Management of nonindigenous species includes prevention, early detection and rapid response and control. Early detection and rapid response depend on prioritizing and monitoring sites at risk for arrival or secondary spread of nonindigenous species. Such monitoring efforts require sufficient biosecurity budgets to be effective and meet management or policy directives for reduced risk of introduction. Such consideration of risk reduction is rarely considered, however. Here, we review the concepts of acceptable level of risk (ALOR) and associated costs with respect to nonindigenous species and present a framework for aligning risk reduction priorities with available biosecurity resources. We conclude that available biosecurity resources may be insufficient to attain stated and desired risk reduction. This outcome highlights the need to consider policy and management directives when beginning a biosecurity program to determine the feasibility of risk reduction goals, given available resources. The wide-ranging impacts of nonindigenous species have spurred development of a suite of management efforts that can be applied at various stages of the invasion process e.g., ,2. TheseRisk assessment can be used at each stage to prioritize vectors and sites for monitoring or control efforts. Continued development of programs within each of the three components occurs from international  to localBallast water exchange (BWE), the replacement of coastal water with open-ocean water during navigation, has been the primary approach to limiting this vector. Yet efficiency of BWE in removing organisms, while often greater than 80%, can be as low as 50% . AttemptThis compromise between acceptable risk and logistical constraints is reflected in the concept of acceptable level of risk (ALOR). Acceptable level of risk is used in a variety of fields, including environmental management, food safety and biomedical research e.g., ,17,18. TAn example of an explicit application of the ALOR concept is seen in Australia’s biosecurity policy. In an extensive review of their biosecurity strategy, Australian policy states that an ALOR of zero is unattainable and unaffordable . To achiDespite the widespread use of ALOR in policy, integrating ALOR into the design of management programs can be challenging . For exaII. Type II errors are the quantitative equivalent of ALOR—both represent the acceptable “miss rate” that a species of concern will not be intercepted. In contrast, Type I errors are the probability of incorrectly rejecting a true null hypothesis, represented by α and denoted here as EI.This approach relegates consideration of ALOR to the end of the process, or nowhere at all. Tied to the fate of ALOR in biosecurity decision-making is the acceptable rate of Type II errors. In a statistical context, Type II errors are the probability of incorrectly accepting a false null hypothesis, represented by β and denoted here as EFor the proposed framework and associated case study, the statistical definition of Type I and II errors are extended into a management context. That is, a Type I error represents over-management of a particular entity , and Type II error represents under-management of a particular entity . The costs of these management-based Type I and Type II errors are the costs of over-management (Type I costs) and the costs of damage from under-management (Type II costs).Costs of under-management (Type II errors) can be very high, particularly in areas of conservation management or when measured over long time frames . This isa priori consideration has been suggested by several authors [II/EI) is compared to the ratio of the costs of each error type (k = CII/CI). A demonstration of how this framework can be used to aid biosecurity decision-making is presented below.Bringing consideration of under-management (Type II errors) alongside over-management (Type I errors) is therefore crucial to designing programs that reflect policy decisions regarding risk and that account for the impact costs of introductions. Such  authors , 25, 26.Effective eradication or control is most successful when population density and abundance are low ,27. TherHere, we use monitoring in an EDRR context to demonstrate the decision-support framework. Specifically, we use a pilot macrophyte survey program for inland lakes by the Michigan Department of Environmental Quality (MI DEQ) Water Resources Division. The survey protocol includes a visual inspection around the lake perimeter, snorkel search and rake toss. We integrate the MI DEQ monitoring program with results from an assessment of Michigan water bodies at risk of introduction via the recreational boating vector to apply the decision-support framework for two scenarios . ScenariWe developed parameter values based on sources related to monitoring for EDRR efforts in the Great Lakes (summarized in Myriophyllum spicatum L. (Eurasian watermilfoil) in Wisconsin lakes. This finding of a 13% decline is applicable to this case study for several reasons. First, while the MI DEQ program targets macrophytes other than Eurasian watermilfoil, nuisance macrophytes generally have similar impacts  [Type I and II costs reflect the respective costs of over- and under-management, respectively. Type I costs represent the costs of monitoring. For the current budget scenario, it was based on the MI DEQ monitoring budget for macrophytes  . Type IIreation) ). Secondreation) . To detereation) , this yiThe cost of species introductions related to control efforts was determined by contacting twelve Michigan lake groups that fund control of aquatic macrophytes through improvement boards, special assessment districts or donations  ; S1 MapAnalyses of at-risk lakes were based on a dataset that gathered locations of boater trips and associated boater behaviours in Michigan . This daOf 391 locations in Michigan captured by the survey, 166 (42%) were categorised as “low”, “moderate”, “high” or “very high” risk scores for incoming trips and had some risk of species introduction. We applied this proportion to the 1157 Michigan lakes larger than 100 acres (a size that makes them reasonably subject to boat traffic and therefore subject to introduction risk). This suggests 491 lakes (water bodies) in Michigan are exposed to the risk of introduction. Thus, the total Type II costs used in the case study was US$64,615,600 .The parameters used for the current budget scenario (Scenario 1) were Type II and Type I costs. From these, we determined the ratio of acceptable Type II to Type I error rates, k’, suggested by the cost ratio, k. The parameters used for the ALOR budget scenario (Scenario 2) were the ratio of Type II to Type I error rates and Type II costs. From these, we determine the Type I costs suggested by the desired ALOR by harmonizing the k and k’ ratios from .II:CostI, US$64,615,600/US$64,137), was 1007:1. This implies a willingness to under manage (accept Type II errors) 1007 times more often than over manage (Type I errors), and, by extension a very high ALOR . For the ALOR budget scenario (Scenario 2), the Type I costs suggested by a very low ALOR was US$323,078,000 . These outcomes demonstrate an enormous discrepancy between the desired (in this case mandated) level of protection and the resources currently available to achieve this protection.For the current budget scenario (Scenario 1), k  would reduce the optimal budget to $6.46 million . We acknowledge that a US$323 million budget is likely unrealistic, at least in the short term. In this case, when an increase in CI = CostII/k’, US$64,615,600/2, where k’ = ErrorII/ErrorI = 0.01/0.005). Similar to the ALOR, setting the acceptable Type I error rate is a decision that will depend on the situation and stakeholders involved. As stated in Methods, we use 0.05 due to the precedence in statistical analysis, but the desired value may vary in a management context. Finnoff et al. [The second option  would reduce the optimal budget to $32.3 million .Given the drawbacks of increasing ALOR (costs of under-management and public perception), explicitly identifying the consequences of insufficient budgets may improve the likelihood of policy makers and stakeholders to ameliorate those shortfalls. It should be noted that many monitoring programs include more than one species. For example, the current MI DEQ monitoring program includes 12 macrophyte species, which with this adjusted budget of US$6.46 million would equate to US$538,000 per species. Other single-species efforts cost up to US$10 million . A second option is to enhance prevention through active management by installing permanent and mandatory boat wash stations at these lakes [The third option to reconcile the difference between available funds and desirable risk outcomes is to reduce the overall risk by reducing the likelihood of an incursion. For the recreational boating vector, this would require reducing the number of lakes at risk through decisions such as closing associated boat launches to prevent spread by recreational boaters ,40. Whilse lakes . A less-se lakes .Decisions on how (best) to effectively leverage available resources are often based on cost-benefit analyses e.g., ,12. StudIn addition to moving consideration of the errors and costs of under-management to the forefront of program design, this framework has several other important qualities. First, it can be applied to other components of nonindigenous species management—prevention efforts , control , and experimental design –and also adapted for use in other environmental fields such as species conservation or habitat restoration. Along with these other applications is the ability to modify the parameter values. That is, acceptable Type I and II error rates will depend on the situation and associated stakeholder priorities. Second, it provides a transparent process that can strengthen the relationship and potential collaboration between research and management sectors. Defining ALOR will facilitate the translation of management objectives into experimental design, and in turn, improve uptake of those research outcomes for decision-making . FinallyThe largest limitation in this case study, and likely in other applications, is the uncertainty surrounding the cost analysis. Ideally, a full cost analysis would include not only market costs, but also the many non-market costs (impacts) to environmental, social and cultural values e.g., reduced biodiversity and tourism; see also . HoweverButomus umbellatus L., flowering rush) and therefore will not decrease property value. A final assumption is that all lakes in this study were either not already invaded by a macrophyte  or that an additional introduced macrophyte would lead to an additional 13% property value decline.This study makes several assumptions. First, it assumes the costs of a new introduction would be similar to the cost of currently established species (primarily Eurasian watermilfoil). This is unknown, but given similar mechanisms of impact, this is likely. Second, it assumes the monitoring program protocol leads to 100% detection rates. While this is unlikely, we were unable to find published data on detection rates for inland lake macrophyte monitoring. Thus, applying lower detection rates would be unjustified and lead to a less conservative outcome. In additional to further cost analyses, we suggest this as an area for future research, as it is an important component in optimizing surveillance effort e.g., . A thirdIn conclusion, the incorporation of ALOR, and by extension this framework, to support decision-making within the realm of biosecurity ensures that management plans account for policy directives, as well as environmental, economic and sociocultural impacts. This case study demonstrates the resources necessary to realize near-zero risk outcomes. Using this framework to transparently and objectively reveal the discrepancies between available resources and acceptable risk will facilitate discussion between policy, management and research sectors, leading to improved biosecurity risk management.S1 Map(PNG)Click here for additional data file.S1 Supplementary Information(PDF)Click here for additional data file.S2 Supplementary Information(DOCX)Click here for additional data file.S3 Supplementary Information(PDF)Click here for additional data file.S4 Supplementary Information(DOCX)Click here for additional data file.


