Using single-worm RNA sequencing to study C. elegans responses to pathogen infection.

BACKGROUND: Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. RESULTS: In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. CONCLUSIONS: We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations.

Imaging of Lung Cancer Staging.

Lung cancer is a leading cause of cancer-related mortality worldwide. Imaging is integral in accurate clinical staging to stratify patients into groups to predict survival and determine treatment. The eighth edition of the tumor, node, and metastasis (TNM-8) staging system proposed by the International Association for the Study of Lung Cancer in 2016, accepted by both the Union for International Cancer Control and the American Joint Committee on Cancer, is the current standard method of staging lung cancer. This single TNM staging is used for all histologic subtypes of lung cancer, including nonsmall cell lung cancer, small cell lung cancer, and bronchopulmonary carcinoid tumor, and it addresses both clinical and pathologic staging. Familiarity with the strengths and limitations of imaging modalities used in staging, the nuances of TNM-8, its correct nomenclature, and potential pitfalls are important to optimize patient care. In this article, we discuss the role of computed tomography (CT) and positron emission tomography/CT in lung cancer staging, as well as current imaging recommendations pertaining to TNM-8.

High time resolution measurements of droplet evaporation kinetics and particle crystallisation.

A refined technique for observing the complete evaporation behaviour of free-falling droplets, from droplet generation to complete solvent evaporation, with ultra-high time resolution is introduced and benchmarked. High-resolution phase-delay stroboscopic imaging is employed to simultaneously resolve the evolving droplet morphology, geometric and aerodynamic diameters, throughout the evaporative lifetime with a user-controlled < µs timescale. This allows rapid, complex morphological changes, such as crystallisation events, to be clearly observed and the corresponding mechanisms to be inferred. The dried particles are sampled for offline SEM analysis and the observed morphologies compared to the inflight imaging. Density changes can be calculated directly from the deviation between the geometric and aerodynamic diameters. The full capabilities of the new technique are demonstrated by examination of the different evaporation behaviours and crystallisation mechanisms for aqueous sodium chloride droplets evaporating under different ambient relative humidity (RH) conditions. The crystallisation window, defined as the time taken from initial to complete crystallisation, is shown to be RH dependent, extending from 0.03 s at 20% RH and 0.13 s at 40% RH. The different crystallisation mechanisms observed during the experiments are also clearly reflected in the final structure of the dry particles, with multi-crystal structures produced at low RH compared to single-crystal structures at higher RH. It is anticipated that this technique will unlock measurements which explore the evaporation behaviour and crystallisation mechanisms for rapid, complex droplet drying events, and with increasingly non-ideal solutions, relevant to industrial applications.

Inactivation of Bacillus anthracis and Bacillus atrophaeus spores on different surfaces with ultraviolet light produced with a low-pressure mercury vapor lamp or light emitting diodes.

AIMS: To obtain quantitative efficacy data of two ultraviolet light (UVC) technologies for surface inactivation of Bacillus anthracis Ames and Bacillus atrophaeus spores. METHODS AND RESULTS: Spores were deposited onto test coupons and controls of four different materials, via liquid suspension or aerosol deposition. The test coupons were then exposed to UVC light from either a low-pressure mercury vapor lamp or a system comprised of light emitting diodes, with a range of dosages. Positive controls were held at ambient conditions and not exposed to UVC light. Following exposure to UVC, spores were recovered from the coupons and efficacy was quantified in terms of log10 reduction (LR) in the number of viable spores compared to that from positive controls. CONCLUSIONS: Decontamination efficacy varied by material and UVC dosage (efficacy up to 5·7 LR was demonstrated). There was no statistical difference in efficacy between the two species or between inoculation methods. Efficacy improved for the LED lamp at lower relative humidity, but this effect was not observed with the mercury vapor lamp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study will be useful in determining whether UVC could be used for the inactivation of B. anthracis spores on different surface types.

Is testosterone linked to human aggression? A meta-analytic examination of the relationship between baseline, dynamic, and manipulated testosterone on human aggression.

Testosterone is often considered a critical regulator of aggressive behaviour. There is castration/replacement evidence that testosterone indeed drives aggression in some species, but causal evidence in humans is generally lacking and/or-for the few studies that have pharmacologically manipulated testosterone concentrations-inconsistent. More often researchers have examined differences in baseline testosterone concentrations between groups known to differ in aggressiveness (e.g., violent vs non-violent criminals) or within a given sample using a correlational approach. Nevertheless, testosterone is not static but instead fluctuates in response to cues of challenge in the environment, and these challenge-induced fluctuations may more strongly regulate situation-specific aggressive behaviour. Here, we quantitatively summarize literature from all three approaches (baseline, change, and manipulation), providing the most comprehensive meta-analysis of these testosterone-aggression associations/effects in humans to date. Baseline testosterone shared a weak but significant association with aggression (r = 0.054, 95% CIs [0.028, 0.080]), an effect that was stronger and significant in men (r = 0.071, 95% CIs [0.041, 0.101]), but not women (r = 0.002, 95% CIs [-0.041, 0.044]). Changes in T were positively correlated with aggression (r = 0.108, 95% CIs [0.041, 0.174]), an effect that was also stronger and significant in men (r = 0.162, 95% CIs [0.076, 0.246]), but not women (r = 0.010, 95% CIs [-0.090, 0.109]). The causal effects of testosterone on human aggression were weaker yet, and not statistically significant (r = 0.046, 95% CIs [-0.015, 0.108]). We discuss the multiple moderators identified here (e.g., offender status of samples, sex) and elsewhere that may explain these generally weak effects. We also offer suggestions regarding methodology and sample sizes to best capture these associations in future work.

