Low Shedders Research Articles

How to best assess shedder status: a comparison of popular shedder tests.

"Shedder status" describes the inherent variation between individuals to leave touch DNA on a surface through direct contact. Depending on the amount and quality of DNA or cellular deposition, individuals are typically deemed high, intermediate, or low shedders. Although many shedder tests have been described, variability in study design and categorisation criteria has limited the ability of researchers to accurately compare results, as well as accrue the necessary population data. As activity level reporting becomes more common, the need for reliable and standardised testing increases. To assess reproducibility, this study compared shedder status data generated by six participants using three different shedder tests, as modified from the literature. This involved DNA quantification and profiling of a handprint made on a glass plate, DNA quantification and profiling of a grip mark made on a plastic conical tube, and cell scoring of a Diamond™ Dye-stained fingermark. All participants washed and dried their hands fifteen minutes before each deposit. To assess the impact of behaviour on shedder designation, participants either refrained from activity or went about their daily tasks during this wait. The shedder status of participants changed between tests, as DNA-based testing often generated lower shedder statuses than cell scores. Further, when different categorisation methods were applied to a single test, intra-person variability increased as the number of shedder designations increased from two (low/high) to five (low/low-intermediate/intermediate/intermediate-high/high). Moving forward, the utilisation of a single shedder test and standardised categorisation criteria is needed to employ shedder testing in forensic casework.

Cell-free DNA Concentration as a Biomarker of Response and Recurrence in HER2-Negative Breast Cancer Receiving Neoadjuvant Chemotherapy.

We previously demonstrated the clinical significance of circulating tumor DNA (ctDNA) in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy (NAC). Here, we compared its predictive and prognostic value with cell-free DNA (cfDNA) concentration measured in the same samples from the same patients. 145 patients with hormone receptor (HR)-positive/HER2-negative and 138 triple-negative breast cancer (TNBC) with ctDNA data from a previous study were included in the analysis. Associations of serial cfDNA concentration with residual cancer burden (RCB) and distant recurrence-free survival (DRFS) were examined. In TNBC, we observed a modest negative correlation between cfDNA concentration 3 weeks after treatment initiation and RCB, but none of the other timepoints showed significant correlation. In contrast, ctDNA was significantly positively correlated with RCB at all timepoints (all R > 0.3 and P < 0.05). In the HR-positive/HER2-negative group, cfDNA concentration did not associate with response to NAC, but survival analysis showed that high cfDNA shedders at pretreatment had a significantly worse DRFS than low shedders (hazard ratio, 2.12; P = 0.037). In TNBC, the difference in survival between high versus low cfDNA shedders at all timepoints was not statistically significant. In contrast, as previously reported, ctDNA at all timepoints was significantly correlated with DRFS in both subtypes. In TNBC, cfDNA concentrations during therapy were not strongly correlated with response or prognosis. In the HR-positive/HER2-negative group, pretreatment cfDNA concentration was prognostic for DRFS. Overall, the predictive and prognostic value of cfDNA concentration was more limited than that of ctDNA.

Abstract A014: Cell-free DNA concentration as a biomarker of response and recurrence in HER-2 negative breast cancer receiving neoadjuvant chemotherapy