Genital viral load (GVL) is the main determinant of sexual transmission of human immune-deficiency virus (HIV). The effect of antiretroviral therapy (ART) on local cervico-vaginal immunological factors associated with GVL is poorly described. We aimed to identify the risk factors of detectable GVL, and the impact of ART on HIV genital shedding and its correlates in a cohort of HIV-infected women, attending HIV care in Kigali, Rwanda.All participants were evaluated for GVL, plasma viral load (PVL), CD4 count, various sexually-transmitted infections (STIs) at baseline and at month 12. Genital concentration of 19 cytokines and mRNA expression of APOBEC3G and BST2, two host HIV restriction factors, were evaluated at baseline in all participants. Cytokine levels were re-assessed at month 12 only in participants eligible for ART at baseline. Risk factors of GVL ≥40copies/mL at baseline and month 12 were assessed using logistic regression. Effect of 12-month ART on various local and systemic immunological parameters was examined using a paired t-test and McNemar as appropriate.N. gonorrhea infection (95% CI 1.32-2.15) and at month 12 with MIP-1β (95% CI 0.96-21.32) after controlling for baseline GVL, PVL and month 12 IL-8.96 of the 247 women enrolled in the study were eligible for ART. After 12 months of ART, PVL and GVL decreased to undetectable level in respectively 74 and 88% of treated participants. ART did not affect cytokine levels. HIV genital shedding occurred only when PVL was detectable. At baseline, GVL was independently associated with IL-1β after controlling for PVL, age and Suppressive ART does not necessarily reduce genital level of immune activation. Minimizing all conditions favoring genital inflammation, including active detection and treatment of STIs, might reduce the risk of HIV transmission as supplement to the provision of potent ART. Sub-Saharan Africa bears 70% of the worldwide prevalence of HIV infections, with the majority of new infections occurring in women . The conin vitro and therefore represent promising candidates for innate immune-based therapeutic strategies [Vif), which triggers the ubiquitination and degradation of APOBEC3G via the proteasomal pathway [Vpu) from infected cells. APOBEC3G and BST2 are expressed by immune cells from various tissues, including the vaginal mucosa. Whether the local expression of these innate viral restriction factors plays a role in the modulating effect of cytokines and vaginal pathogens on HIV genital shedding has never been studied.Conversely, cytokines such as IFN-α and IP-10 might also reduce HIV replication through the regulation of the cellular expression of intrinsic anti-viral factor APOBEC3G and tetherin (BST2). Host restriction factors have been demonstrated to inhibit HIV-1 replication rategies , 18. APO pathway . BST2 im pathway . BST-2 rPVL suppression to undetectable level is the hallmark of potent antiretroviral therapy (ART) However, ART-induced virological suppression in the plasma does not always correlate with a GVL below the level of detection –23, reprWe investigated the factors associated with having a detectable GVL as well as the impact of ART on GVL and genital inflammation, in a cohort of HIV-infected women in Rwanda.The SEARCH study has been described previously , 27 and The SEARCH study was designed to enroll at least 100 HIV-infected women immediately eligible for ART according to the Rwandan national guidelines   to partners, and 2) to get regular Pap smears to prevent cervical cancer.The treatment of STIs followed the syndromic management guidelines because laboratory results were not available in real time. Syndromic treatments were not recorded in the frame of this study. Positive herpes serology and positive Human Papilloma Virus (HPV) PCR were not treated; women were given their test results and given advise on 1) how to prevent transmission of All patients were seen at baseline, and every three months for 6 to 24 months thereafter. Patients initiating ART received additional visits in the first trimester of the follow up, visits at week 2, month 1 and month 2. All patients were interviewed for information on socio-demographics, sexual and contraceptive behavior as well HIV and reproductive health history.+T cell counts on EDTA blood samples were always done on the day of sample collection. All other blood samples were stored as aliquots of plasma, serum or buffy coats at −80°C until further processing and testing.Blood samples were collected at all clinic visits. They were hand-carried from the clinic to the laboratory next door within 4 hours after collection. CD4Endocervical specimens were collected every 6 months during pelvic exams. A swab was vigorously agitated in Amplicor specimen transport medium , and a cytobrush in PreservCyt medium .Cervico-vaginal lavages (CVL) were obtained by irrigating the left and right fornix and cervical os twice using 5mL normal saline. The liquid was subsequently aspirated after 30 seconds. The CVL fluid was immediately placed on ice or at 4 –8°C and centrifuged at 1,000 rpm for 10 min to separate the liquid phase from the cells. The cell pellet was re-suspended in 1 ml of PBS, centrifuged again, and cells were stored at -80°C. Cells pellets and aliquots of supernatant were stored at -80°C until further processing.GVL, cytokine, and APOBEC3G and BST2 gene expression testing was done at the Academic Medical Centrum (AMC) in Amsterdam, the Netherlands. All other laboratory tests were carried out at the National Reference Laboratory in Kigali, Rwanda,+T cell counts  were measured every 3 months for women who did not yet qualify for ART and every 6 months for those on ART. PVL testing  was done at ART initiation and every 12 months thereafter. The lower limit of detection was 40 HIV RNA copies/mL. Women were tested for pregnancy using an hCG urine dipstick test at baseline and every 6 months. Participants were tested for Herpes simplex type 2 (HSV-2) using HerpeSelect test kits  at baseline, for syphilis by RPR confirmed by TPHA  at baseline and every 6 months, and for Neisseria gonorrhea and Chlamydia trachomatis by PCR  at baseline and every 12 months.CD4p70, IL-17, IFN-α,), anti-inflammatory cytokines , chemokines , adaptive immune mediators  and growth factors , were selected based on their potential involvement in the inflammation process of the genital tract. Each standard curve was fitted using Bio-Plex manager 6.0 software and was based on 11 standards (8 recommended plus 3 higher dilutions of the standards).CVL supernatants were shipped to the AMC in Amsterdam on dry ice, thawed, and 500μL of CVL was mixed to 500μL of phosphate buffer saline. The GVL was determined by nucleic acid amplification using COBAS/Ampliprep/COBAS Taqman v2.0 according to the manufacturer instructions . Quantification of cytokines in CVLs was done on diluted samples (4x) using a Luminex-based multiplex system  per manufacturer’s instructions. Nineteen pro- . The concentration of the isolated RNA was measured on a nanodrop (ND1000 Isogen Lifescience). cDNA was prepared from 500ng of RNA using the Transcription RT Reaction Buffer as recommended by the manufacturer (Roche Transcriptor First Strand cDNA Synthesis Kit). The qPCR was performed with a Lightcycler 480 using specific primer pairs for APOBEC3G and BST2  and SYBREthical approval was obtained from the Rwandan National Ethics Committee. All study participants provided written informed consent prior to enrollment, were free to withdraw from the study at any time, and were transferred to publicly-funded HIV treatment programs at the end of their study participation. Participants with curable genital infections were treated according to the national treatment guidelines.The PVL and GVL data were dichotomized based on the lower detection limit (< 40 copies/ml) of the viral load assay. Individuals with < 40 copies/ml of virus were categorized as having low (non-detectable) level of virus and those with ≥ 40 copies/ml of virus as having high (detectable) level of virus.Cytokines concentrations above the highest and below the lowest cytokine concentration of each respective standard curve were identified as out of range (OOR) values. Cytokines for which < 15% of the readings were OOR values were analyzed as continuous variables. Cytokines giving readings between 15 and 50% OOR values were analyzed as binary variables (present/absent). Cytokines with more than 50% OOR read-out values were removed from the analysis. For cytokines that qualified for analysis, the OOR values below the lowest point of standard curve were assigned a value of one half of the lowest concentration of the standard curve and OOR values above the highest point of the standard curve were assigned a value of 1.5 times the highest concentration of the standard curve of the respective cytokines. The concentrations of APOBEC3G, BST2 and cytokines qualifying for analysis as continuous variables were log (base 10) transformed.http://www.r-project.org/).All binary and categorical variables were summarized as counts (proportions) and continuous variables as mean . Differences at baseline between low and high GVL groups were assessed by t-test for continuous variables and Fisher’s exact test for proportions. Logistic regression was used to assess factors associated with GVL at baseline with GVL categorized as absent (undetectable) or present (detectable). In the multivariable model, all variables with P value < 0.2 in the bivariable analyses were considered, and model selection was performed using stepwise backward elimination method. The final model was selected using the Akaike Information Criteria (AIC). Similar analyses were performed to determine the effect of ART on GVL (present/absent) upon 12 months of treatment after controlling for the baseline level of genital viral load and other factors. For the final model, assumption of normality was assessed using residual plots and linearity for continuous variables was assessed by plotting log odds of predicted values against the continuous variables. All statistical analyses