Carriage of antimicrobial-resistant bacteria in a high-density informal settlement in Kenya is associated with environmental risk-factors.

BACKGROUND: The relationship between antibiotic use and antimicrobial resistance varies with cultural, socio-economic, and environmental factors. We examined these relationships in Kibera, an informal settlement in Nairobi-Kenya, characterized by high population density, high burden of respiratory disease and diarrhea. METHODS: Two-hundred households were enrolled in a 5-month longitudinal study. One adult (≥ 18 years) and one child (≤ 5 years) participated per household. Biweekly interviews (n = 1516) that included questions on water, sanitation, hygiene, and antibiotic use in the previous two weeks were conducted, and 2341 stool, 2843 hand swabs and 1490 drinking water samples collected. Presumptive E. coli (n = 34,042) were isolated and tested for susceptibility to nine antibiotics. RESULTS: Eighty percent of presumptive E. coli were resistant to ≥ 3 antibiotic classes. Stool isolates were resistant to trimethoprim (mean: 81%), sulfamethoxazole (80%), ampicillin (68%), streptomycin (60%) and tetracycline (55%). Ninety-seven households reported using an antibiotic in at least one visit over the study period for a total of 144 episodes and 190 antibiotic doses. Enrolled children had five times the number of episodes reported by enrolled adults (96 vs. 19). Multivariable linear mixed-effects models indicated that children eating soil from the household yard and the presence of informal hand-washing stations were associated with increased numbers of antimicrobial-resistant bacteria (counts increasing by 0·27-0·80 log10 and 0·22-0·51 log10 respectively, depending on the antibiotic tested). Rainy conditions were associated with reduced carriage of antimicrobial-resistant bacteria (1·19 to 3·26 log10 depending on the antibiotic tested). CONCLUSIONS: Antibiotic use provided little explanatory power for the prevalence of antimicrobial resistance. Transmission of resistant bacteria in this setting through unsanitary living conditions likely overwhelms incremental changes in antibiotic use. Under such circumstances, sanitation, hygiene, and disease transmission are the limiting factors for reducing the prevalence of resistant bacteria.

Mobile phone-based surveillance for animal disease in rural communities: implications for detection of zoonoses spillover.

Improving the speed of outbreak detection and reporting at the community level are critical in managing the threat of emerging infectious diseases, many of which are zoonotic. The widespread use of mobile phones, including in rural areas, constitutes a potentially effective tool for real-time surveillance of infectious diseases. Using longitudinal data from a disease surveillance system implemented in 1500 households in rural Kenya, we test the effectiveness of mobile phone animal syndromic surveillance by comparing it with routine household animal health surveys, determine the individual and household correlates of its use and examine the broader implications for surveillance of zoonotic diseases. A total of 20 340 animal and death events were reported from the community through the two surveillance systems, half of which were confirmed as valid disease events. The probability of an event being valid was 2.1 times greater for the phone-based system, compared with the household visits. Illness events were 15 times (95% CI 12.8, 17.1) more likely to be reported through the phone system compared to routine household visits, but not death events (OR 0.1 (95% CI 0.09, 0.11)). Disease syndromes with severe presentations were more likely to be reported through the phone system. While controlling for herd and flock sizes owned, phone ownership was not a determinant of using the phone-based surveillance system, but the lack of a formal education, and having additional sources of income besides farming were associated with decreased likelihood of reporting through the phone system. Our study suggests that a phone-based surveillance system will be effective at detecting outbreaks of diseases such as Rift Valley fever that present with severe clinical signs in animal populations, but in the absence of additional reporting incentives, it may miss early outbreaks of diseases such as avian influenza that present primarily with mortality. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Livestock vaccinations translate into increased human capital and school attendance by girls.