Abstract Purpose: Cell-free DNA (cfDNA) shed into the blood by dying cells and its subset—circulating tumor DNA (ctDNA) exclusively released by tumors—are promising non-invasive biomarkers for predicting treatment response and survival. We sought to determine the clinical significance of cfDNA concentration in comparison to that of circulating tumor DNA (ctDNA) in patients with high-risk HER2-negative breast cancer receiving neoadjuvant chemotherapy (NAC) in the I-SPY2 trial. Methods: 283 patients with early-stage breast cancer (145 hormone receptor (HR)-positive/HER2-negative and 138 triple-negative) who had ctDNA data from a previous study (Magbanua et al. Cancer Cell 2023) were included in the analysis. CfDNA concentration in plasma was measured at pretreatment, during, and after NAC before surgery. Associations of cfDNA concentration (as a continuous and dichotomous variable using the median as cutoff) with clinicopathologic variables, response, and survival were examined. In addition, ctDNA data from the same cohort using a personalized tumor-informed test was available for comparative analysis (Magbanua et al., Cancer Cell 2023). The response endpoint was residual cancer burden (RCB) after NAC. The survival endpoint was distant recurrence-free survival (DRFS). Results: There were no significant differences in cfDNA concentration between the HR-positive/HER2-negative and TNBC groups at any time point. In the TNBC group, but not in HR-positive/HER2-negative group, high pretreatment cfDNA concentration was significantly associated with larger tumors (clinical stage T3/T4, Wilcoxon p=0.023) and higher grade (grade 3, Fisher p=0.0134). TNBC patients with no residual cancer (RCB-0 or pathologic complete response) after NAC had significantly higher cfDNA concentration 3 weeks after treatment initiation than those with extensive residual cancer after NAC (RCB-III, Kruskal-Wallis adjusted p=0.034). In the HR-positive/HER2-negative group, cfDNA concentration did not associate with response to NAC, but survival analysis showed that high cfDNA shedders at pretreatment had a significantly worse DRFS than low shedders (hazard ratio 2.12, 95% confidence interval 1.05-4.60, p=0.037). However, in the TNBC group, the difference in survival between high vs. low cfDNA shedders at all time points was not statistically significant. In contrast, as previously reported, ctDNA at all time points was significantly correlated with metastatic recurrence and death in both HR-positive/HER2-negative and TNBC groups (Magbanua et al., Cancer Cell 2023). Conclusions: In TNBC, cfDNA concentration reflected tumor burden, aggressiveness, and early response to NAC. In the HR-positive/HER2-negative group, pretreatment cfDNA concentration was prognostic for DRFS. Overall, the predictive and prognostic value of cfDNA concentration was more limited than that of ctDNA. Thus, ctDNA should be the measurement of choice when planning clinical interventions. Citation Format: Mark Jesus M. Magbanua, Ziad Ahmed, Rosalyn W. Sayaman, Lamorna Brown-Swigart, Gillian L. Hirst, Christina Yau, Denise M. Wolf, Wen Li, Amy L. Delson, Jane Perlmutter, W. Fraser Symmans, Douglas Yee, Nola M. Hylton, Laura J. Esserman, Angela M. DeMichele, Hope S. Rugo, Laura J. van ’t Veer. Cell-free DNA concentration as a biomarker of response and recurrence in HER-2 negative breast cancer receiving neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A014.

Active surveillance and associated epidemiological risk factors of caprine paratuberculosis in selected district of Odisha, India.

Caprine paratuberculosis (PTB) is a progressive, debilitating and production-limiting disease that causes significant economic losses and raises public health concerns. The goal was to study active surveillance and associated epidemiological risk factors of caprine PTB in selected district of Odisha, India. The 818 goats of various ages, sexes and breeds were randomly screened in ten different districts for a year based on history, clinical signs and fecal smear examination using the Ziehl-Neelsen stain, yielding an overall prevalence of 38.75%, with clinical and sub-clinical PTB at 8.06 and 30.68%, respectively. A molecular tool, IS900 polymerase chain reaction, was also used to confirm the disease. With Mycobacterium avium subsp. paratuberculosis (MAP) bacilli and endoparasite infections, the majority of affected goats (69.08%) were low shedders. Puri coastal district had the highest prevalence (52.29%) followed by Sambalpur (48.61%), while Khordha had the lowest prevalence (26.41%). Caprine PTB was more common in goats over 2 years old (51.23%), in the Ganjam breed (42.30%), in females (39.17%) and in goats housed on earthen floors (55.83%) according to chi-square analysis. The current study concluded that higher (30.68%) observations of subclinical PTB were cause of real concern due to its insidious spread as well as its zoonotic significance with potential human consequences, which requires immediate attention at all levels. Because of the public health importance of this hidden killer disease, the current findings would be useful in developing a roadmap for implementing prevention and control policies, prompting provision for adequate funding with elaborative research.