were performed using R version 3.1.1 . Other STIs were less frequent, with only Neisseria gonorrhea being significantly more frequent in women with detectable GVL . Although the two groups did not differ in terms of clinical stage of HIV disease (p = 0.306), CD4 count and PVL levels reflected a more advanced disease progression in the group with detectable GVL compared to the group with undetectable GVL  and PVL is log10 4.51 versus 3.53 RNA copies/mL, respectively (p < 0.0001)).Participants with detectable versus undetectable GVL at baseline were similar in terms of education levels, social status and utilization of family planning methods . Women wp70, IL-17, IFN-γ, MIP-1α, RANTES, TNF-α and IFN-α) had more than 50% OOR values and were removed from the analysis , but negatively correlated in the blood compartment .10 3.37 mRNA copies/106 cells in both groups, p = 0.39) or APOBEC3G mRNA in genital cells .The relationships between IFN-α and IFN-γ levels and the genital expression of HIV restriction factors could not be investigated due low levels of interferons in CVL supernatants. The concentration of soluble genital IP-10, which is induced by interferons, was not significantly correlated to levels of genital BST2 or APOBEC3G mRNA (data not shown). IP-10 levels in CVL supernatant did not differ in samples with high versus low levels of BST2 mRNA  that had initiated ART and 38 of 147 26%) women not eligible for treatment had their genital HIV viral load measured at month 12. Among treated participants, the mean genital viral load decreased from 2.34 logp<0.000, . In thisp<0.000,  while CDp<0.000, . Eight o% women n10 RNA copy/mL, p = 0.068 for GVL and from 3.5 to 3.58 log10 RNA copies/mL, p = 0.444 for PVL, data not shown) while CD4 count significantly decreased from baseline to month 12 . Among treated patients, IP-10 was the single cytokine of which average concentration significantly decreased from 3.44 to 2.64 log10 pg/mL (p = 0.001) between baseline and month 12. In this group, levels of all other measurable cytokines remained comparable between baseline and month 12  indicated that risk factors for detectable GVL at baseline were baseline PVL (adjusted OR (aOR) = 2.47 95% CI: 1.66–3.67) IL-1β concentration in CVLs , after adjusting for age and N. gonorrhea  consisted of PVL at month 12  and MIP-1β at month 12 , after controlling for the baseline levels of GVL, PVL and IL-8 .N. Gonorrhea infection.Twelve months of ART successfully reduced PVL and GVL to undetectable levels in the majority of participants but had no effect on genital levels of inflammatory markers. All patients with detectable GVLs also had detectable PVLs. In contrast, no women with undetectable PVLs had detectable GVLs, suggesting that PVL, which is an essential marker of disease progression is the most important determinant of GVL –4. GVL wAlthough the proportion of detectable HIV genital shedding decreased substantially after one year ART, it remained strongly associated with detectable PVL at month 12. These observations contrast with previous findings reporting HIV genital shedding in up to a third of women with undetectable PVL , 26. It Interestingly, the highly effective ART had no effect on cytokine levels in genital secretion. Cytokine levels remained largely unchanged at month 12, showing even less variation as compared to the untreated group. Much of the inflammation in the genital tract may not be primarily related to GVL. The decrease of some cytokine levels observed in both treated and non-treated participants at month 12, could relate to the syndromic treatment of STIs during routine HIV care. Interestingly, the significant reduction of IP-10 concentrations among treated participants at month 12, suggest that ART had a significant reduction effect only on anti viral IFN-(induced) responses. For the other cytokines evaluated, treatment-induced changes might have been be masked by genital inflammation caused, for instance by concurrent STIs. The possible role of non viral infections in fueling mucosal inflammation is corroborated by findings from a similar study conducted in India , wherebyOur findings have the following implications. First, they corroborate the hypothesis that providing effective highly active ART reduces the risk for sexual HIV transmission . SecondlAlthough residual genital inflammation did not translate into GVL in this particular cohort, our results suggests that treating STIs, reducing local inflammation, combating inappropriate hygiene practices or restoring the balance of the vaginal flora would contribute to the normalization of genital cytokine secretion profile and the risk reduction of HIV genital shedding. Additional studies are warranted to explore the potential benefit of local anti-inflammatory treatment and/or topical administration of commensal bacteria to the genital tract. Whether more proactive detection and treatment of STIs have additional advantages, as compared to the syndromic STI treatment approach currently recommended by the Rwandan ART program , need toAt baseline, the CVL concentrations of only a few cytokines and growth factors  were elevated in women with detectable GVL. These findings are in line with previous reports identifying the same immune factors as correlates of GVL in the context of genital infection and/or local immune activation .N. gonorrhea infection, while the effects of IL-8, MIP-1α, VEGF and GCSF were attenuated. Our data suggests that, compared to the other cytokines studied, the secretion of IL-1β might be more closely associated to genital infections not assessed in this cohort or to inflammation related to non-infectious events. Our observations are in line with the identification of IL-1β as a primary pro-inflammatory mediator secreted in the inflammasome in response to various triggers including bacterial as well as viral infections [At baseline levels of IL-1β were still associated with GVL after adjusting for PVL, age and fections . Notablyfections –42 and fConversely, a number of cytokines, including IL-6, MCP-1, IL-1α and IL-1RA generally found to be associated with GVL  had compThe precise cytokine profile predicting genital shedding was different between our study and others, on the one hand and between baseline and month 12, on the other hand. This could reflect the multi-factorial causes of HIV genital shedding in the context of a fluctuating interaction between local STIs, cytokine secretion and inflammation.N. gonorrhea infection on HIV genital shedding at baseline corroborates previous reports associating this genital infection to increased local replication of HIV, local immune activation and cytokine secretion [The effect of ecretion , 11, 12 ecretion –45, whilin vitro, their effect in vivo, might be detectable only at supra-physiological expression induced by supplementation of IFN-α [The exploration of the association between intrinsic anti-viral factors, GVL and IFN-induced cytokines remains inconclusive. Although we report the possibility that that low level genital BST-2 expression might be associated with high GVL, neither cellular mRNA expression of BST2 nor APOBEC 3G correlated with soluble IP-10 in the genital tract. These findings could be related to first, the lack of direct link between levels of cell-associated BST2 and APOBEC3G and levels of extra-cellular HIV RNA and soluble IP-10, in clinical blood and genital samples. Secondly, restriction factors expressed in heterogeneous population of blood and mucosal cells might poorly correlate with VL, since only a fraction of the cells analyzed represent targets and source for HIV. Thirdly, despite the clear anti-viral properties of APOBEC3G and BST2 of IFN-α .Our work has several limitations. The follow-up period included only two time points at a year interval. The possible occurrence and consequences of blips of viral replication in the plasma and the genital tract at intermediate time points could not be investigated. Quite a number of samples were not available in sufficient volume to perform the whole panel of testing, thereby reducing the statistical power of the calculation. An important consequence might be that the frequency of HIV genital shedding episodes during suppressive ART might have been under-estimated in this study. Bacterial vaginosis and yeast infections, likely to be frequent in this setting were not assessed in this cohort. In addition, the treatment of STIs followed a syndromic approach since laboratory results were not available in real time, with the risk of missing asymptomatic women. Hence, an accurate triangulation between the presence of STI, elevated inflammation and HIV shedding in the genital tract could not be done. Finally, 8 of the 19 cytokines in the panel evaluated yielded very low concentrations, and could not be analyzed. The CVL sampling method based on vaginal douches using 10mL of PBS may have diluted some cytokines levels below the detection limit, possibly resulting in only a fragmentary determination of the mucosal inflammation at baseline and/or month 12.In conclusion, the present set of data clearly indicates that although HIV genital shedding primarily depends on plasma viral load it is also influenced by other factors, including local concentration of certain cytokines. The failure of ART to alter cytokines levels, underscores the persistent risk of HIV genital shedding and HIV transmission among patients with undetectable plasma viral load. Further investigation assessing HIV genital shedding at closer intervals and in larger cohorts are warranted to further delineate the possible role of cytokines on the risk of HIV transmission during suppressive ART. The causative role of various STI or vaginal washing practices in promoting genital inflammation and GVL needs to be determined. These data could inform the design and assess the relative effectiveness of various approaches to reduce the odds of HIV transmission during suppressive ART in the context of the Rwandan HIV program.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLS)Click here for additional data file.


