Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of Histoplasma from equine clinical samples.

Histoplasma capsulatum var. farciminosum (HCF) is a dimorphic fungus that causes epizootic lymphangitis in equids. Current diagnostic approaches, including culture, microscopy, and clinical presentation, lack speed, sensitivity, and specificity when diagnosing clinical cases. In this study, equine blood and pus samples on Whatman FTA cards from Senegal (n = 3), The Gambia (n = 19), Ethiopia (n = 16), and Mali (n = 13) were tested using a real-time PCR (qPCR) protocol. The assay was optimized and tested for its suitability to detect and quantify HCF in blood and pus loaded onto Whatman FTA cards at sampling. Whatman FTA cards were tested for their suitability for use with qPCR and were found to recover DNA more efficiently than from direct extraction. Using TaqMan fluorescent probes and specific primers, the assay demonstrated 100% analytical specificity when detecting multiple strains of Histoplasma and no false positives with off-target organisms. The assay's diagnostic performance was measured against an existing nested internal transcribed spacer PCR protocol using a receiver operating characteristic curve. The test was found to have a diagnostic specificity and sensitivity of 100% and 71.4%, respectively, when analyzing pus samples using a cycle threshold (Ct) cutoff determined by Youden's index (27.75). Blood sample cutoff Ct value was proposed at 34.55. Further optimization is required to improve the performance of the protocol when applied to blood samples. This study has, for the first time, demonstrated the ability to detect and quantify the DNA of Histoplasma spp. in equine blood and pus samples with a high degree of accuracy, providing a platform to further investigate the pathogenesis and epidemiology of this disease. IMPORTANCE: Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease in situ. This paper reports, for the first time, the validation and application of a qPCR to detect Histoplasma directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.

Prevalence, molecular characterization, and antimicrobial resistance profiles of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 on dairy cattle farms in Jordan.

This study determined the prevalence, pulsed-field gel electrophoresis profiles, and antimicrobial resistance profile of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 isolates from dairy cattle farms in Jordan. Samples from bulk tank milk (n = 305), cattle feces (n = 610), and rectoanal mucosal swabs (n = 610) were collected from 61 dairy cattle farms. We confirmed 32 L. monocytogenes, 28 S. enterica, and 24 E. coli O157:H7 isolates from the samples. The farm-level prevalence (at least 1 positive sample per farm) of L. monocytogenes, S. enterica, and E. coli O157:H7 was 27.9, 19.7, and 23.0%, respectively. The prevalence of L. monocytogenes, S. enterica, and E. coli O157:H7 in bulk tank milk was 7.5, 1.6, and 3.3%, respectively. The prevalence of L. monocytogenes and S. enterica in fecal samples was 1.5 and 3.8%, respectively, and the prevalence of E. coli O157:H7 in rectoanal mucosal swabs was 2.3%. Based on disk diffusion testing, all L. monocytogenes, S. enterica, and E. coli O157:H7 isolates exhibited resistance to at least 1 antimicrobial class. Multidrug resistance (resistance to 3 or more classes of antimicrobials) was exhibited by 96.9% of L. monocytogenes, 91.7% of E. coli O157:H7, and 82.1% of S. enterica isolates. Moreover, 93.8, 79.2, and 57.1% of the L. monocytogenes, E. coli O157:H7, and S. enterica isolates, respectively, were resistant to 5 or more antimicrobial classes. More than 50% of L. monocytogenes isolates were resistant to ampicillin, clindamycin, penicillin, erythromycin, quinupristin-dalfopristin, streptomycin, teicoplanin, linezolid, vancomycin, kanamycin, and tetracycline. More than 50% of S. enterica and E. coli O157:H7 isolates were resistant to ampicillin, cephalothin, nalidixic acid, kanamycin, streptomycin, amoxicillin-clavulanic acid, and tetracycline. The prevalence of the studied pathogens this study was comparable to reports from other countries. The isolated pathogens exhibited a high degree of antimicrobial resistance, suggesting that the bacterial flora of dairy cattle in Jordan are under intense antimicrobial selection pressure. Additional research is required to determine the causes and drivers of resistance, and to develop approaches to mitigating antimicrobial resistance.

Seroprevalence, risk factors and spatial distribution of West Nile virus in Jordan.