Assessing the consistency of shedder status under various experimental conditions

Shedder status is defined as the propensity of an individual to leave DNA behind on touched items or surfaces and has been suggested as one of the major factors influencing DNA transfer. However, little is known about whether shedder status is a constant property of an individual across multiple measurements or when the environmental conditions are changed. We have assessed DNA depositions of six males on 20 occasions to acquire a reference data set and to classify the participants into high, intermediate, or low shedders. This data set was also used to investigate how the probability of a correct shedder status classification changed when the number of DNA deposition measurements increased. Individual sweat rates were measured with a VapoMeter and data regarding hygiene routines were collected through a questionnaire on each sampling occasion. Next, we investigated how changes in the experimental conditions such as seasonal variation, hygiene routines, the temperature of the touched object, and repeated handling of an object influenced the DNA shedding. Additionally, we assessed DNA collected from the face and from T-shirts worn by the six participants to explore whether shedder status may be associated with the relative amount of DNA obtained from other body parts. Our results indicate that shedder status is a stable property across different seasons and different temperatures of handled objects. The relative DNA amounts obtained from repeatedly handled tubes, worn T-shirts, and from faces reflected the shedder status of the participants. We suggest that an individual’s shedder status is highly influenced by the DNA levels on other body parts than hands, accumulating on the palms by frequently touching e.g., the face or previously handled items harboring self-DNA. Assessing physiological differences between the participants revealed that there were no associations between DNA shedding and individual sweat rates.

Evaluation of Fecal Egg Count Tests for Effective Control of Equine Intestinal Strongyles.

The American Association of Equine Practitioners strongly advocates evidence-based intestinal strongyle control in horses. It recommends targeted treatment of all heavy egg shedders (>500 eggs per gram (EPG) of feces), while the low shedders (0-200 EPG) are left untreated. As 50-75% of adult horses in a herd are low shedders, preventing them from unnecessary anthelmintic exposure is critical for tackling resistance. There are various fecal egg count (FEC) techniques with many modifications and variations in use, but none is identified as a gold standard. The hypothesis of the study was that the diagnostic performance of 12 commonly used quantitation methodologies (three techniques with four variants) differs. In this regard, method comparison studies were performed using polystyrene beads as proxy for intestinal strongyle eggs. Mini-FLOTAC-based variants had the lowest coefficient of variation (CV%) in bead recovery, whereas McMaster variants had the highest. All four variants of Mini-FLOTAC and the NaNO3 1.33 specific gravity variant of modified Wisconsin followed a linear fit with R2 > 0.95. In contrast, the bead standard replicates for modified McMaster variants dispersed from the regression curve, causing a lower R2. The Mini-FLOTAC method seems less influenced by the choice of floatation solution and has better repeatability parameters and linearity for bead standard recovery. For FEC tests with high R2 (>0.95) but that underestimated the true bead count, a correction factor (CF) was determined to estimate the true count. Finally, the validity of CF was analyzed for 5 tests with R2 > 0.95 to accurately quantify intestinal strongyle eggs from 40 different horses. Overall, this study identified FEC methodologies with the highest diagnostic performance. The limitations in standardizing routine FEC tests are highlighted, and the importance of equalization of FEC results is emphasized for promoting uniformity in the implementation of parasite control guidelines.

Selective Anthelmintic Treatment in Horses in Sweden Based on Coprological Analyses: Ten-Year Results.

In Sweden, routine deworming has been used for several decades; however, to slow down the development of anthelmintic resistance, selective treatment is currently recommended. As part of a monitoring programme, equestrian premises submitted faecal samples to the National Veterinary Institute (SVA) twice per year between 2008 and 2017. Analyses for strongyles (small and large), tapeworms and ascarids, followed by premise-specific advice regarding deworming and parasite control strategies, were provided. In total, 43,330 faecal samples, collected from 26,625 horses on 935 premises in springtime (March to June), were analysed by quantitative or semi-quantitative flotation. Moreover, Strongylus vulgaris was detected by larval culture or PCR. Between 4 and 11% of individual horses tested positive for S. vulgaris and 3-10% were shedding tapeworm eggs. There were recurrent high and low egg shedders; 75% of horses with S. vulgaris appeared to have been recently introduced into the herd; the proportion of S. vulgaris-positive premises increased when individual samples rather than pooled samples were used. Based on the results of S. vulgaris diagnostics and strongyle egg-shedding level, 59% of the horses did not need to be dewormed.

