previously collected urine samples (N = 390) from 114 preselected proven parasitological and/or clinical S. haematobium positive Kenyan schoolchildren were analyzed by a Schistosoma internal transcribed spacer-based real-time PCR after 14 years of storage. Pre-treatment day-to-day fluctuations of PCR and microscopy over three consecutive days were measured for 24 children using intra-class correlation coefficient. A combined ‘gold standard’ (PCR and/or microscopy positive) was used to measure sensitivity and negative predictive value (NPV) of several diagnostic tools at baseline, two and 18 months post-treatment with praziquantel.

Principal Findings

All 24 repeatedly tested children were PCR-positive over three days with little daily variation in median Ct-values, while 83.3% were found to be egg-positive for S. haematobium at day 1 and 75.0% at day 2 and 3 pre-treatment, signifying daily fluctuations in microscopy diagnosis. Of all 114 preselected schoolchildren, repeated microscopic measurements were required to detect 96.5% versus 100% of positive pre-treatment cases by single PCR. At two months post-treatment, microscopy and PCR detected 22.8% versus 69.3% positive children, respectively. Based on the ‘gold standard’, PCR showed high sensitivity (>92%) as compared to >31% sensitivity for microscopy, both pre- and post-treatment.

Conclusions/Significance

Detection and quantification of Schistosoma DNA in urine by real-time PCR was shown to be a powerful and specific diagnostic tool for detection of S. haematobium infections, with less day-to-day variation and higher sensitivity compared to microscopy. The superior performance of PCR before, and two and 18 months post-treatment provides a compelling argument for PCR as an accurate and reproducible tool for monitoring treatment efficacy.     

Development of a Real-Time PCR Probe for Quantification of the Heterotrophic Dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in Environmental Samples

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of Cryptoperidiniopsis brodyi in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to C. brodyi. The C. brodyi-specific assay was used in conjunction with Pfiesteria piscicida- and Pfiesteria shumwayae-specific real-time PCR assays to investigate the temporal variations of C. brodyi, P. piscicida, and P. shumwayae abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that C. brodyi occurred in all seasons at very low densities, mostly below 25 cells liter⁻¹, with higher abundance (maximum, 112 cells liter⁻¹) in April and May. P. piscicida was detected only once, in May 2005 at 60 cells liter⁻¹. P. shumwayae was not detected during the survey.     

Longevity of Mycobacterium bovis in Raw and Traditional Souring Milk as a Function of Storage Temperature and Dose

Background

Unpasteurised fresh and souring dairy products form an essential component of household diets throughout many rural communities in southern Africa. The presence of milk-borne zoonotic pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis and zoonotic tuberculosis in humans, constitute a public health threat, especially in remote areas with poor disease surveillance in livestock and highly compromised human health due to HIV/AIDS.

Methods

In this study we used culture to determine the longevity of M. bovis in experimentally inoculated fresh and naturally souring milk obtained from communal cattle in the KwaZulu-Natal province of South Africa. The effect of bacterial load and storage temperature on the survival of M. bovis was evaluated by spiking mixtures of fresh milk and starter soured milk (aMasi) culture with three concentrations of bacteria (10², 10⁴, 10⁷ colony forming units/ml), followed by incubation under controlled laboratory conditions that mimicked ambient indoor (20°C) and outdoor (33°C) temperatures and periodic sampling and testing over time (0-56 days).

Results

M. bovis cultured from samples of the fresh and souring milk was identified by PCR analysis. At the highest spiking concentration (10⁷cfu/ml), M. bovis survived for at least 2 weeks at 20°C; but, at all concentrations in the 33°C treatment, M. bovis was absent by three days after inoculation. Logistic regression analysis was used to assess the effects of bacterial concentration and time since inoculation, as well as determine the potential half-life of M. bovis in raw souring milk. Given the most favourable tested conditions for bacterial survival (20°C), approximately 25% of mycobacteria were alive after one day of storage (95% CI: 9-53%), giving an estimated half-life of M. bovis in raw souring milk of approximately 12 hours (95% CI: 7-27 hours).

Conclusions

This study demonstrates that M. bovis may survive in fresh and souring milk for periods of time that represent a risk of exposure to people consuming these products, as well as domestic or wild animal populations that have reported opportunities to consume homemade unpasteurised dairy products. The temperature at which the milk is soured and stored substantially affects the survival time of M. bovis.     