We read with interest the paper by De Pascale and colleagues  and the TGC is a small lipophilic antibiotic with a high distribution volume in critically ill patients ,4. The rDe Pascale and colleagues are right to state that more pharmacokinetic investigation is needed before high-dose TGC can be recommended for treatment of multidrug-resistant bacterial infections . We stro


Among attendees there were virologists, immunologists, neurologists and epidemiologists – all of them concerned on how to stop the rapid progress of Zika virus in Brazil and in other countries in the Americas.Last March, many experts were brought together in Geneva in a meeting organized by the World Health Organization (WHO) to discuss the dramatically increase of Zika virus infection in the Americas.,3 in such a degree that WHO declared a public health emergency situation of international concern.The Zika virus, in the past an agent responsible for occasional disease in humans, is today responsible for outbreaks in Brazil and Latin America of microcephaly, Guillain-Barré syndrome, myelitis and meningoencephalitis cases,-7 related to maternal infection by Zika virus that works as an excellent model for discussion of a number of bioethical issues, such as the dilemma of healthcare professionals to assist pregnant woman who will have a baby with microcephaly, the autonomy principles of mother and fetus, the discussion on authorization of abortion in cases of fetal malformation , patient-physician relationship models, and resources replacement and equality in access to health system.However, the particular increase of microcephaly casesIn bioethics, there is no absolute and immutable truth. However, there is a number of concepts and a set of actions that aim to identify and help to solve a number of ethical dilemmas appearing daily in relation to patient care.For this reason, we consider the context itself  and its aggravating and relieving factors before take a decision that, sometimes, is not ideal, but for a specific case, seems to be more adequate.Bioethics approach requires a continuous behavior both in medical practice and in behavior as citizens. Sometimes such roles are overlap such as in the case of women who inquiry physicians about Zika virus infection. This situation to discuss only about delay pregnancy, sometimes so expected by the woman, however, when the pregnancy occurs, there is the need to discuss issues related with risk of have a baby with microcephaly.Today, it seems to be easy to discuss with women to not become pregnant. Especially due to the uncontrolled outbreak and news that appear every day, this seems a clever behavior, but we cannot deny that we are interfering in the free will of future mothers.Obstetricians and infectious disease specialists are able to assess statistical risk of fetal malformation appearance that is generally considered remote and occasional, and they do not usually influence patient’s decision to become pregnant. However, what about the risk of microcephaly?Other cases of microcephaly associated with other factors are well known and continue to occur. Such cases are similar or more prevalent than Zika virus. These factors are alcoholism, poor controlled gestational diabetes and other perinatal infections as toxoplasmosis, rubella, cytomegalovirus and syphilis.,5 whom had symptoms compatible to Zika virus and gave birth of microcephaly babies. The virus identification in the tissue sample of children who born with microcephaly lead the Brazilian Ministry of Health to confirm the relationship between the virus and microcephaly, a fact that has been causing concern to pregnant women.However, since November 2015, temporal relationship between Zika outbreak and alarming increase of microcephaly prevalence has indicated a strong relationship among these events. Confirmation of virus presence in the amniotic fluid of two pregnant women from Paraiba State (Brazil),Aedes aegypti . Pregnant women should consult their physician before travel to risk areas.Officially, the Brazilian Ministry of Health reinforces pregnant women to perform qualified prenatal test, and prescribed tests, and also they must report their physician any clinical changes, in addition to continue with preventive measures against The Brazilian Ministry of Health recommends to health professionals careful evaluation of cephalic perimeter and gestational age during prenatal, and also notification of suspected cases of microcephaly at birth. Because health professionals are always in contact with population, they must reinforce measures to combat the mosquitoes spreading the virus. a similar position was stated by WHO directors.However, in the age of Dr. Google consult, facts can become more controversial. The Centers for Disease Control (CDC) in newsletter published last March 11, recommended pregnant women to not travel for areas where Zika virus transmission is occurring,More controversial than cancel a travel is discuss if women living in areas with Zika outbreak must postpone pregnant, which sometimes was intensively planned. One scenario is to delay pregnancy at 25-years-old, a different one is request this delay for women older than 40 years .Who can guarantee that this outbreak will pass quickly? Recently, the CDC issued guidance recommending men and women diagnosed with Zika to wait before decided to have a child. According to CDC, women must wait for at least 8 months after beginning of symptoms to become pregnant. For men the recommendation is to wait at least 6 months.In case of exposition to risk areas where Zika virus circulates recommendation to try conception is 8 weeks. Such guidance are based on limiting data available about virus permanence in the blood and semen. To date, there is no evidence that fetus conceived after disappearance of virus in the bloodstream is at risk to develop the infection.Some Brazilians’ experts remember that such recommendations of CDC applies to American context, where no outbreak has occurred yet, and they can be used as safety measures for persons diagnosed with Zika from now on. An agreement among Brazilian scientists is that CDC decisions about the time to become pregnant are personal, complex and must be individualized case by case, and also based on guidance of an obstetrician.,4Data analysis of literature indicates that number of microcephaly notifications in children who born from mothers infected with Zika virus in Brazil has increased more than 20 times if compared to notification from previous years. A total of 3,741 cases are still under investigation.Since the beginning of investigations by the Brazilian Ministry of Health in October 2015, a total of 7,150 notifications were recorded until April 16, 2016. Of the cases already finished, 2,241 were excluded and 1,168 microcephaly cases and other nervous system changes suggesting congenital infection were confirmed.Of 1,168 cases, 192 have been already confirmed in laboratory tests for Zika virus. However, the Brazilian Ministry of Health emphasizes that such data do not represent adequately the total number of cases related to the virus, considering that there was infection by Zika virus in the majority of mothers who had babies with microcephaly.Until April 16, 246 fetal or neonatal deaths were recorded (3.4% of notified cases) with suspicion of microcephaly and/or central nervous system change after delivery or during pregnancy (abortion or stillbirth). In 51 cases, microcephaly was confirmed. Others 165 cases continue under investigation, 30 were excluded.It is know that most of pregnant women exposed to Zika virus will give birth to babies without microcephaly, but the real risk of occurrence of malformation still generating controversial information even in the medical literature. .Data analysis of Zika virus outbreak reached the French Polynesia between 2013 and 2014 suggesting risk of occurrence of microcephaly in about 1% of cases in which the infection occurred in the first trimester of pregnancy, reported prevalence of microcephaly significantly higher  in the Brazilian States where active transmission of Zika virus occurs. A recent cohort study carried out by group from Rio de Janeiro, Brazil, included 88 pregnant women with at least 5 days of pruriginous skin rash. In 72 women (82%) the infection by Zika virus was confirmed and it might occurred between 5th and 38th weeks of pregnancy.Kleber de Oliveira et al.The most concern aspect of this study was that 29% of obstetric ultrasound performed in 42 pregnancies with positive test for Zika (58%) show very significant abnormalities as delayed intrauterine growth with or without microcephaly, ventricular calcifications, oligohydramnios and arterial flow changes, and two fetal deaths.,14Although medical community have advanced in little period of time concerning Zika virus infection, studies lack to answer a variety of questions, such as vertical transmission rate and data on infected fetuses who show complications.In April 2016, CDC, based on rigorous review of scientific evidence, confirmed the relationship of Zika virus and occurrence of microcephaly, attributing to the virus the brain damages identified in fetuses.As a matter of fact, all this insecurity of having a child with microcephaly have relight the controversial debate about women right of control over their body and, as a consequence, to abort.How to deal with birth of a child with severe microcephaly? What is the future impact of this sequela? Are there conditions for full inclusion of this child in the society? Does the mother have the right to abort?This mother, under strong emotional impact and, sometimes, abandoned by her partner, is in condition to decide autonomously? in addition to beneficent of nonmaleficence and justice.The principle related with autonomy constitutes one of the four fundamental premises of bioethical proposed by Beauchamp et al., proposed an alterity model, .However, we can clearly understand the bioethical point of view over such complex subject, and also the different approaches it involves. Other important author on bioethics, Levinas, invertes the golden rule “treat others the same way you want them to treat you”. The others discoveries are what impose the adequate conduct. When the golden rule “Your rights end where mine begin” does not make sense anymore, what become to be valid is “my rights are guaranteed by others rights”.LévinasIn this context of individual values of each patient, many times different from our own values, pregnant women’s opinion must be always respected, because some of them, due to their believes, are contrary to interrupt the pregnancy, other pregnant women with different views must have their option respected as well.As a support for bioethical decision-making is recommended the deep assessment and knowledge about ethical and legal behavior that support the physician, specially: code of medical ethics that guides the fundamental principles of medicine, including physicians rights, professional responsibility, relationship with patient and families, among other topics; the Brazilian civil rights law that regulates legal relationship between individuals in several levels, the Criminal code that defines what is considered crime and which responsibilities each citizen has.It is never too late to remember how devastating are the consequences of microcephaly over underserved population. Not only for lack of access of children to needed therapies on the future  but also because of terminate of unintended pregnancy in unfavorable socioeconomic conditions is often related to high rates of maternal morbidity and mortality.Aedes aegypti, Zika virys and microcephaly of their babies?What about the young, single and poor girls living in risk areas who do not know and/or cannot prevent unintended pregnancies, particularly do not know how to plan such pregnancies and end up becoming victims of their pregnancy, of  “the internal conflict of a healthcare professional facing a terminal disease, a mother willing to abort, or maintenance of confidentiality in risk situations that involve others, is part of him/her, and immune to the law.”As stated by Professor Segre


To the Editor: Zika virus is an emerging arbovirus spread by Aedes aegypti mosquitoes and belongs to the genus Flavivirus of the Spondweni serocomplex , where viral RNA was extracted from it by using the QIAamp Viral RNA Mini Kit . We used real-time RT-PCR for diagnosis, using the Superscript III system  and primers and probes previously reported (http://www.megasoftware.net) from aligned nucleotide sequences. The maximum-likelihood statistical algorithm and the Tamura-Nei substitution model with 1,000 replicates for bootstrap were used. Phylogenetic analyses showed that the partially sequenced strain MEX/InDRE/14/2015 belongs to the Zika virus Asian lineage and is closely related to those reported from Brazil and Suriname in 2015  as a reference. The strain MEX/InDRE/14/2015bears the mutation markers K546R, K642R, and E561K, which cause the differentiation of the Asian lineage from the clades representing the African lineage . In addiIn summary, we identified Zika virus in a traveler who returned from Colombia to Mexico in October 2015. A partial sequence of the NS5 gene showed that the isolate from this patient was closely related to those described elsewhere in the Western Hemisphere belonging to the Asian lineage, particularly to 2 strains identified in Brazil and Suriname during 2015.