143 Retrospective Look at Cow-Calf Performance in Spring-Calving Dexter Cattle Scored for Winter Hair Shedding Level

Abstract Delayed winter hair coat loss in beef cows through the spring and summer may contribute to heat stress and reduce herd performance. This study evaluated the performance history of cows in the years that preceded scoring and during the first year, cows were scored for hair shedding levels. Hair shedding levels of purebred Dexter cows (n = 72; mean age = 5.6 years; mean body weight = 321.6 kg) in the university herd were recorded based on the percentage of their body exhibiting winter hair loss (0% = thick winter coat, 100% = short summer coat) at eight biweekly evaluation periods from April to August 2019. Hair shedding levels were observed by two herd managers. Average hair shedding levels for the eight periods were 9%, 31%, 58%, 73%, 83%, 93%, 96%, and 96%, respectively. Cow reproductive performance from 2016 to 2020 (reflective of 2015 to 2019 breeding seasons) and calf growth performance from 2016 to 2019 were analyzed. Cows were exposed to Dexter bulls by natural service annually for an average of 56 days in July and August. Spring-born calves (n =39) were weighed at birth, at the midpoint of the preweaning period (3.5 mo of age), and at weaning (7 mo of age). Hair shedding levels averaged across periods 3 (May 20-21) and 4 (June 3-4) were used to classify cows as low shedders (0 to 50%; LoShed; n = 26) or high shedders (51 to 100%; HiShed; n = 46). Calf weights were tested using SAS MIXED models. Along with dam hair shedding group, dam lactation history, dam hair color, and calf sex were also used as model terms. Cow reproductive rates (n = 239 matings) were tested using SAS GLIMMIX models that included hair shed class, lactation history, and hair color. The HiShed cow group had greater (P &amp;lt; 0.01) winter hair shedding levels compared with the LoShed cow group (86 ± 2% vs. 30 ± 3%). Birth weight was not affected (P = 0.39) by dam shedding level. The midpoint calf weights tended to be heavier (P = 0.07) for HiShed cows than for LoShed cows (91.9 ± 3.7 vs. 84.9 ± 4.6 kg). Calf weaning weights were heavier (P &amp;lt; 0.05) for HiShed cows than for LoShed cows (139.8 ± 7.0 vs.26.3 ± 8.2 kg). Calving rates and weaning rates were greater (P &amp;lt; 0.05) for HiShed cows (86.9 ± 5.3%; 86.8 ± 5.2%) than for LoShed cows (78.9 ± 8.1%; 77.3 ± 8.2%). Results indicated that cow groups based on high or low winter hair shedding level had historically different reproductive rates and calf weaning weights. The high shedding cow group weaned heavier calves and had greater levels of reproductive success.

Pre-exposure cognitive performance variability is associated with severity of respiratory infection

Using data from a longitudinal viral challenge study, we find that the post-exposure viral shedding and symptom severity are associated with a novel measure of pre-exposure cognitive performance variability (CPV), defined before viral exposure occurs. Each individual’s CPV score is computed from data collected from a repeated NeuroCognitive Performance Test (NCPT) over a 3 day pre-exposure period. Of the 18 NCPT measures reported by the tests, 6 contribute materially to the CPV score, prospectively differentiating the high from the low shedders. Among these 6 are the 4 clinical measures digSym-time, digSym-correct, trail-time, and reaction-time, commonly used for assessing cognitive executive functioning. CPV is found to be correlated with stress and also with several genes previously reported to be associated with cognitive development and dysfunction. A perturbation study over the number and timing of NCPT sessions indicates that as few as 5 sessions is sufficient to maintain high association between the CPV score and viral shedding, as long as the timing of these sessions is balanced over the three pre-exposure days. Our results suggest that variations in cognitive function are closely related to immunity and susceptibility to severe infection. Further studying these relationships may help us better understand the links between neurocognitive and neuroimmune systems which is timely in this COVID-19 pandemic era.