To fulfill the United Nation's Sustainable Development Goals (SDGs), it is useful to understand whether and how specific agricultural interventions improve human health, educational opportunity, and food security. In sub-Saharan Africa, 75% of the population is engaged in small-scale farming, and 80% of these households keep livestock, which represent a critical asset and provide protection against economic shock. For the 50 million pastoralists, livestock play an even greater role. Livestock productivity for pastoralist households is constrained by multiple factors, including infectious disease. East Coast fever, a tick-borne protozoal disease, is the leading cause of calf mortality in large regions of eastern and Southern Africa. We examined pastoralist decisions to adopt vaccination against East Coast fever and the economic outcomes of adoption. Our estimation strategy provides an integrated model of adoption and impact that includes direct effects of vaccination on livestock health and productivity outcomes, as well as indirect effects on household expenditures, such as child education, food, and health care. On the basis of a cross-sectional study of Kenyan pastoralist households, we found that vaccination provides significant net income benefits from reduction in livestock mortality, increased milk production, and savings by reducing antibiotic and acaricide treatments. Households directed the increased income resulting from East Coast fever vaccination into childhood education and food purchase. These indirect effects of livestock vaccination provide a positive impact on rural, livestock-dependent families, contributing to poverty alleviation at the household level and more broadly to achieving SDGs.

Relations between Household Livestock Ownership, Livestock Disease, and Young Child Growth.

BACKGROUND: In resource-limited settings in which child malnutrition is prevalent, humans live in close proximity to household livestock. However, the relation between household livestock and child nutrition represents a considerable knowledge gap. OBJECTIVE: We assessed whether household livestock ownership or livestock disease episodes were associated with growth in young children in western Kenya. METHODS: We incorporated monthly anthropometric measurements for children <5 y of age into an ongoing linked human and animal surveillance cohort in rural western Kenya. Using linear mixed models adjusted for age, sex, and household wealth, we tested whether baseline household livestock ownership was related to baseline child height for age or prospective growth rate. We also evaluated whether livestock disease episodes were associated with child growth rate over 11 mo of follow-up. RESULTS: We collected data on 925 children over the course of follow-up. Greater household livestock ownership at baseline was not related to baseline child height-for-age z score (adjusted ß: 0.01 SD; 95% CI: -0.02, 0.04 SD) or child growth rate (adjusted ß: 0.02 cm/y; 95% CI: -0.03, 0.07 cm/y). Livestock disease episodes were not significantly associated with child growth across the entire cohort (adjusted ß: -0.007 cm/mo; 95% CI: -0.02, 0.006 cm/mo). However, children in households with livestock digestive disease between June and November gained less height than did children in households that did not report livestock disease (ß: -0.063 cm/mo; 95% CI: -0.112, -0.016 cm/mo). Children <2 y of age in households with livestock digestive disease gained less weight than did those who did not report disease (ß: -0.033 kg/mo; 95% CI: -0.063, -0.003 kg/mo). CONCLUSION: In this cohort of young children in western Kenya, we did not find an association between ownership of livestock and child growth status. However, disease episodes in household livestock may be related to a lower child growth rate in some groups.

Linking human health and livestock health: a "one-health" platform for integrated analysis of human health, livestock health, and economic welfare in livestock dependent communities.

BACKGROUND: For most rural households in sub-Saharan Africa, healthy livestock play a key role in averting the burden associated with zoonotic diseases, and in meeting household nutritional and socio-economic needs. However, there is limited understanding of the complex nutritional, socio-economic, and zoonotic pathways that link livestock health to human health and welfare. Here we describe a platform for integrated human health, animal health and economic welfare analysis designed to address this challenge. We provide baseline epidemiological data on disease syndromes in humans and the animals they keep, and provide examples of relationships between human health, animal health and household socio-economic status. METHOD: We designed a study to obtain syndromic disease data in animals along with economic and behavioral information for 1500 rural households in Western Kenya already participating in a human syndromic disease surveillance study. Data collection started in February 2013, and each household is visited bi-weekly and data on four human syndromes (fever, jaundice, diarrhea and respiratory illness) and nine animal syndromes (death, respiratory, reproductive, musculoskeletal, nervous, urogenital, digestive, udder disorders, and skin disorders in cattle, sheep, goats and chickens) are collected. Additionally, data from a comprehensive socio-economic survey is collected every 3 months in each of the study households. FINDINGS: Data from the first year of study showed 93% of the households owned at least one form of livestock (55%, 19%, 41% and 88% own cattle, sheep, goats and chickens respectively). Digestive disorders, mainly diarrhea episodes, were the most common syndromes observed in cattle, goats and sheep, accounting for 56% of all livestock syndromes, followed by respiratory illnesses (18%). In humans, respiratory illnesses accounted for 54% of all illnesses reported, followed by acute febrile illnesses (40%) and diarrhea illnesses (5%). While controlling for household size, the incidence of human illness increased 1.31-fold for every 10 cases of animal illness or death observed (95% CI 1.16-1.49). Access and utilization of animal source foods such as milk and eggs were positively associated with the number of cattle and chickens owned by the household. Additionally, health care seeking was correlated with household incomes and wealth, which were in turn correlated with livestock herd size. CONCLUSION: This study platform provides a unique longitudinal dataset that allows for the determination and quantification of linkages between human and animal health, including the impact of healthy animals on human disease averted, malnutrition, household educational attainment, and income levels.