A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants

Background

Diagnosis of urogenital schistosomiasis by microscopy and serological tests may be elusive in travelers due to low egg load and the absence of seroconversion upon arrival. There is need for a more sensitive diagnostic test. Therefore, we developed a real-time PCR targeting the Schistosoma haematobium-specific Dra1 sequence.

Methodology/Principal Findings

The PCR was evaluated on urine (n = 111), stool (n = 84) and serum samples (n = 135), and one biopsy from travelers and migrants with confirmed or suspected schistosomiasis. PCR revealed a positive result in 7/7 urine samples, 11/11 stool samples and 1/1 biopsy containing S. haematobium eggs as demonstrated by microscopy and in 22/23 serum samples from patients with a parasitological confirmed S. haematobium infection. S. haematobium DNA was additionally detected by PCR in 7 urine, 3 stool and 5 serum samples of patients suspected of having schistosomiasis without egg excretion in urine and feces. None of these suspected patients demonstrated other parasitic infections except one with Blastocystis hominis and Entamoeba cyst in a fecal sample. The PCR was negative in all stool samples containing S. mansoni eggs (n = 21) and in all serum samples of patients with a microscopically confirmed S. mansoni (n = 22), Ascaris lumbricoides (n = 1), Ancylostomidae (n = 1), Strongyloides stercoralis (n = 1) or Trichuris trichuria infection (n = 1). The PCR demonstrated a high specificity, reproducibility and analytical sensitivity (0.5 eggs per gram of feces).

Conclusion/Significance

The real-time PCR targeting the Dra1 sequence for S. haematobium-specific detection in urine, feces, and particularly serum, is a promising tool to confirm the diagnosis, also during the acute phase of urogenital schistosomiasis.     

Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)n and (A/T)n microsatellites

During microsatellite polymerase chain reaction (PCR), insertion–deletion mutations produce stutter products differing from the original template by multiples of the repeat unit length. We analyzed the PCR slippage products of (CA)_n and (A)_n tracts cloned in a pUC18 vector. Repeat numbers varied from two to 14 (CA)_n and four to 12 (A)_n. Data was generated on approximately 10 single molecules for each clone type using two rounds of nested PCR. The size and peak areas of the products were obtained by capillary electrophoresis. A quasi- likelihood approach to the analysis of the data estimated the mutation rate/repeat/PCR cycle. The rate for (CA)_n tracts was 3.6 × 10^–3 with contractions 14 times greater than expansions. For (A)_n tracts the rate was 1.5 × 10^–2 and contractions outnumbered expansions by 5-fold. The threshold for detecting ‘stutter’ products was computed to be four repeats for (CA)_n and eight repeats for (A)_n or ~8 bp in both cases. A comparison was made between the computationally and experimentally derived threshold values. The threshold and expansion to contraction ratios are explained on the basis of the active site structure of Taq DNA polymerase and models of the energetics of slippage events, respectively.     

Determination of chlorpromazine in human breast milk and serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of chlorpromazine in serum and human breast milk. Chlorpromazine in serum and human breast milk was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction, and a reversed-phase HPLC separation technique was developed. Chlorpromazine and levomepromazine as the internal standard were detected by ultraviolet absorbance at 254 nm. Determination was possible for chlorpromazine in the concentration range 10–300 ng/ml. The recoveries of chlorpromazine added to serum and human breast milk were 80.1–87.6 and 80.3–84.4%, respectively, with coefficients of variation of less than 10.2 and 7.8%. The method is applicable to drug level monitoring in the serum and human breast milk of patients treated with chlorpromazine.     