PSIV-B-18 Effects of Winter Hair Shedding Level and Service Sire Breed on cow-Calf Performance of a Spring-Calving Herd in the Southeastern United States

Abstract Over three years, the effects of winter hair shedding and service sire breed on cow-calf performance were assessed. Dexter cows were scored each spring for percentage of winter hair loss (0% = thick coat, 100% = smooth coat). Cows were exposed by natural service to Dexter (DEX) and Mashona (MAS) bulls in July. Calves were weighed at birth, 3.5 mo of age, and at weaning (7 mo). Based on hair scores in early June of each year, cows were classified as low shedders (LoS; 0 to 50%; n = 67) or high shedders (HiS; 51 to 100%; n = 130). Hair shed interacted (p &amp;lt; 0.05) with sire breed to affect pregnancy rates. HiS x MAS had a greater (p &amp;lt; 0.05) pregnancy rate than HiS x DEX (92.8 ± 3.8 vs. 60.9 ± 7.7%). Breeds did not differ (p &amp;gt; 0.05) within LoS. Pregnancy rate was greater (p &amp;lt; 0.05) for HiS than LoS (81.6 ± 4.8 vs. 63.1 ± 7.7%). Hair shed scores did not affect (p &amp;gt; 0.05) ADG. MAS calves had greater (p &amp;lt; 0.05) birth to 3.5 mo and birth to weaning ADG (0.77 ± 0.06 and 0.58 ± 0.05 kg/d, respectively) than DEX calves (0.64 ± 0.06 and 0.48 ± 0.05 kg/d, respectively). Across two complete production years, shedding group and service sire breed interacted (p &amp;lt; 0.05) for calf weight weaned per cow exposed. Within MAS, HiS weaned more weight (p &amp;lt; 0.05) than LoS (157.08 ± 24.27 vs. 48.47 ± 28.90 kg). Hair shedding was not important within DEX. Within HiS, MAS cows weaned more weight (p &amp;lt; 0.05) than DEX cows (157.08 ± 24.27 vs. 84.01 ± 24.01 kg). Service sire breed was not important within LoS. Hair shedding level and service sire breed influenced cow-calf performance.

Shedding persistency and intensity patterns of feline coronavirus (FCoV) in feces of cats living in breeding catteries in the Czech Republic

Infection with feline coronavirus (FCoV) is a major problem in multiple-cat households, where many cats are kept together in a small space such as catteries and shelters. Sixty cats from 19 breeding catteries included in the study were evaluated for their shedding persistency and intensity patterns using qPCR identification of FCoV in feces. Cats were identified based on shedding persistency as non-shedders (NS) if all four samples negative, intermittent shedders (IS) when at least one positive and one negative sampling followed by another positive sampling, persistent shedders (PS) if all four samples positive and shedders with unclear status (US) if the shedding patterns could not be determined based on only 4 samples. There were 11 NS (18%), 15 IS (25%) and 15 PS (25%) and in 19/60 cats (32%), the shedding patterns could not be determined based only on four samplings. The intensity of shedding was evaluated based on the total number of FCoV particles shed during the 12 months of the study. There were 11 non-shedders (18%), 2 very low intensity shedders (3%), 9 low intensity shedders (15%), 25 medium intensity shedders (42%) and 13 high intensity shedders (22%). Intermittent shedders were shedding significantly lower FCoV particles compared to the persistent shedders (p = 0.0082). Permanent shedders represent the most important source of FCoV infection in multi-cat households and identifying permanent shedders in is the key to minimize the viral load in the environment to control FCoV in a shelters and breeding catteries.

Differential gene expression reveals host factors for viral shedding variation in mallards (Anas platyrhynchos) infected with low-pathogenic avian influenza virus.