Tick passage results in enhanced attenuation of Babesia bovis.

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype.

Targeted surface expression of an exogenous antigen in stably transfected Babesia bovis.

Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine delivery platform.

Improved diagnostic performance of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5-glutathione S-transferase fusion protein as antigen.

The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (<30%I) using the commercial rMSP5-MBP cELISA with MBP adsorption, resulting in 97.8% specificity. This specificity was higher than some previous reports, so to improve the specificity of the commercial cELISA, a new recombinant antigen designated rMSP5-glutathione S-transferase (GST) was developed, eliminating MBP from the antigen and obviating the need for MBP adsorption. Using the rMSP5-GST cELISA, only 1 of 358 Anaplasma-negative sera, which included the 139 sera with significant (≥30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested polymerase chain reaction. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera. In summary, the rMSP5-GST cELISA was a faster and simpler assay with higher specificity, comparable sensitivity, and improved resolution in comparison with the rMSP5-MBP cELISA with MBP adsorption.

Seroprevalence of Coxiella burnetii in Washington State domestic goat herds.

A caprine herd seroprevalence of Coxiella burnetii infection was determined by passive surveillance of domestic goat herds in Washington State. Serum samples (n=1794) from 105 herds in 31 counties were analyzed for C. burnetii antibodies using a commercially available Q fever antibody enzyme-linked immunosorbent assay (ELISA) test kit. The sera were submitted to the Washington Animal Disease Diagnostic Laboratory for routine serologic screening over an approximate 1-year period from November, 2010, through November, 2011. To avoid bias introduced by testing samples from ill animals, only accessions for routine screening of nonclinical animals were included in the study. A standard cluster sampling approach to investigate seroprevalence at the herd level was used to determine optimal study sample size. The results identified C. burnetii antibodies in 8.0% of samples tested (144/1794), 8.6% of goat herds tested (9/105), and 25.8% of counties tested (8/31). Within-herd seroprevalence in positive counties ranged from 2.9% to 75.8%. Counties with seropositive goats were represented in the western, eastern, southeastern, and Columbia basin agricultural districts of the state. To our knowledge this is the first county-specific, statewide study of C. burnetii seroprevalence in Washington State goat herds. The findings provide baseline information for future epidemiologic, herd management and public health investigations of Q fever.

Loss of neurovirulence is associated with reduction of cerebral capillary sequestration during acute Babesia bovis infection.

BACKGROUND: Severe neurological signs that develop during acute infection by virulent strains of Babesia bovis are associated with sequestration of infected erythrocytes in cerebral capillaries. Serial passage of virulent strains in cattle results in attenuated derivatives that do not cause neurologic disease. We evaluated whether serial passage also results in a loss of cerebral capillary sequestration by examining brain biopsies during acute disease and at necropsy. FINDINGS: Cerebral biopsies of spleen intact calves inoculated intravenously with a virulent or attenuated strain pair of B. bovis were evaluated for capillary sequestration at the onset of babesiosis and during severe disease. In calves infected with the virulent strain, there was a significant increase in sequestration between the first and second biopsy timepoint. The attenuated strain was still capable of sequestration, but at a reduced level, and did not change significantly between the first and second biopsy. Necropsy examination confirmed the second biopsy results and demonstrated that sequestration identified at necropsy reflects pathologic changes occurring in live animals. CONCLUSIONS: Loss of neurovirulence after serial in vivo passage of the highly virulent T2Bo strain of B. bovis in splenectomized animals is associated with a significant reduction of cerebral capillary sequestration. Previous genomic analysis of this and two other strain pairs suggests that this observation could be related to genomic complexity, particularly of the ves gene family, rather than consistent gene specific differences. Additional experiments will examine whether differential gene expression of ves genes is also associated with reduced cerebral sequestration and neurovirulence in attenuated strains.