Road traffic injuries are the eighth leading cause of death globally and the most affected are young people aged 15–29. By 2030 road traffic deaths will become the fifth leading cause of death unless urgent action is taken. Motorcyclists are among the most vulnerable road users and in Uganda they contribute 41% of all road traffic injuries. This paper establishes factors associated with the injuries of commercial motorcycle riders also known as boda-boda riders in Kampala, Uganda’s capital city.The study was matched case-control with a case being a boda-boda rider that was seen at one of the 5 major city hospitals with a road traffic injury while a control was a boda-boda rider that was at the parking stage where the case operated from before the injury. The sample size was 289 riders per arm and data collection took 7 months. A structured questionnaire was used to collect data on background and exposing factors. Being matched case-control data conditional logistic regression was used in the analysis.Factors independently associated with injury among motorcyclists were younger age group, being a current alcohol drinker , lower engine capacity (<100cc), riding experience of less than 3 years, not changing a motorcycle in past 1 year , riding for a longer time in a day  and sharing a motorcycle . Other factors associated with injury were low level of knowledge of traffic rules, being stopped by police for checks on condition of motorcycle/license/insurance, working till late.More road safety sensitization is required among riders to raise awareness against sharing motorcycles, working for a longer time and alcohol consumption. Police enforcement of drink-driving laws should include riders of commercial motorcycles. Investigate the validity of motorcycle riding licenses and test the riding competency of all who got licenses in last 3 years. Road traffic injuries are the eighth leading cause of death globally and the leading cause of death among young people aged 15–29 . Road trThe commonest injuries among motorcyclists are lower-extremity and head injuries. Other a the United Nations General Assembly (UNGA) adopted a resolution [Following the publication of the 2009 global status report on road safety solution  that decStudies carried out in several countries have established that factors that have an influence on motorcycle crashes include age, ethnicity, income, education, motorcycle license insurance status, self reported alcohol consumption in the 12 hours preceding the crash, years of on-road riding experience, kilometers travelled on the specific motorcycle at interview, posted speed limit, and weather conditions. Other f(boda-boda) are a major cause or agent of road traffic injuries and a significant economic burden as each costs an estimated range of USD 300–369 in treatment [In Uganda, motorcycle taxis reatment –20. A streatment . The cosreatment . About 1reatment .Boda-bodas are filling a gap in the transport system in Uganda. They operate where more conventional transport services are uneconomical or physically impossible . Therefoboda bodas has raised road safety concerns as the number of injuries attributed to them continues to rise. This study aims at establishing the factors associated with motorcycle injuries in Kampala city. In the process it also establishes whether factors associated with motorcycle injuries in studies elsewhere are mirrored in Uganda.The increase in number of The study was carried out at Mulago national referral hospital and four other major city hospitals, namely, Kibuli, Nsambya, Rubaga and Mengo. Mulago is a public facility offering free services while the rest are private hospitals where patients have to pay for services. Owing to free services offered at Mulago the hospital is overstretched with large number of patients for limited facilities such as in-patient beds. Data coThe research was designed as matched case-control study with cases being all those boda-boda riders who were injured on the road and brought to the study hospitals. The controls were boda-boda riders at the same boda-boda packing stage as the cases. Therefore, the matching was by packing stage. The ratio of cases to controls was 1:1. It was assumed that both controls and cases were exposed to similar conditions that could influence their risk of injury.The population under study comprised boda-boda riders in Kampala city. They were grouped into two: the cases that were injured at the time of the study and controls that were at parking stages where the cases operated from. Since the hospitals are the largest in Kampala city, comprising of both public and private facilities, it is believed that the cases and the selected controls represent all boda-boda riders in the city.The sample size was 289 motorcyclists per arm. Using an OPEN EPI software which applies the formula by Fleiss, aiming The method of selecting a control was informed by the pilot test which found that sampling the eligible boda-boda riders on a stage was ineffective. Boda-boda riders were found to be impatient at the sight of a potential client, and some would abandon the interview midway if a client arrived. Some control riders were not willing to participate in the study because they were unsure of the outcome of the study despite several re-assurances. Shortly before the study Kampala Capital City Authority had registered all boda-boda riders with the aim of streamlining their operations. Some thought the data collection was linked to the registration process which would eventually limit their operations. For these reasons, it was decided that the first of the eligible controls to read through the consent form and consent for the interview would be interviewed.Controls had to be known usual boda-boda operators at the stage while the cases had to have been brought to hospital after a boda-boda related injury. Cases would have been stabilized after initial emergency treatment. Both cases and controls must have been operating within the boundaries of Kampala city. The exclusion criteria were refusal to participate or complete the interview and deteriorating condition of the cases.The main place of data collection was the emergency department and therefore the nurse in-charge of the department was instrumental in ascertaining the cases. The data collection was by face-to-face interviews using a structured questionnaire. Each control was interviewed within 24 hours after interview of the case. The interviewers were knowledgeable in the local language. At the boda-boda packing stage the first contact was the stage leader. At each stage the data collector worked with the stage leader to identify eligible riders.The main outcome variable was a boda-boda related injury. The key exposure variables were taking alcohol, frequency of alcohol consumption, Alcohol Use Disorders Identification Test (AUDIT) score and helmet use. Major confounding variables included background characteristics  engine capacity, ownership of motorcycle and experience of riding, helmet and reflective jacket use, training, time of start and end of riding in a day, model of the motorcycle and ownership of the motorcycle, alcohol and drug use and knowledge of road safety rules. Inclusion of each of the variables in the study is premised on results of previous studies carried out in different parts of the world as shown in the background section.Data on most variables were obtained from face-to-face interviews. Data on boda-boda related injury were obtained through observation, enquiries from the nurse in-charge at the emergency department and interview of the rider. The data on exposure and confounding variables were obtained through face-to-face interviews with the boda-boda riders. The data on AUDIT were obtained using a tool developed with support of World Health Organization. The tooSelection of controls had potential for bias towards the educated riders and those who may not have had many clients. This is because those who accepted to read and sign the consent forms were more likely to have been educated. In addition, those who accepted to be interviewed may have been those who were either not busy or hoped to get money for compensation of their time. To counteract this bias, research assistants were trained to explain the study well and to use local language. The tool had local translation as well.A rider who has not had an injury at around the same time his colleague is in hospital may not be the best control because he may have had an injury days or weeks before or after. However, these control riders at the stages provide the best base for comparisons and they are also easy to get.Thirdly, those who go to hospitals may not be the best representation of those who are involved in road crashes and eventual injury. For example, there are those will only go to hospitals when they are severely injured. Then the fact that most of the injured motorcycle riders were recruited from the public hospital is another cause for selection bias. The hospital is the only one that offers free services. It is possible that boda-boda riders, who are generally low income earners, were either taken to, or preferred to go where they could access free services. The effort to collect data from other major but paying hospitals was meant partly to address the bias created by use of one source of data.Recall bias and social desirability bias from interviewer-administered surveys are also important potential limitations. For example riders may hesitate to report they take alcohol given that it impairs decision making during riding.Residual confounding may arise from misclassification of measured covariates as well as unmeasured variables. Examples of unmeasured variables are distance covered, fatigue, road features and environmental factors.All data were double entered using EPIDATA V3 software and later exported to STATA V12 for cleaning and analysis. The data entry screens had been fitted with range and consistency checks.Data from a few of the variables needed further treatment before use in analysis. Key among these were the AUDIT score and background characteristics. The audit score is a summative one and it involved adding up scores for all the ten questions. The score was categorized into low (0–7), medium (8–15) and high (16–40) as informed by documents on AUDIT score. Other cTo establish factors associated with injury, bivariate and multivariable conditional logistic regression analyses were carried out with the outcome as case/control. Conditional logistic regression was used because the cases and controls were matched. Model building principles  were folclogit command to fit the fit the model:Specifically, the analysis was carried out in STATA V12 using the Wherem = number of confounder adjustment variablesα + α2xs2 +…….+ αmxm = variables for many small strata which are not of interest per se[st per se. Strata For each exposure variables Using the model building methods outlined above three statistical models were developed. The first model examined the relationship between boda-boda related injury and background characteristics, the second was the grand model that highlighted all factors associated with the injury while the third model only showed how the AUDIT score, index for knowledge of recommended riding practices and enforcement of traffic rules are related to boda-boda related injury. None of these factors featured in the second model because their relationship with boda-boda injury was grossly confounded by other variables yet they represent whole sections of the tool used.The minimal data set underlying the main multivariable findings in the study is accessible through a link to a STATA file named The investigators first secured ethical approval from Makerere University School of Public Health Institutional Research Review Board (IRB) and the National Council of Science and technology (NCST).Written consent was requested from each eligible rider after a clear explanation of the research project, its goal, and objectives and eventual possible benefits to road users. The data collectors assured the study participants of the confidentiality of the interviews and the security of the data, which could only be accessed by the researchers on this study. All the riders interviewed provided written informed consent.The total number of cases that were potentially eligible for the study was 325 and it included 15 who died at emergency department shortly after arrival . Those sThe study participants were equally distributed across the five divisions of Kampala city. The age ranged from 18 to 56 years . Most ofExamination of crude odds ratios of getting injured in different categories compared to base categories shows that the odds of getting injured significantly reduced with higher age groups , were lower among the married , lower among the Muslims  and highest among high income earners  . The odds of injury did not significantly change by education level.In a model with only background (socio-demographic) characteristics, it was found that factors associated with injury were lower age group, not being married and having a higher income . The bacAfter controlling for all key variables, having an injury was strongly associated with younger age group, taking alcohol, lower engine capacity (<100cc), having a riding permit, having few years of riding experience (less than 3 years), not changing a motorcycle in past 1 year, riding for longer hours and sharing a motorcycle . FactorsResults from the study have shown that injured motorcyclists were more likely to be younger, unmarried and earned a higher income compared to those who were not injured. Further on, the results show that factors independently associated with injury among the riders were alcohol consumption, lower engine capacity, having a driving permit, fewer years of riding experience, not changing a motorcycle in previous year, riding for a longer time in a day and sharing a motorcycle. Other factors that were significantly associated with boda-boda related injury but after adjusting for only baseline characteristics were low knowledge of road safety rules, being checked by police and having a higher alcohol use disorder test scoreThe difference in socio-demographic factors between the cases and controls in the study are consistent with results from several studies. For example, the protective effect of higher age was established in case control studies in New Zealand, Malaysia and Pakistan , 32–33. Results on association of road injuries among motorcycle riders with alcohol consumption and short riding experience concur with findings of studies carried out in several countries including the USA, New Zealand and France , 36–38. Higher odds of getting injured among those who have been stopped for routine checks by police may be explained by a possibility that those who are checked by police are more likely to be traffic offenders who are at high risk of getting injured. The issue of higher odds of injury among those with motorcycle riding permits needs further investigation because it’s unexpected. On cross tabulation with length of time of riding a motorcycle it was found that this unique relationship was most evident among those who had ridden motorcycles for less than 3 years. There has been a police campaign to test the riding competency of all commercial motorcyclists and this could have encouraged a rush for fake riding licenses.Higher engine capacity has been associated with higher risk of injury among motorcyclists in a number of studies but this study has found different results. Examples of these studies are those by Langley et al in New Zealand , Cheng eThe association of road traffic injury with sharing motorcycles, not owning a motorcycle that one rides have also been established in several studies . The assAssociation of working long hours, working till late and starting very early morning with injuries has featured in other studies in different countries , 36–38. Significant association of injury with lower level of knowledge of road safety rules, higher AUDIT score and having been stopped by traffic police officers can be explained by information on other factors discussed. Higher AUDIT score imply alcohol consumption which is already associated with higher odds of injury. Being stopped by police may signify a problem with riding motorcycle like influence of alcohol or risk taking which may cause an injury. The police may also stop a rider if he demonstrates ignorance of road safety rules which also can lead to injury.The results of this study can be generalized to all major urban centers and other busy heavily populated areas in the country. Such places tend to have high concentration of commercial motorcyclists and are expected to have more riders injured on the road.A rider who has not had an injury at around the same time his colleague is in hospital may not be the best control because he may have had an injury days or weeks before or after. In such circum stances the control is supposed to be among the cases. Selecting a control as a person who has not had an injury for a specified length of time may be better but such benefit could be eroded by inaccuracies.Major factors independently associated with road traffic injuries among boda-boda riders include young age, alcohol consumption, sharing motorcycles, working late hours, being young, lower engine capacity (below 100cc), short riding experience (less than 3 years), not changing a motorcycle in a year, and having a riding permit. Other factors found important include lower knowledge of road traffic rules and responsibilities, having higher score of alcohol use disorder score (AUDIT)The research team makes a range of recommendations arising from the study results. Firstly there should be tougher controls for issuance of riding permits to young people aged less than 25 years. Secondly, current random drink-driving checks for motorists by traffic police should be extended to boda-boda riders. Investigators on this study are not aware of drink-riding checks carried out by the traffic police. Fourthly, consider screening for alcohol disorders among riders before they are hired or given permits.Other recommendations call for more effort for road safety sensitization programs for boda-boda riders, investigation of validity of riding permits and more research. Sensitization programs should raise more awareness against sharing motorcycles, working long hours, working at times when visibility is low, and alcohol consumption. Information from the police force shows that there is an existing sensitization program for boda boda riders. The forces do also partner with the NGOs to carry out sensitization of the riders but they(riders) are too many. The resS1 Datasethttps://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/RSOQ5E.The data set is in STATA V12 format and has the variables case/control, age group, alcohol consumption, engine capacity, having a driving permit, length of experience of riding motorcycle, changing motorcycle in previous year, hours driven in a day, sharing a motorcycle and matched case identification number. The data set has been uploaded in the public repository Harvard dataverse and its URL is (DTA)Click here for additional data file.