Intraspecific variation in pathogen shedding impacts disease transmission dynamics; therefore, understanding the host factors associated with individual variation in pathogen shedding is key to controlling and preventing outbreaks. In this study, ileum and bursa of Fabricius tissues of wild-bred mallards (Anas platyrhynchos) infected with low-pathogenic avian influenza (LPAIV) were evaluated at various post-infection time points to determine genetic host factors associated with intraspecific variation in viral shedding. By analysing transcriptome sequencing data (RNA-seq), we found that LPAIV-infected wild-bred mallards do not exhibit differential gene expression compared to uninfected birds, but that gene expression was associated with cloacal viral shedding quantity early in the infection. In both tissues, immune gene expression was higher in high/moderate shedding birds compared to low shedding birds, and significant positive relationships with viral shedding were observed. In the ileum, expression for host genes involved in viral cell entry was lower in low shedders compared to moderate shedders at 1 day post-infection (DPI), and expression for host genes promoting viral replication was higher in high shedders compared to low shedders at 2 DPI. Our findings indicate that viral shedding is a key factor for gene expression differences in LPAIV-infected wild-bred mallards, and the genes identified in this study could be important for understanding the molecular mechanisms driving intraspecific variation in pathogen shedding.

Parasite faecal egg counts in equine veterinary practice

SummaryWidespread occurrence of anthelmintic resistance in ascarid and strongylid parasites has led to recommendations of routine treatment efficacy screening with the faecal egg count reduction test. Furthermore, faecal egg counts are used for classifying mature horses into low, moderate and high strongylid egg shedders, and to monitor for ascarids in foals, weanlings and yearlings. The plethora of egg counting techniques is surrounded by confusion and misconception regarding their use, diagnostic performance and interpretation. This article aims to explain general concepts of evaluating and assessing faecal egg count test performance and address some of the most common misconceptions. The multiplication factor, which is used to convert the number of eggs counted under the microscope to eggs per gram of faeces, is a theoretically derived number, which does not reflect sensitivity. Accuracy and precision are quantitative performance parameters, which are not synonymous. Accuracy is a measure of how close a technique measures to the true count, and has implications for the classification of strongylid egg shedders, but will not affect anthelmintic treatment efficacy evaluation or ascarid monitoring. Precision is a measure of how close repeated counts are to each other, and affects anthelmintic efficacy estimates and strongylid shedding classification. Faecal egg counts do not correlate with worm burdens and do not inform on parasitic involvement in disease. Techniques using test tubes with cover slips placed on top tend to perform with low accuracy and precision in comparison to techniques using counting chambers. Veterinarians should consider the performance of the technique employed for determining faecal egg counts, whether they offer these in house or use a referral diagnostic service. Furthermore, they should consider the training and experience of personnel and protocols in place for diagnostic quality assurance.

Determination of shedder status: A comparison of two methods involving cell counting in fingerprints and the DNA analysis of handheld tubes

The shedder status of an individual may be important to consider in the context of DNA transfer, persistence and recovery and in Bayesian networks where a person’s shedder status may have an impact on the outcome. In this study we compared two methods to determine shedder status: the handheld tube (HH) method and a fluorescent cell count (CC) method. A poor association was observed between the numbers of detected cells in a fingerprint using the CC method and the strength of the DNA result with the HH method. The 20 participants were classified into low (25%), medium (50%) and high (25%) shedders based on the HH method. While the low and high shedders showed a good consistency between the replicates, the medium shedders varied more and have to be considered more carefully as they may act as either a high or a low shedder in an event of DNA transfer.