Acute and persistent infection by a transfected Mo7 strain of Babesia bovis.

A Mo7-derived Babesia bovis line stably transfected with the gfp-bsd gene was inoculated into two four to five months old calves, while two additional calves were inoculated with Mo7 parasites. Similar mild clinical signs were detected in all calves. B. bovis rap-1 was identified in the bloodstream by PCR four days post inoculation (dpi), and consistently over ten months thereafter. Transfusion of blood from experimentally infected calves into four naïve splenectomized calves at 212 dpi resulted in acute disease in recipients, confirming persistent infection in the four donor animals. The proportion of GFP expressing parasites recovered from a splenectomized recipient calf is undistinguishable from transfected parasites that were maintained in long term culture under blasticidin selection. Furthermore, the sequences of transfected genes in recovered parasites remained unaltered. Together, the data demonstrates that exogenous B. bovis transgenes can be expressed and remain stable throughout acute and persistent infection in calves.

Expression and strain variation of the novel "small open reading frame" (smorf) multigene family in Babesia bovis.

Small open reading frame (smorf) genes comprise the second largest Babesia bovis multigene family. All known 44 variant smorf genes are located in close chromosomal proximity to ves1 genes, which encode proteins that mediate cytoadhesion and contribute to immune evasion. In this study, we characterised the general topology of smorf genes and investigated the gene repertoire, transcriptional profile and SMORF expression in two distinct strains, T2Bo and Mo7. Sequence analysis using degenerate primers identified additional smorf genes in each strain and demonstrated that the smorf gene repertoire varies between strains, with conserved and unique genes in both. Smorf genes have multiple semi-conserved and variable blocks, and a large hypervariable insertion in 20 of the 44 genes defines two major branches of the family, termed smorf A and smorf B. A total of 32 smorf genes are simultaneously transcribed in T2Bo strain B. bovis merozoites obtained from deep brain tissue of an acutely infected animal. SMORF peptide-specific antiserum bound in immunoblots to multiple proteins with a range of sizes predicted by smorf genes, confirming translation of smorf gene products from these transcripts. These results indicate that the smorf multigene family is larger than previously described and demonstrate that smorf genes are expressed and are undergoing variation, both within strains and in a lineage-specific pattern independent of strain specificity. The function of these novel proteins is unknown.

A network control theory approach to modeling and optimal control of zoonoses: case study of brucellosis transmission in sub-Saharan Africa.

BACKGROUND: Developing control policies for zoonotic diseases is challenging, both because of the complex spread dynamics exhibited by these diseases, and because of the need for implementing complex multi-species surveillance and control efforts using limited resources. Mathematical models, and in particular network models, of disease spread are promising as tools for control-policy design, because they can provide comprehensive quantitative representations of disease transmission. METHODOLOGY/PRINCIPAL FINDINGS: A layered dynamical network model for the transmission and control of zoonotic diseases is introduced as a tool for analyzing disease spread and designing cost-effective surveillance and control. The model development is achieved using brucellosis transmission among wildlife, cattle herds, and human sub-populations in an agricultural system as a case study. Precisely, a model that tracks infection counts in interacting animal herds of multiple species (e.g., cattle herds and groups of wildlife for brucellosis) and in human subpopulations is introduced. The model is then abstracted to a form that permits comprehensive targeted design of multiple control capabilities as well as model identification from data. Next, techniques are developed for such quantitative design of control policies (that are directed to both the animal and human populations), and for model identification from snapshot and time-course data, by drawing on recent results in the network control community. CONCLUSIONS/SIGNIFICANCE: The modeling approach is shown to provide quantitative insight into comprehensive control policies for zoonotic diseases, and in turn to permit policy design for mitigation of these diseases. For the brucellosis-transmission example in particular, numerous insights are obtained regarding the optimal distribution of resources among available control capabilities (e.g., vaccination, surveillance and culling, pasteurization of milk) and points in the spread network (e.g., transhumance vs. sedentary herds). In addition, a preliminary identification of the network model for brucellosis is achieved using historical data, and the robustness of the obtained model is demonstrated. As a whole, our results indicate that network modeling can aid in designing control policies for zoonotic diseases.