Nutritional and ecological evaluation of dairy farming systems based on concentrate feeding regimes in semi-arid environments of Jordan

The objective of this study was to evaluate the nutritional and ecological aspects of feeding systems practiced under semi-arid environments in Jordan. Nine dairy farms representing the different dairy farming systems were selected for this study. Feed samples (n = 58), fecal samples (n = 108), and milk samples (n = 78) were collected from the farms and analysed for chemical composition. Feed samples were also analysed for metabolisable energy (ME) contents and in vitro organic matter digestibility according to Hohenheim-Feed-Test. Furthermore, fecal nitrogen concentration was determined to estimate in vivo organic matter digestibility. ME and nutrient intakes were calculated based on the farmer’s estimate of dry matter intake and the analysed composition of the feed ingredients. ME and nutrient intakes were compared to recommended standard values for adequate supply of ME, utilizable crude protein, rumen undegradable crude protein (RUCP), phosphorus (P), and calcium (Ca). Technology Impact Policy Impact Calculation model complemented with a partial life cycle assessment model was used to estimate greenhouse gas emissions of milk production at farm gate. The model predicts CH₄, N₂O and CO₂ gases emitted either directly or indirectly. Average daily energy corrected milk yield (ECM) was 19 kg and ranged between 11 and 27 kg. The mean of ME intake of all farms was 184 MJ/d with a range between 115 and 225 MJ/d. Intake of RUCP was lower than the standard requirements in six farms ranging between 19 and 137 g/d, was higher (32 and 93 g/d) in two farms, and matched the requirements in one farm. P intake was higher than the requirements in all farms (mean oversupply = 19 g/d) and ranged between 3 and 30 g/d. Ca intake was significantly below the requirements in small scale farms. Milk nitrogen efficiency N-_eff (milk N/intake N) varied between 19% and 28% and was mainly driven by the level of milk yield. Total CO₂ equivalent (CO₂ equ) emission ranged between 0.90 and 1.88 kg CO₂/kg ECM milk, where the enteric and manure CH₄ contributed to 52% of the total CO₂ equ emissions, followed by the indirect emissions of N₂O and the direct emissions of CO₂ gases which comprises 17% and 15%, respectively, from total CO₂ equ emissions. Emissions per kg of milk were significantly driven by the level of milk production (r² = 0.93) and of eDMI (r² = 0.88), while the total emissions were not influenced by diet composition. A difference of 16 kg ECM/d in milk yield, 9% in N-_eff and of 0.9 kg CO₂ equ/kg in ECM milk observed between low and high yielding animals. To improve the nutritional status of the animals, protein requirements have to be met. Furthermore, low price by-products with a low carbon credit should be included in the diets to replace the high proportion of imported concentrate feeds and consequently improve the economic situation of dairy farms and mitigate CO₂ equ emissions.     

Impact of Transmammary-Delivered Meloxicam on Biomarkers of Pain and Distress in Piglets after Castration and Tail Docking

To investigate a novel route for providing analgesia to processed piglets via transmammary drug delivery, meloxicam was administered orally to sows after farrowing. The objectives of the study were to demonstrate meloxicam transfer from sows to piglets via milk and to describe the analgesic effects in piglets after processing through assessment of pain biomarkers and infrared thermography (IRT). Ten sows received either meloxicam (30 mg/kg) (n = 5) or whey protein (placebo) (n = 5) in their daily feedings, starting four days after farrowing and continuing for three consecutive days. During this period, blood and milk samples were collected at 12-hour intervals. On Day 5 after farrowing, three boars and three gilts from each litter were castrated or sham castrated, tail docked, and administered an iron injection. Piglet blood samples were collected immediately before processing and at predetermined times over an 84-hour period. IRT images were captured at each piglet blood collection point. Plasma was tested to confirm meloxicam concentrations using a validated high-performance liquid chromatography-mass spectrometry method. Meloxicam was detected in all piglets nursing on medicated sows at each time point, and the mean (± standard error of the mean) meloxicam concentration at castration was 568.9±105.8 ng/mL. Furthermore, ex-vivo prostaglandin E₂ (PGE₂) synthesis inhibition was greater in piglets from treated sows compared to controls (p = 0.0059). There was a time-by-treatment interaction for plasma cortisol (p = 0.0009), with meloxicam-treated piglets demonstrating lower cortisol concentrations than control piglets for 10 hours after castration. No differences in mean plasma substance P concentrations between treatment groups were observed (p = 0.67). Lower cranial skin temperatures on IRT were observed in placebo compared to meloxicam-treated piglets (p = 0.015). This study demonstrates the successful transfer of meloxicam from sows to piglets through milk and corresponding analgesia after processing, as evidenced by a decrease in cortisol and PGE₂ levels and maintenance of cranial skin temperature.