146 Fecal egg count reduction in juvenile and mature horses administered fenbendazole or moxidectin/praziquantel dewormer

Parasite resistance is a concern in horses and can be monitored by performing fecal egg counts (FECs). Unfortunately, FECs are not performed regularly, which could contribute to developing anthelmintic resistance as it may lead to deworming too frequently or not using an effective dewormer for the horse. This study aims to evaluate the ability of pellet and paste fenbendazole (FBZ) and a paste moxidectin/praziquantel (MOX/PZQ) dewormer to reduce parasite populations in juvenile and mature horses in River Falls, Wisconsin. To evaluate dewormers, horses with an initial FEC ≥50 eggs per gram (EPG) were used. For FBZ, horses were blocked by shedding level as low (<200 EPG), moderate (200 – 500 EPG), and high (>500 EPG) shedders and assigned to the pellet (n = 21) or paste (n = 23) FBZ treatment. Two-weeks post-treatment, another FEC was taken and a fecal egg count reduction (FECR) was calculated. Eight-weeks following this study, another FEC was performed and horses with ≥50 EPG were administered a MOX/PZQ paste (n = 46). Two-weeks post-treatment, another FEC was taken and subsequent FECR was calculated. Results from this study indicated no differences were observed for horses receiving pellet or paste FBZ with a FECR of 57.4% and 65.7%, respectively ( P ≥ 0.05). Additionally, no difference in the FECR for juvenile versus mature horses or shedding level was observed regardless of the dewormer used ( P ≥ 0.05). While both FBZ and MOX/PZQ reduced FECs when comparing the pre- and post-treatment EPG ( P ≤ 0.01), the average FECR was 60.4% and 98.5% for horses receiving FBZ and MOX/PZQ, respectively. These results demonstrate parasite resistance to FBZ was observed in this herd while MOX/PZQ was still effective in reducing FECs.

Development and validation of a loop-mediated isothermal amplification assay-a rapid and sensitive detection tool for Mycobacterium avium subsp. paratuberculosis in small ruminants.

The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop-mediated isothermal amplification (LAMP) system, as a time-saving and user-friendly method in contrast to real-time PCR. For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10fgμl-1 . After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698T , the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real-time PCR results. An internal amplification control was incorporated into the assay to exclude false-negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real-time PCR. Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended. This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies.

An investigation of shedding and super-shedding of Shiga toxigenic Escherichia coli O157 and E.coli O26 in cattle presented for slaughter in the Republic of Ireland.

Shiga toxigenic Escherichia coli (STEC) are an important group of pathogens and can be transmitted to humans from direct or indirect contact with cattle faeces. This study investigated the shedding of E.coli O157 and O26 in cattle at the time of slaughter and factors associated with super-shedding (SS) animals. Rectoanal mucosal swab (RAMS) samples were collected from cattle (n=1,317) at three large Irish commercial beef abattoirs over an 18month period, and metadata were collected at the time of sampling regarding farm of origin, animal age, breed and gender. RAMS swabs were examined for the presence and numbers of E.coli O157 and O26 using a previously developed quantitative real-time PCR protocol. Samples positive by PCR were culturally examined and isolates analysed for the presence of stx subtypes, eae and phylogroup. Any samples with counts >104 CFU/swab of STEC O157 or O26 were deemed to be super-shedders. Overall, 4.18% (55/1,317) of RAMS samples were positive for STEC O157, and 2.13% (28/1,317) were classified as STEC O157 SS. For STEC O26, 0.76% (10/1,317) of cattle were positive for STEC O26, and 0.23% (3/1,317) were classified as super-shedders. Fewer STEC shedders and SS were noted among older animals (>37months). There was a seasonal trend observed for STEC O157, with the highest prevalence of shedding and SS events in the autumn (August to October). The majority of E.coli O157 (50/55) isolates had stx2 and were eae positive, with no significant difference between SS and low shedders (LS). Interestingly, all STEC O26 (n=10) were eae negative and had varied stx profiles. This study demonstrates that, while the overall shedding rates are relatively low in cattle at slaughter, among positive animals there is a high level of SS, which may pose a higher risk of cross-contamination during slaughter.

