To evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania ...infantum in the canine reservoir.
A stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household.
A total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs.
The synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas.
We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance ...genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance-only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.
Footrot is an infectious bacterial disease of sheep that causes lameness. The causal agent is Dichelobacter nodosus. There is debate regarding the role of Fusobacterium necrophorum in disease ...initiation. This research used an observational longitudinal study of footrot, together with quantitative PCR (qPCR) of bacterial load of D. nodosus and F. necrophorum, to elucidate the roles of each species in the development of disease. All feet of 18 a priori selected sheep were monitored for five weeks assessing disease severity (healthy, interdigital dermatitis (ID) and severe footrot (SFR)) and bacterial load. A multinomial model was used to analyse these data.
Key unadjusted results were that D. nodosus was detected more frequently on feet with ID, whereas F. necrophorum was detected more frequently on feet with SFR. In the multinomial model, ID was associated with increasing log10 load of D. nodosus the week of observation (OR=1.28 (95% CI=1.08–1.53)) and the week prior to development of ID (OR=1.20 (95% CI=1.01–1.42). There was no association between log10 load2 of F. necrophorum and presence of ID (OR=0.99 (95% CI=0.96–1.02))). SFR was associated with increasing log10 load of D. nodosus the week before disease onset (OR=1.42 (95% CI=1.02–1.96)) but not once SFR had occurred. SFR was positively associated with log10 load2 of F. necrophorum once disease was present (OR=1.06 (95% CI=1.01–1.11)). In summary, there was an increased risk of increasing D. nodosus load the week prior to development of ID and SFR and during an episode of ID. In contrast, F. necrophorum load was not associated with ID before or during an episode, and was only associated with SFR once present. These results contribute to our understanding of the epidemiology of footrot and highlight that D. nodosus load plays the primary role in disease initiation and progression, with F. necrophorum load playing a secondary role. Further studies in more flocks and climates would be useful to confirm these findings. This study identifies that D. nodosus load is highest during ID. This supports previous epidemiological findings, which demonstrate that controlling ID is the most effective management strategy to prevent new cases of ID and SFR.
The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater ...and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 10(19) bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.
The relationships between heterogeneities in host infection and infectiousness (transmission to arthropod vectors) can provide important insights for disease management. Here, we quantify ...heterogeneities in Leishmania infantum parasite numbers in reservoir and non-reservoir host populations, and relate this to their infectiousness during natural infection. Tissue parasite number was evaluated as a potential surrogate marker of host transmission potential.
Parasite numbers were measured by qPCR in bone marrow and ear skin biopsies of 82 dogs and 34 crab-eating foxes collected during a longitudinal study in Amazon Brazil, for which previous data was available on infectiousness (by xenodiagnosis) and severity of infection.
Parasite numbers were highly aggregated both between samples and between individuals. In dogs, total parasite abundance and relative numbers in ear skin compared to bone marrow increased with the duration and severity of infection. Infectiousness to the sandfly vector was associated with high parasite numbers; parasite number in skin was the best predictor of being infectious. Crab-eating foxes, which typically present asymptomatic infection and are non-infectious, had parasite numbers comparable to those of non-infectious dogs.
Skin parasite number provides an indirect marker of infectiousness, and could allow targeted control particularly of highly infectious dogs.
A study was made of the efficiency of three different extractants, 0.1
M sodium pyrophosphate (pH 7), 67
mM phosphate buffer (pH 6) and 0.5
M potassium sulphate (pH 6.6), in recovering the protein ...quantity and the β-glucosidase enzyme activity from two natural forest soils: (1) an Inceptisoil located in Tuscany (Italy) in a mild Mediterranean climate, and (2) a Lithic Calcixeroll soil located in Murcia (south-east of Spain) in a dry-semiarid climate. The pyrophosphate was used to determine the activity of extracellular-humic-bound proteins, while the phosphate buffer and potassium sulphate were used to extract dissolved extracellular proteins. The latter extractant, after chloroform fumigation, was also used to measure total proteins in soil. A preliminary screening, using SDS-PAGE in one dimension, was also carried out in order to optimize the separation condition of soil proteins extracted with different buffers. To remove the interfering co-extracted substances (humic acid) a purification step using a column packed with insoluble polyvinylpyrrolidone was performed. The highest β-glucosidase activity was recovered in the pyrophosphate extract, thus confirming its capability of extracting humic-bound β-glucosidase enzyme in a stable and active form. The extractants performed differently with the two soil types and band patterns obtained with SDS-PAGE were extractant-specific, demonstrating that each was selective for a particular class of proteins. Surprisingly, protein bands were also obtained using pyrophosphate, in spite of the very dark extract colour due to the presence of humic substances.
Functionally important proteins at the interface of cell and soil are of potentially low abundance when compared with commonly recovered intracellular proteins. A novel approach was developed and ...used to extract the metaexoproteome, the subset of proteins found outside the cell, in the context of a soil enriched with the nitrogen-containing recalcitrant polymer chitin. The majority of proteins recovered was of bacterial origin and localized to the outer membrane or extracellular milieu. A wide variety of transporter proteins were identified, particularly those associated with amino-acid and phosphate uptake. The metaexoproteome extract retained chitinolytic activity and we were successful in detecting Nocardiopsis-like chitinases that correlated with the glycoside hydrolase family 18 (GH18) chi gene data and metataxonomic analysis. Nocardiopsis-like chitinases appeared to be solely responsible for chitinolytic activity in soil. This is the first study to detect and sequence bacterial exoenzymes with proven activity in the soil enzyme pool.