Significant net uncompensated magnetizationin BFO, an insulating superlattice, is demonstrated using polarized neutronreflectometry. Remarkably, the magnetization enables magnetic field to changethe dielectric properties of the superlattice, which we cite as an exampleof synthetic magnetoelectric coupling. Importantly, controlled creation ofmagnetic moment in BFO is a much needed path toward design and implementationof integrated oxide devices for next generation magnetoelectric data storageplatforms.Given the paucity of single phase multiferroic materials (with large ferromagneticmoment), composite systems seem an attractive solution to realize magnetoelectriccoupling between ferromagnetic and ferroelectric order parameters. Despitehaving antiferromagnetic order, BiFeO The ability to control magnetization, M, via electric fields or alternativelyelectric polarization, P, via magnetic fields enables a myriad of technologicalinnovations in information storage, sensing, and computing. For example, Oersted-fieldsthat are presently used to switch the magnetic state of commercial magnetictunnel junctions are spatially extended and require modest current to produce.These attributes limit the areal density of magnetic tunnel junctions. Becauseelectrostatic fields can be confined and require very little current to produce,integration of a multiferroic composite—a system of different constituentswith coupled M and P order parameters—into a magnetic tunnel junctionmight enable the “single memory solution”—non-volatile memorythat is more energy efficient, faster, higher capacity and more affordablethan competing technologies.3 (BFO) is a single phase multiferroic material which exhibitsmagnetoelectric coupling between antiferromagnetic4i.e., BFO lacks a “magnetichandle”. Calculations performed by Ederer and SpaldinB/Fe(~15 kA/m) in BFO—corresponding to a magnetization about twoorders smaller than that of a ferromagnet in a tunnel junction. The DM interactionis unlikely to be a useful magnetic handle.BiFeOVarious groups78910saturation magnetization with an electric field has proven elusive.To date the magnitude of exchange bias, |HE| has been smaller thanthe coercive field, Hc, of the FM, so the saturation magnetizationcannot be fully reversed at zero magnetic field. Because HE isproportional to the component of the sub-lattice magnetization parallel tothe FM magnetizationE|.The approach has met with some success; however, complete switching ofthe uncompensated magnetizationof BFO layers in a superlattice structure. The size of the uncompensated magnetizationsuggests that when a very thin BFO film is sandwiched between LSMO, BFO becomes ferri-magnetic.The temperature dependence of the ferrimagnetic order parameter in BFO isthe same as that of the ferromagnetic order parameter of LSMO, suggestingthat LSMO induces the uncompensated magnetization in BFO. In addition, wedemonstrate control over the dielectric constant of the superlattice with magneticfield. We also propose a means to extend our discovery above room temperature.Here, we propose a much different approach to realize a magnetoelectricmultiferroic. Namely, we demonstrate intimate coupling (though not exchangebias) between the magnetization of LSMO with the n/(BFO)m]Nsuperlattice on a (001) SrTiO3 (STO) substrate by pulsed laser(KrF) deposition, where n = 6, m = 5 unit cells and N = 8 is the number LSMO/BFObilayers. Evidence for chemically and structurally well-defined interfacesover lateral dimensions of tens of nm was obtained using high angle annulardark field (HAADF) Z-contrast microscopy [See 2+. Further,we found no evidence for structural phases other than LSMO, BFO and STO usingx-ray diffraction [See We grew a [(LSMO)copy See  online acopy See  online. tion See  online.0H, after cycling ±0.5 T. The hysteresisloop is shifted by −20 mT. The shift is three times larger thanthat observed by Wu et al.140H from ±7 T.The result is shown by the red symbols  and BFO (triangles) magnetizationsare shown in B/Fe at 10 K)is much larger than can be attributed to the DM interactionPolarized neutron reflectivity data of the superlattice were acquired atthe Spallation Neutron Source, Oak Ridge National Laboratory. Briefly, thereflectivity, R, was measured with two neutron beam polarizations—oneparallel (+) and one opposite (−) to the 0.5 T applied fieldas a function of wavevector transfer, Q, (the difference between the incidentand specularly reflected neutron wavevectors). R (Q)See  online w0H at a rate of 0.1%/T. In contrast, the capacitanceof the 20 nm thick BFO film is unchanged with field at 10 and 300 K.Both samples were insulating during the measurements [magneticfield.Whereas urements . Our con± (Q)  detected no neutronspin dependence of R (Q) See  online. 2+tends to make the film metallic, thus, compromising magnetoelectric couplinget al.2+, epitaxial strain or suppression oftilts, the electronic and magnetic structures and properties of BFO in thesuperlattice are likely different than those of the 20 nm thick BFOfilm or bulk BFO. We have explored computational models of BFO/LSMO heterostructureswith thinner layers of BFO and LSMO (1–3 unit cells) and tilt-free octahedrain BFO along the growth direction. Uncompensated magnetization on the BFO-sideof the BFO/LSMO interface was found using density functional theory.The origin of uncompensated magnetization in BFO films has been the subjectof some controversy1617162 vs. SrO, influences thesign of exchange coupling across the LSMO/BFO interface2 terminated LSMO layers are anti-ferromagneticallycoupled to spins in the adjacent BFO layer. Our results are consistent with anti-ferromagneticallycoupled spins across MnO2 terminated layers. Growth of LSMO filmson TiO2-terminated (001) STO substrates yields MnO2terminated LSMO layersTermination of the LSMO layer, MnOc = 179 ± 1 K for the two films are notsignificantly different. Suppression of Tc well below 350 Kis typical for LSMO films less than 30 nm thicknessonly the LSMO layers to achieve Tc~ 350 K—typical of bulk LSMO.)The temperature dependencies of the LSMO and BFO magnetic order parameterswere fitted to the usual form  curves, . ThevalUncompensated magnetization in the BFO component of the superlattice providesan opportunity for magnetic field to affect the electronic properties of BFO,provided the uncompensated magnetization is coupled to the sub-lattice magnetizationof BFO. The observed change of capacitance with field may be due to correlationbetween measurement of capacitance and field-induced-change of resistance,magnetostriction, or ideally magnetoelectric coupling.In order to determine the influence of a change of resistance on capacitance,we measured the capacitance of a test capacitor as a function of a variableresistor placed in parallel with the capacitor. We found a threefold increasein resistance produced a 0.3% increase in the measured capacitance of thecircuit. The magnitude of the change is smaller than the effect we see. Inaddition, the capacitance increased with resistance in our test, yet we observedthe resistance of the sample to decrease slightly with increasing B . Therefo−5 . The saturation magnetizationof the superlattice is achieved for fields of not more than 1 T, thus,magnetostrictive effects are at least two orders of magnitude too small toexplain the change of capacitance (induced by the change in distance betweenelectrodes).For strongly magnetic materials the magnetostriction at saturation is oforder 10ferri-magnetic ultra-thin BFO layers. An applied magneticfield aligns the net ferri-magnetic moment, which in turn establishesa preference for a subset of easy planes of the sub-lattice magnetization.Domains with electric polarization normal to the preferred easy planes ofthe sub-lattice magnetization grow at the expense of all other polarizationdomains. Thus, application of field alters the net electric polarization ofthe superlattice, which manifests itself as an increased dielectric constantin the capacitance value we measured. Previously, complete switching of saturationmagnetization with an electric field has proven elusive. Similar difficultiesmay be encountered in our reverse measurement—one changing the dielectricconstant with a magnetic field.We propose that magnetoelectric coupling intrinsic to BFO films and bulkBFO persists in c ~ 350 K comparableto bulk LSMO) while confining BFO layers to a thickness of 5 unit cells.In conclusion, we have developed a composite of two materials, neitherindependently exhibit dielectric response to magnetic field, but when fashionedinto a superlattice, the dielectric constant changes by 0.1%/Tesla. The superlatticeconsists of 8 repetitions of 6 unit cells of LSMO grown on 5 unit cells ofBFO. Below 179 K, LSMO is ferromagnetic and BFO exhibits net uncompensatedmagnetization with the magnetization of BFO opposite to that of the LSMO.The magnetic order parameters have the same dependence with temperature, suggestingan intimate relationship. While uncompensated magnetization in the BFO layersis intimately linked to the LSMO magnetization, its detailed origin is unknown.Nevertheless, we have discovered a new means to produce synthetic magnetoelectriccoupling in a nanocomposite at 10 K. Synthetic magnetoelectric couplingmight be possible at room temperature in a superlattice consisting of ~75unit cell thick LSMO layers (to achieve TP.J. performed the capacitance measurements, Q.W., A.G., V.L. and M.R.F.performed the neutron measurements and analysis, M.R. and M.V. performed electronmicroscopy, C.U. performed magnetometry, Z.B. and Q.X.J. made the samples,T.A. and J.Z. contributed theoretical insights. P.J. and M.R.F. wrote themanuscript with contributions from all authors.