Evaluation of three commercial PCR kits for the direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine faeces

Mycobacterium avium subsp. paratuberculosis which is more commonly referred to as MAP is the causative agent of Johnes’s disease in ruminants. While cultivation of MAP from faecal samples remains the reference standard diagnostic test for the disease, faecal culture is expensive, slow and not widely available in Ireland. The current study evaluated three commercial kits that combine both DNA extraction and real-time PCR amplification of specific targets for direct MAP detection in cattle faeces.In total, 100 positive samples were tested which consisted of 25 high shedders, 25 medium shedders, 25 low shedders and 25 very low shedders. Also included were 100 negative faecal samples obtained from two Irish herds known to be free of Johne’s disease. The kits evaluated in this study showed significant differences in identifying MAP DNA from animals shedding various concentrations of the bacterium. Kit C had the highest specificity, followed by kits B and A. Sensitivity in kits A, B and C was 73.5%, 81% and 93%, respectively, and specificity was 99%, 97% and 100%, respectively. Sensitivity decreased with lower concentrations of MAP in faeces but this was more significant for kits A and B than for kit C. The results of this study demonstrate that PCR can provide an accurate and rapid detection of MAP faecal shedding and kit C was selected for further evaluation of the role of PCR in the confirmation of animal infection in cattle.

The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario

In court questions are often raised related to how trace DNA was deposited, directly during the crime or innocently for instance by secondary transfer. It is therefore of interest to have knowledge of the probability of transfer or secondary transfer in different situations. Factors that could influence transfer probabilities are background DNA and the shedder status of the involved persons. In this study, we have classified participants as high or low DNA shedders. We observed DNA transfer in a simulated attack scenario, and demonstrated that shedder status has a significant influence of transfer rates. We have examined the background DNA in samples from T-shirts worn in an area with frequent human traffic and detected multiple contributors. We further demonstrated that DNA from co-workers of a T-shirt wearer can be secondarily transferred from the environment and detected in samples, and that the composition of background DNA is correlated with the shedder status of the wearer. Finally, we have illustrated the inference with the results of transfer probabilities and a fictive case with the use of a Bayesian network.

Comparison of commercial DNA extraction kits and quantitative PCR systems for better sensitivity in detecting the causative agent of paratuberculosis in dairy cow fecal samples

Mycobacterium avium ssp. paratuberculosis (MAP) causes ruminant paratuberculosis (Johne’s disease) worldwide. Oral-fecal contamination is the most important mode of transmission of paratuberculosis, so eradicating MAP-shedding animals could prevent disease propagation. Fecal culture, a well-known method for MAP diagnosis, requires costly specialized media and a long incubation time that sometimes ends in disappointing bacterial contamination. To facilitate the efforts of control programs, we evaluated the performance of direct fecal quantitative PCR (qPCR) assays for their sensitivity and robustness for MAP detection. Commercial kits use different strategies for extracting DNA, combined with qPCR systems, to detect the presence of MAP in fecal samples. In this study, we compared the sensitivity of 3 commercially available DNA extraction kits (A, B, and C) combined with 2 qPCR systems (T and V) for the detection of MAP in infectious cows. A total of 49 dairy cows from 5 herds were sampled twice a year for 3 yr and diagnosed using fecal culture and ELISA. Eight replicates of their fecal samples from the first sampling were tested using each DNA extraction method and qPCR detection system. Although all 3 of the commercial DNA extraction kits have been previously described as very efficient for the diagnosis of paratuberculosis, kit B provided the highest sensitivity. Indeed, 89% of the cows declared positive for paratuberculosis by both fecal culture and ELISA were identified with kit B, whereas only 23 and 43% of the cows were identified with kits A and C, respectively. Interestingly, kit B was able to detect some low-MAP shedders. The qPCR detection system also played a critical role: system T yielded qPCR with the highest sensitivity. The results of this study suggest that DNA extraction kit B combined with detection system T provides the best amplification of MAP DNA from fecal samples with the highest sensitivity and specificity. Although 1 DNA extraction and qPCR analysis should be adequate to confirm that an animal with diarrhea or other signs of paratuberculosis is positive, detecting low shedders at the highest sensitivity should include repetitive testing. This study demonstrates the importance of repetitions using the most appropriate method for extracting DNA from fecal samples, combined with a compatible qPCR system for identifying MAP-shedding animals.