•First FISH study of ovine FR to examine pathogen spatial distribution and load.•D. nodosus cell counts are significantly associated with both ID and SFR.•F. necrophorum cell counts are significantly ...associated with SFR.•Highlights FISH as a useful tool for studying microbial populations in situ.
Analysis of bacterial populations in situ provides insights into pathogen population dynamics and potential reservoirs for disease. Here we report a culture-independent study of ovine footrot (FR); a debilitating bacterial disease that has significant economic impact on sheep farming worldwide. Disease begins as an interdigital dermatitis (ID), which may then progress to separation of the hoof horn from the underlying epidermis causing severe footrot (SFR). Dichelobacter nodosus is the causative agent of ovine FR, however, the role of Fusobacterium necrophorum and other bacteria present in the environment and on the feet of sheep is less clear. The objective of this study was to use fluorescence in situ hybridisation (FISH) to detect, localise and quantify D. nodosus, F. necrophorum and the domain Bacteria from interdigital skin biopsies of healthy, ID- and SFR-affected feet. D. nodosus and F. necrophorum populations were restricted primarily to the epidermis, but both were detected more frequently in feet with ID or SFR than in healthy feet. D. nodosus cell counts were significantly higher in feet with ID and SFR (p<0.05) than healthy feet, whereas F. necrophorum cell counts were significantly higher only in feet with SFR (p<0.05) than healthy feet. These results, together with other published data, indicate that D. nodosus likely drives pathogenesis of footrot from initiation of ID to SFR; with D. nodosus cell counts increasing prior to onset of ID and SFR. In contrast, F. necrophorum cell counts increase after SFR onset, which may suggest an accessory role in disease pathogenesis, possibly contributing to the severity and duration of SFR.
•Lambs’ feet were D. nodosus-negative at birth.•However D. nodosus was detected on lambs’ feet within 5–13h of birth.•Multiple pgrA and MLVA alleles were detected on the feet of ewes and lambs.•D. ...nodosus on lambs’ feet originated from sources other than just their mother's feet.•The environment plays a key role in D. nodosus transmission between ewes and lambs.
Dichelobacter nodosus (D. nodosus) is the essential causative agent of footrot in sheep. The current study investigated when D. nodosus was detectable on newborn lambs and possible routes of transmission. Specific qPCR was used to detect and quantify the load of D. nodosus in foot swabs of lambs at birth and 5–13h post-partum, and their mothers 5–13h post-partum; and in samples of bedding, pasture, soil and faeces. D. nodosus was not detected on the feet of newborn lambs swabbed at birth, but was detected 5–13h after birth, once they had stood on bedding containing naturally occurring D. nodosus. Multiple genotypes identified by cloning and sequencing a marker gene, pgrA, and by multi locus variable number tandem repeat analysis (MLVA) of community DNA from swabs on individual feet indicated a mixed population of D. nodosus was present on the feet of both ewes and lambs. There was high variation in pgrA tandem repeat number (between 3 and 21 repeats), and multiple MLVA types. The overall similarity index between the populations on ewes and lambs was 0.45, indicating moderate overlap. Mother offspring pairs shared some alleles but not all, suggesting lambs were infected from sources(s) other than just their mother's feet. We hypothesise that D. nodosus is transferred to the feet of lambs via bedding containing naturally occurring populations of D. nodosus, probably as a result of transfer from the feet of the group of housed ewes. The results support the hypothesis that the environment plays a key role in the transmission of D. nodosus between ewes and lambs.
Dichelobacter nodosus (D. nodosus) is the causative agent of footrot in sheep; one of the most important health and welfare issues of sheep worldwide. For control programmes to be effective, it is ...essential that the transmission cycle of D. nodosus is understood and bacterial reservoirs in the environment are better defined. This study evaluated the survival of D. nodosus in different soils using soil microcosms. Cultivation independent and dependent methods were used to detect D. nodosus over 40 days from seeding in soil. A D. nodosus specific probe was used for quantification by qPCR and viability was assessed by cell permeability to an intercalating dye, PMA, and by culture. Survival varied dramatically depending on soil type, matric potential (MP) and temperature. Our findings indicate that D. nodosus survival was higher at 5 °C compared with 25 °C in all soils and significantly longer at both temperatures in clay soil (>44% clay) compared with other soil types. Survival under all conditions was longer than 30 days for both culture independent and dependent methods, this is substantially longer than previous studies and, if this is an infectious dose, longer than the current recommendation of resting a field for 14 days to prevent onward infection.
•Viable Dichelobacter nodosus was detected for 40 days off host in ventilated soil microcosms.•D. nodosus survival rate was affected by matric potential, temperature and soil type.•D. nodosus survived for 40 days both at 5 °C and 25 °C in four contrasting soils.•D. nodosus survival was higher at 5 °C compared with 25 °C in all soils.•D. nodosus survival was significantly longer in clay soil (>44% clay).
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