Drosophila melanogaster. The transgenic construct, Munin, expresses the yeast transcription factor Gal4, tethered to a transmembrane anchor via a linker that can be cleaved by a viral protease. In infected cells, liberated Gal4 will then transcribe any gene that is linked to a promoter with a UAS motif, the target for Gal4 transcription. For instance, infected cells will glow red in the offspring of a cross between the Munin stock and flies with a UAS-RFPnls transgene (expressing a red fluorescent protein). In such flies we show that after natural infection, via the faecal-oral route, 5–15% of the midgut cells are infected, but there is little if any infection elsewhere. By contrast, we can detect infection in many other tissues after injection of virus into the body cavity. The same principle could be applied for other viruses and it could also be used to express or suppress any gene of interest in infected cells.We have created a transgenic reporter for virus infection, and used it to study Nora virus infection in  Drosophila melanogaster2 up to 1010 viral genomes per fly2Nora virus is a currently unclassified picorna-like virus that naturally infects the fruit fly Virus detection is crucial in all virus research and several methods have been used for this purpose. Viruses can be detected by their specific pathogenesis in human and animal models, changes in morphology and viability of cultured cells, detection of viral nucleic acid or use of antibodies and other molecular markers456789Drosophila melanogaster, for example the popular Gal4-UAS systemSaccharomyces cerevisiae, which acts on a yeast-specific promoter motif, the upstream activating sequence (UAS). Tens of thousands of such “driver” fly stocks are available for the Gal4-UAS expression system, which in a combinatorial way allow expression of virtually any gene in any tissue. A common application is to express an RNA hairpin that feeds into the fruit fly’s RNA interference (RNAi) pathway, in order to suppress the expression of an endogenous Drosophila gene.Several techniques are developed to modify gene expression in Drosophila driver stock with constitutive and ubiquitous expression of a target to the viral protease. The target is the Gal4 protein fused to a transmembrane anchor, via a linker that has a cleavage site for the virus-encoded protease , under the control of the Munin reporter. To test the Munin reporter we established a persistent Nora virus infection in the fly stock. The flies were thus environmentally exposed to the virus via faeces deposited in the fly vial and they ingested the virus with the food. We dissected adult flies one week after eclosion and examined their abdominal organs for RFPnls expression by fluorescence microscopy. Our previous results suggested that Nora virus is an enteric virusnls expression varies considerably between the infected cells  or enteroendocrine cells (about 10%). Enterocytes are morphologically distinguishable from the other cell types by their big nuclei12nls in cells with a big nucleus, indicating that Nora virus has tropism for enterocytes  VK00013 flies, which have a predestined landing site at 3L:19197458We constructed the Munin reporter gene by inserting the first 216 bp (encoding the first two transmembrane helices) of the w1118; P{w[+mC] = UAS-RedStinger}4/CyO stocknls in this work. The flies were reared on standard mashed potato-agar fly foodWolbachia infections.A Drosophila Nora virus strain Umea 2007 was derived from an infectious cDNA clonew; +; RelishE23 fliesw; +; RelishE23 flies in 0.5 ml PBS and filtering the suspension through a 0.45 μm filter. Using an Inject + matic microinjector , we injected approximately 100 nl of the virus suspension into the thorax of each fly via the thin cuticle under the wing hinge. Infected this way, the flies show an 100–1000 fold increase of Nora virus RNA within ten days after injectionFlies were infected by environmental exposure to the virus via contamination of the food by the faeces of their virus-infected parents, or by microinjection of a virus-containing suspension into the blood. For the latter, Nora virus suspension was prepared by homogenising approximately 100 infected Drosophila C virus strain of uncertain origin that has been kept in our lab. The region around the protease cleavage site was sequenced (see Drosophila C virus strain EB (accession AF014388). The virus was propagated in Drosophila S2 cells until 50% of the cells had lysed. The suspension was freeze-thawed three times to completely lyse the cells, diluted 105 times in phosphate-buffered saline, pH 7.5 , and filtered through a 0.22 μm filter before injection into flies as described above.We used a nced see  and founNora virus titres were determined by one-step quantitative RT-PCR as described2Dissected organs were fixed in 3.5% paraformaldehyde for 10 min at room temperature, permeabilised with 0.1% Triton X-100 for 10 min, incubated in CytoPainter Phalloidin iFlour 488 (Abcam) for 30 min, and mounted in ProLong Gold antifade reagent with DAPI (Life Technologies). Images were acquired in Zeiss ApoTome.2 and Zeiss Lumar.V12 microscopes, and the contrast was enhanced with the Adobe Photoshop software. Images of control and infected animals were always processed the same way. Contrast was equally enhanced in In the Norse mythology, Hugin and Munin is a pair of ravens that fly over the worlds every day. They see everything that happens and they bring all news to the god Odin, who is thereby kept informedHow to cite this article: Ekström, J.-O. and Hultmark, D. A Novel Strategy for Live Detection of Viral Infection in Drosophila melanogaster. Sci. Rep.6, 26250; doi: 10.1038/srep26250 (2016).


Campylobacter fetus subsp. venerealis and its glycerine-tolerant variant Campylobacter fetus subsp. venerealis biovars intermedius. The disease can be economically important when present in cattle herds, causing poor reproductive performance, embryo mortality and abortion. Sensitive and specific diagnostic tests are required in the diagnosis of infection and to inform and monitor disease control. Current tests include bacterial culture and fluorescent antibody testing of preputial sheath washings and an enzyme-linked immunosorbent assay and an agglutination test on vaginal mucus, although the predictive values of these tests can be inadequate in field investigations.Bovine venereal campylobacteriosis is caused by Artificial insemination is often considered as a simple control method for bovine venereal campylobacteriosis, but is impractical for many beef suckler herds where breeding takes place at pasture. Commercial vaccines are unavailable in the UK, while the efficacy of autogenous vaccines using a bacterial isolate from infected animals on a specific farm is at best unproven. Hence, for some infected herds, the development of an alternative control strategy based on segregation of potentially infected and uninfected animals in combination with culling or treatment would be desirable. This approach requires meticulous records and herd health management.In this paper we highlight difficulties in diagnosing bovine venereal campylobacteriosis and demonstrate the benefits of good record keeping when investigating poor reproductive performance in a beef suckler herd and establishing a herd-specific approach to bio-containment of the infectious cause.Bovine venereal campylobacteriosis is an economically important disease that should be considered in investigations of suckler herd subfertility problems. Control of the disease based on segregation of potentially infected and uninfected animals in combination with extensive culling can be achieved without the use of artificial insemination or vaccination, but requires meticulous records and strict adherence to herd biosecurity practices. Campylobacter contains two important pathogens of animals affecting mainly the reproductive and gastrointestinal tracts [Campylobacter fetus subsp. venerealis (Cfv) which has been isolated from the reproductive tract of cattle and internal organs of aborted foetuses [Campylobacter fetus subsp. fetus (Cff) is transmitted orally and colonises the intestines of cattle and sheep, inducing enteritis and abortion mostly in sheep and sporadically in cattle [Campylobacter fetus subsp. venerealis biovars intermedius (Cfvi) [The genus l tracts . Bovine foetuses -3. Campyn cattle ,3. Both s (Cfvi) ,5.Bovine venereal campylobacteriosis is transmitted mainly by natural service, but infection may also be spread during artificial insemination (AI) using semen from infected bulls or through contaminated equipment . Direct Campylobacter fetus infection [Campylobacter spp. survival rate [The first steps in the investigation of a potential bovine venereal campylobacteriosis problem are to review the herd reproductive history, to conduct a biosecurity audit and to establish the presence or absence of associated clinical signs. Fluorescent antibody tests (FATs) are commonly used for antigen detection in preputial washings and have a reported sensitivity and specificity of 92.6% and 88.9% respectively . An enzynfection ,14. Prepval rate ,15. Moleval rate ,17.C. fetus have been reported [There is anecdotal evidence of spontaneous recovery from bovine venereal campylobacteriosis occurring in young bulls. Successful local and systemic antibiotic therapy has been reported in bulls less than 3 years old, while culling of older bulls is usually recommended ,9. Strepreported ,19. Treareported .C. fetus has been detected in vaginal washings from cows more than 1 year after infection [Potentially infected breeding bulls are frequently culled following the confirmation of bovine venereal campylobacteriosis in beef suckler herds, accompanied by a temporary switch from natural mating to AI. nfection , hence nnfection . These hnfection  but vaccnfection .In September 2012 high barren rates prompted an investigation into the cause of the subfertility in a beef suckler herd in south-east Scotland. The herd of predominantly spring calving cows, mostly commercial crossbreeds and some pedigree Angus, used natural service with Charolais, Simmental and Angus bulls. A small number of pedigree Angus cows were mated during the winter to calve in the following autumn. Store calves were finished off-site and the farm also sold breeding bulls.st PD) and those females that were found non-pregnant at 1st PD were rescanned 6 weeks after removal of the second bull (2nd PD). In total 22% of heifers and cows were found barren, a figure noticeably higher than in previous years. The pregnancy and barren rates for the different groups that prompted the disease investigation are shown in Table During the summer of 2012, 169 breeding females split into 6 groups (A – F) had been mated to 8 different bulls. The