Attenuation of virulence in an apicomplexan hemoparasite results in reduced genome diversity at the population level.

BACKGROUND: Virulence acquisition and loss is a dynamic adaptation of pathogens to thrive in changing milieus. We investigated the mechanisms of virulence loss at the whole genome level using Babesia bovis as a model apicomplexan in which genetically related attenuated parasites can be reliably derived from virulent parental strains in the natural host. We expected virulence loss to be accompanied by consistent changes at the gene level, and that such changes would be shared among attenuated parasites of diverse geographic and genetic background. RESULTS: Surprisingly, while single nucleotide polymorphisms in 14 genes distinguished all attenuated parasites from their virulent parental strains, all non-synonymous changes resulted in no deleterious amino acid modification that could consistently be associated with attenuation (or virulence) in this hemoparasite. Interestingly, however, attenuation significantly reduced the overall population's genome diversity with 81% of base pairs shared among attenuated strains, compared to only 60% of base pairs common among virulent parental parasites. There were significantly fewer genes that were unique to their geographical origins among the attenuated parasites, resulting in a simplified population structure among the attenuated strains. CONCLUSIONS: This simplified structure includes reduced diversity of the variant erythrocyte surface 1 (ves) multigene family repertoire among attenuated parasites when compared to virulent parental strains, possibly suggesting that overall variance in large protein families such as Variant Erythrocyte Surface Antigens has a critical role in expression of the virulence phenotype. In addition, the results suggest that virulence (or attenuation) mechanisms may not be shared among all populations of parasites at the gene level, but instead may reflect expansion or contraction of the population structure in response to shifting milieus.

Development of a bead-based multiplex PCR assay for the simultaneous detection of multiple Mycoplasma species.

We describe the development and analytical validation of a 7-plex polymerase chain reaction assay coupled to a bead-based liquid suspension array for detection of multiple ruminant Mycoplasma spp. The assay employs a combination of newly designed and previously validated primer-probe sets that target genetic loci specific for Mycoplasma bovis, Mycoplasma mycoides cluster, Mycoplasma mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subspecies capripneumoniae (Mccp). Analytical sensitivity for the targeted Mycoplasma species ranged from 10 fg to 1 pg of purified gDNA extracted from broth cultures (approximately 8-800 MmmSC genome equivalents). In silico comparison of primers and probes, and analytical assessment with a range of near-neighbor Mycoplasma species and multiple bacterial respiratory pathogens demonstrated 100% analytical specificity of the assay. To assess assay performance and diagnostic specificity, 192 bovine respiratory samples were analyzed by incorporating a high throughput DNA extraction platform. The assay correctly classified all samples as negative for MmmSC or Mccp. All 33 field samples confirmed as positive for M. bovis by sequencing the uvrC gene were positive in the assay. The results from this study indicate that the bead-based liquid suspension array will provide a reliable, analytically sensitive and specific platform to simultaneously interrogate ruminant respiratory samples for multiple Mycoplasma species, including M. mycoides cluster organisms that are exotic to the United States. Sequential addition of primer-probe sets to the assay did not significantly impact analytical sensitivity of individual primer-probe combinations, suggesting that expanding the assay to include more Mycoplasma species will not compromise overall performance.

A Babesia bovis gene syntenic to Theileria parva p67 is expressed in blood and tick stage parasites.