Background: This is the first countrywide study of the seroprevalence, risk factors and spatial distribution of West Nile virus (WNV) in Jordan. Methods: A cross-sectional sample of 801 participants was administered a comprehensive questionnaire survey and tested for WNV immunoglobulin G antibodies. Results: The point seroprevalence rate for WNV infection was 8.61% (95% confidence interval 6.8 to 10.8). Multivariate regression analysis demonstrated that sex, age, climate, income and drinking water source were significantly associated with seropositivity (p≤0.05). Males had 1.73 greater odds of infection compared with females. Compared with 15 to 29-year-olds, adults 30-49 y old and adults ≥50 y old had 2.0 and 3.1 greater odds of infection, respectively. Individuals living in the Jordan Valley and Badia had 22.2 and 7.2 times greater odds of infection, respectively, compared with individuals living in the highlands. Households with an income of

Evaluating the Efficacy of Knowledge-Transfer Interventions on Animal Health Knowledge of Rural Working Equid Owners in Central Ethiopia: A Cluster-Randomized Controlled Trial.

The objectives of this study were to evaluate the efficacy of several knowledge-transfer interventions about donkey health, utilizing a cluster-randomized controlled trial (c-RCT), on the long-term knowledge change (~6 months post intervention) of Ethiopian rural working equid owners. Knowledge transfer interventions included: an audio programme, a village meeting and a diagrammatic hand-out, which were also compared to a control group, which received no intervention. All interventions addressed identical learning objectives. Thirty-two villages were randomly selected and interventions randomly assigned to blocks of eight villages. All participants in a village received the same intervention, and knowledge levels were assessed by questionnaire administration both pre and post intervention. Data analysis included multilevel linear and logistic regression models (allowing for clustering of individuals within villages) to evaluate the change in knowledge between the different knowledge-transfer interventions, and to look at other factors associated with change in knowledge. A total of 516 randomly selected participants completed pre-intervention questionnaires, 476 undertook a post-dissemination questionnaire ~6 months later, a follow-up response rate of 92%. All interventions significantly improved the overall knowledge score on the post intervention questionnaire compared to the control group, with the diagrammatic hand-out [coefficient (coef) 10.0, S.E. = 0.5] and the village meeting (coef 8.5, S.E = 0.5) having a significantly greater impact than the audio programme (coef 4.0, S.E = 0.5). There were differences in learning across interventions, learning objectives, age and education levels of the participants. Participants with higher levels of formal education had greater knowledge change but this varied across interventions. In conclusion, knowledge of donkey health was substantially increased by a diagrammatic hand-out and the impact of this simple, low-cost intervention should be further evaluated in other communities in low-income countries. This study should assist in the design and development of effective knowledge-transfer materials for adult learning for rural villagers in low-income countries.

Infectious diseases of working equids.

Most working equids reside in low-income countries where they have an essential role in the livelihoods of their owners. Numerous infectious diseases negatively impact the health and productivity of these animals. There are considerable technical, social-behavioral, and institutional impediments globally to reducing the burden of infectious diseases on working equids. One the greatest remaining challenges is the lack of funding for research, resulting from the low priority assigned to working equids by funding bodies. Changing the attitudes of decision makers will require data-driven advocacy, and global networks of collaborators have a vital role in building this more robust evidence base.

Reducing sampling error in faecal egg counts from black rhinoceros (Diceros bicornis).

Faecal egg counts (FECs) are commonly used for the non-invasive assessment of parasite load within hosts. Sources of error, however, have been identified in laboratory techniques and sample storage. Here we focus on sampling error. We test whether a delay in sample collection can affect FECs, and estimate the number of samples needed to reliably assess mean parasite abundance within a host population. Two commonly found parasite eggs in black rhinoceros (Diceros bicornis) dung, strongyle-type nematodes and Anoplocephala gigantea, were used. We find that collection of dung from the centre of faecal boluses up to six hours after defecation does not affect FECs. More than nine samples were needed to greatly improve confidence intervals of the estimated mean parasite abundance within a host population. These results should improve the cost-effectiveness and efficiency of sampling regimes, and support the usefulness of FECs when used for the non-invasive assessment of parasite abundance in black rhinoceros populations.