mating period for both heifers and cows was 9 weeks and bulls had been rotated after 5 weeks. Pregnancy diagnosis (PD) using trans-rectal ultrasonography was carried out 32 days after the first bull was removed .The farm boundaries were stock-proof and the herd was accredited free from bovine viral diarrhoea (BVD) and vaccinated against c to identify persistent infection with BVD and presence of Johne’s disease antibodies. Two of the bought-in heifers were found to be persistently infected with BVD and culled. All bought-in heifers tested seronegative for antibodies against Johne’s disease.The herd had bred its own replacement females for a number of years until the owner decided to purchase 17 replacement heifers at market in April 2012. These females had been placed in quarantine on arrival and tested by the local veterinary diagnostic laboratoryDuring previous years the herd also acquired a number of breeding bulls, each of which had been young virgin animals of equal or higher health status.Data were collected for the body condition scores of all breeding cattle commencing in May 2012, before and at the start of the mating period.Trans-rectal ultrasonography was used to examine the reproductive tracts of each of the non-pregnant females.d [All 8 stock bulls had been submitted to a two-stage pre-breeding evaluation involving physical examination and examination of semen collected via electro-ejaculation using a Lane Pulsator IV Electronic Ejaculatord , 6 weeksc for Campylobacter culture within two hours of collection. At this point bovine venereal trichomonosis was not considered as a likely differential diagnosis since the disease had not been recognised in Britain for many years.In October 2012, Bull 3, Bull 8 and 6 non-pregnant heifers from Group C (3 homebred and 3 purchased in April 2012) were presented for further investigation into the possibility of infectious infertility. Blood samples taken from these 8 animals were submitted for infectious bovine rhinotracheitis (IBR) serology. To assess whether bovine venereal campylobacteriosis was present in the herd, single preputial and vaginal washings were collected from the 2 bulls and 6 heifers, respectively using 30 to 50 ml of warmed saline and disposable sterile syringes, polythene tubing and sample bottles. The samples were labelled with the animals’ identities and submitted to the local Disease Surveillance Centrest or 2nd PD which gave an indication of which bulls they had conceived to, if any.In depth analysis of farm records, in particular from those groups with poorest pregnancy results, was carried out. These records consisted of hand written documents kept up-to-date by the farm manager and provided information on the management tag numbers of all females allocated to groups A to F and the bulls that were used in these respective groups including the dates they were moved in and out. They also showed whether or not the females were found pregnant at 1There was no evidence of vitamin, mineral or trace-element deficiencies and while the summer of 2012 was exceptionally wet and cold resulting in poaching and poor grass growth, the heifers were well grown and adult cattle were consistently in good target body condition score of 3/5 – 3.5/5 throughout the investigation.The majority of the non-pregnant females’ ovaries showed active follicular waves and/or corpora lutea on trans-rectal ultrasonographic examination and there was no evidence of widespread anoestrus or endometritis.The findings of all breeding soundness evaluations performed on the bulls in this herd, either as part of routine herd management procedures or as part of this investigation, were satisfactory.Serological testing for IBR revealed that all bulls and homebred females were positive, whereas the bought-in replacements tested negative indicating that it was unlikely that the virus circulated in Group C prior to the poor reproductive performance problem arising.Campylobacter spp were detected on culture of prepuce and vaginal washings.No In group C all females that were pregnant had conceived to Bull 3 during the first 5 weeks of the mating season. None were found pregnant to Bull 8, a young homebred virgin sire. In Group E, with the exception of one cow, again all females that were found pregnant conceived during the first 5 weeks of the mating season .Heifers and cows from groups C, E and the pedigree Angus heifers that maintained pregnancy to term were kept separate from the other breeding cattle in the herd and re-bred the following mating season, with homebred virgin bulls which were slaughtered at the end of mating.Bulls 3 and 8 were culled.g was used for the sheath lavage. The suspension was inserted into the preputial orifice and massaged along the preputial cavity for one minute. This was repeated every 10 minutes for 1 hour. A streptomycin solutionh was administered via intramuscular injection at a dose rate of 10 mg active ingredient per kg bodyweight.Bull 5 was treated using a combination of local and systemic therapy for 3 consecutive days. A warmed penicillin and streptomycin solutionf for Campylobacter spp culture and FAT. Preputial washings were collected at 30 and 33 days after the last day of treatment. A third washing was collected 30 days after the last previous test.The success of treatment was monitored using preputial washings into TEM and submission of samples to the diagnostic laboratoryThe success of treatment and management interventions was monitored by assessing pregnancy and barren rates following consecutive mating period(s).The results of the disease investigation enabled the formulation of a novel disease management strategy:Campylobacter spp. culture and FAT results, obtained from preputial washings collected on 3 occasions post-treatment. Furthermore, Bull 5 was used to mate virgin heifers only for 9 weeks during the 2013 summer mating period and achieved a pregnancy rate of 93%. A total of 194 females were mated for 9 weeks during the summer of 2013 of which 177 (91%) were found pregnant and 17 (9%) barren. This is similar to pregnancy and barren rates achieved in the herd in the years preceding the disease outbreak described here.Successful treatment of Bull 5 was demonstrated by consistently negative This study shows that the introduction of Cfvi to a beef suckler herd with a group of purchased heifers resulted in 36 of 72 (50%) heifers and cows in 3 groups being barren before the diagnosis was confirmed and preventive management instigated, compared to a target barren rate for this system of 6% . ProfitaC. fetus FAT results during the follow-up investigation. Routine culturing procedures for Cfv appear to have poor sensitivity and consequently the disease could have been missed in the initial investigation. The problems associated with the diagnosis of bovine venereal campylobacteriosis based on the isolation of the organism are complex and include reduced viability of the organism under normal atmospheric conditions and rapid overgrowth of more vigorously multiplying contaminating organisms [Bacterial culture of preputial and vaginal washings was unhelpful in the initial investigation described, and only confirmed the presence of Cfvi in one of three bulls with positive rganisms . Furtherrganisms . There arganisms . A recomrganisms , althougWhile there was evidence of seroconversion against IBR in this herd and the presence of this virus can manifest itself as an increased barren cow rate , it was While bovine venereal campylobacteriosis can be transmitted through AI, in the European Union all licensed bulls used for AI purposes are tested and disease-free . CullingThis novel approach to the control of bovine venereal campylobacteriosis in a beef suckler herd involved culling of heifers and cows that were scanned barren and total segregation of potentially infected and Cfvi-free animals. Barren females were culled in an attempt to reduce the prevalence of the disease and remove those that could be barren due to chronic damage to their reproductive tract. Additionally culling barren heifers and cows and replacing them with bulling heifers is considered more economical than keeping unproductive females until the next mating season. Two of the infected bulls were culled, while a particularly valuable bull was treated and then tested to demonstrate elimination of Cfvi from the prepuce. In this case, the approach proved to be more practical and considerably less expensive than the alternative of adopting an indefinite whole herd AI programme. The success of this approach required meticulous records and strict adherence to herd biosecurity practices.In theory the purchase of virgin heifers and young bulls should offer little or no risk of introducing bovine venereal campylobacteriosis. Bulls 3, 5 and 8 were all homebred and either virgin or had achieved good pregnancy results during previous mating seasons. However, investigation of the ages of some of the purchased heifers in this case showed that they may have been old enough to have been mated and found barren prior to being sold. While this is unproven, it nevertheless highlights the potential risks and responsibilities for both vendors and purchasers of breeding heifers. Whilst 2 of the bought-in heifers were found to be persistently infected with BVD, the strict biosecurity measures adopted in this herd, including isolation and testing of introduced animals, were effective at avoiding the major financial losses that could have resulted from introducing this disease. From January 2014 Scottish legislation specifies that an animal identified positive for BVD virus on the most recent test will be assumed persistently infected and is not allowed to move other than directly to slaughter.This investigation clearly shows the value of accurate record-keeping on farm including the length of the breeding season, bull-to-cow details and pregnancy rates, in addition to information from bull breeding soundness evaluations, bull power, herd health and vaccination status, biosecurity policies and nutritional management. It demonstrates that record-keeping can be particularly important when investigating poor reproductive performance and in the bio-containment of infectious disease.aLeptavoid-H, MSD Animal Health, UK.bBovidec, Novartis, UK.cSAC Consulting, Veterinary Services, Allan Watt Building, Bush Estate, Penicuik, EH26 0QE, UK.dLane Manufacturing, Colorado, USA.eCampyculture RAI 0800.fAnimal Health and Veterinary Laboratories Agency, Starcross, UK.gPen & Strep Suspension for Injection, 200 mg/ml Procain Penicillin, 250 mg/ml Dihydrostreptomycin Sulphate in aqueous suspension, Norbrook, UK.hDevomycin D Solution for Injection, 150 mg/ml Streptomycin Sulphate, 150 mg/ml Dihydrostreptomycin Sulphate in aqueous suspension, Norbrook, UK.