Completion of the Babesia bovis (T2Bo strain) genome provides detailed data concerning the predicted proteome of this parasite, and allows for a bioinformatics approach to gene discovery. Comparative genomics of the hemoprotozoan parasites B. bovis and Theileria parva revealed a highly conserved syntenic block of genes flanking the p67 gene of T. parva, a sporozoite stage-specific vaccine candidate against East Coast fever (ECF). The syntenic gene in B. bovis, designated bov57, encodes a protein of limited amino acid sequence identity (11.8%) to p67. Monoclonal antibodies were produced against recombinant BOV57 and were used to demonstrate expression of BOV57 in merozoite and kinete stages of the T2Bo strain of B. bovis. Transcript levels of bov57 in kinetes were increased 100-fold in comparison to msa-1, a previously identified gene encoding an erythrocyte stage surface protein. Amino acid sequence comparisons between the T2Bo strain and two attenuated and virulent strains from Argentina and Australia revealed a high degree of sequence conservation in BOV57 among these geographically and pathogenically divergent isolates (97% amino acid sequence identity). Additional genomic comparisons show that the bov57 gene locus is also conserved in Babesia bigemina and Babesia equi. While not identifiable through amino acid or nucleotide sequence similarity, the conserved gene order within this locus in multiple piroplasms may suggest a critical function adapted for each species' unique host and life-cycle.

Genotypic diversity of merozoite surface antigen 1 of Babesia bovis within an endemic population.

Multiple genetically distinct strains of a pathogen circulate and compete for dominance within populations of animal reservoir hosts. Understanding the basis for genotypic strain structure is critical for predicting how pathogens respond to selective pressures and how shifts in pathogen population structure can lead to disease outbreaks. Evidence from related Apicomplexans such as Plasmodium, Toxoplasma, Cryptosporidium and Theileria suggests that various patterns of population dynamics exist, including but not limited to clonal, oligoclonal, panmictic and epidemic genotypic strain structures. In Babesia bovis, genetic diversity of variable merozoite surface antigen (VMSA) genes has been associated with disease outbreaks, including in previously vaccinated animals. However, the extent of VMSA diversity within a defined population in an endemic area has not been examined. We analyzed genotypic diversity and temporal change of MSA-1, a member of the VMSA family, in individual infected animals within a reservoir host population. Twenty-eight distinct MSA-1 genotypes were identified within the herd. All genotypically distinct MSA-1 sequences clustered into three groups based on sequence similarity. Two thirds of the animals tested changed their dominant MSA-1 genotypes during a 6-month period. Five animals within the population contained multiple genotypes. Interestingly, the predominant genotypes within those five animals also changed over the 6-month sampling period, suggesting ongoing transmission or emergence of variant MSA-1 genotypes within the herd. This study demonstrated an unexpected level of diversity for a single copy gene in a haploid genome, and illustrates the dynamic genotype structure of B. bovis within an individual animal in an endemic region. Co-infection with multiple diverse MSA-1 genotypes provides a basis for more extensive genotypic shifts that characterizes outbreak strains.

One Health approach to identify research needs in bovine and human babesioses: workshop report.

BACKGROUND: Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks. RESULTS: The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein. CONCLUSIONS: The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.

Transfection systems for Babesia bovis: a review of methods for the transient and stable expression of exogenous genes.

With the recently sequenced Babesia bovis genome, a large pool of genes with unknown function was identified. The ability to complement and knock-out both unknown and previously identified genes would be a valuable tool to better understand gene function in B. bovis parasites. This review describes recent advances in the development of transient and stable transfection systems for B. bovis. Transient transfection constructs were initially generated using the promoter and the 3' region of the rap-1 genes of B. bovis controlling expression of luciferase as a reporter. Successful expression of luciferase in B. bovis parasites using this plasmid introduced by classic electroporation of B. bovis infected erythrocytes was followed by the identification and characterization of stronger promoters, such as the ef-1alpha promoter, using transient transfection techniques. Further refinement of the transient transfection technique included development of the ability to transfect free merozoites using nucleofection, an alternative method to electroporation that results in higher transfection yields and improved viability of transfected parasites. Availability of the transient transfection system was critical for the further development of a stable transfection technique using a plasmid designed to target integration of a gfp-bsd gene into the B. bovisef-1alpha locus. Several parasite lines resistant to the anti-babesial drug blasticidin (bsd) and constitutively expressing the gfp-bsd gene were generated after transfection. Integration of the gfp-bsd cassette into the genome was demonstrated by Southern blot and sequence analysis. Taken together these experiments demonstrated the feasibility to introduce, integrate and express exogenous genes in B. bovis. The stable transfection protocol was reproducible and used to transfect at least two distinct B. bovis strains. Further development of these transfection systems will facilitate functional analysis of B. bovis genes and will improve our understanding of the biology of and immunological response to this parasite.