Formation of Highly Ordered Spherical Aggregates from Drying Microdroplets of Colloidal Suspension.

The formation of highly ordered spherical aggregates of silica nanoparticles by the evaporation of single droplets of an aqueous colloidal suspension levitated (confined) in the electrodynamic quadrupole trap is reported. The transient and final structures formed during droplet evaporation have been deposited on a silicon substrate and then studied with SEM. Various successive stages of the evaporation-driven aggregation of nanoparticles have been identified: formation of the surface layer of nanoparticles, formation of the highly ordered spherical structure, collapse of the spherical surface layer leading to the formation of densely packed spherical aggregates, and rearrangement of the aggregate into the final structure of a stable 3D quasi-crystal. The evaporation-driven aggregation of submicrometer particles in spherical symmetry leads to sizes and morphologies of the transient and final structures significantly different than in the case of aggregation on a substrate. The numerical model presented in the article allows us to predict and visualize the observed aggregation stages and their dynamics and the final aggregates observed with SEM.

B cell lymphoma and myeloma in murine Gaucher's disease.

Multiple myeloma and B cell lymphoma are leading causes of death in Gaucher's disease but the nature of the stimulus driving the often noted clonal expansion of immunoglobulin-secreting B cells and cognate lymphoid malignancy is unknown. We investigated the long-term development of B cell malignancies in an authentic model of non-neuronopathic Gaucher's disease in mice: selective deficiency of ß-glucocerebrosidase in haematopoietic cells [Gba(tm1Karl/tm1Karl)Tg(Mx1-cre)1Cgn/0, with excision of exons 9-11 of the murine GBA1 gene, is induced by poly[I:C]. Mice with Gaucher's disease showed visceral storage of ß-glucosylceramide and greatly elevated plasma ß-glucosylsphingosine [median 57.9 (range 19.8-159) nm; n = 39] compared with control mice from the same strain [median 0.56 (range 0.04-1.38) nm; n = 29] (p < 0.0001). Sporadic fatal B cell lymphomas developed in 11 of 21 GD mice (6-24 months) but only two of eight control animals developed tumours by age 24 months. Unexpectedly, most mice with overt lymphoma had absent or few Gaucher cells but local inflammatory macrophages were present. Eleven of 39 of Gaucher mice developed monoclonal gammopathy, but in the control group only one animal of 25 had clonal immunoglobulin abnormalities. Seven of 10 of the B cell lymphomas were found to secrete a monoclonal paraprotein and the lymphomas stained intensely for pan-B cell markers; reactive T lymphocytes were also present in tumour tissue. In the Gaucher mouse strain, it was notable that, as in patients with this disease, CD138(+) plasma cells frequently surrounded splenic macrophages engorged with glycosphingolipid. Our strain of mice, with inducible deficiency of ß-glucocerebrosidase in haematopoietic cells and a high frequency of sporadic lethal B cell malignancies, faithfully recapitulates human Gaucher's disease: it serves as a tractable model to investigate the putative role of bioactive sphingolipids in the control of B cell proliferation and the pathogenesis of myelomatosis-the most prevalent human cancer associated with this disorder.

A first report of Pseudosuccinea columella (Say, 1817), an alien intermediate host for liver fluke, in Malawi.

Starting in October 2021, quarterly malacological surveys have been undertaken in Malawi, with the sampling of 12 specified freshwater habitats throughout a calendar year. Each survey monitors the presence of aquatic intermediate snail hosts of medical and veterinary importance. In March 2023, the alien lymnaeid species Pseudosuccinea columella was encountered for the first time in the surveys, in Nsanje District. This species identity was later confirmed upon DNA analysis of mitochondrial ribosomal 16S sequences. In July 2023, P. columella was also noted at single sites within Mangochi and Chikwawa Districts, and again in Nsanje District, with an additional location observed. Of particular importance, our sampled location in Mangochi District was directly connected to Lake Malawi, which expands the species list of invasive molluscs in this lake. While P. columella is a well-known intermediate snail host for human and animal fascioliasis, screening collected snails for trematode cercariae, alongside molecular xenomonitoring, did not yield equivocal evidence of active fluke infection. However, the newly recognized presence of this alien intermediate snail host within Lake Malawi, and along the Shire River Valley, flags a new concern in altered local transmission potential for human and animal fascioliasis.

The unseen cloud: a survey of vaping practices and the acquisition of vaping products within the UK.

BACKGROUND: Vaping of cannabinoid-based products and informal acquisition of vaping products were associated with the outbreak of E-cigarette or vaping associated lung injury (EVALI) in the USA. Current prevalence of cannabinoid-based vaping within the UK is not known and literature regarding the acquisition of vaping products is limited. AIM: To estimate the prevalence of nicotine-based, nicotine-free and cannabinoid-based product vaping within the UK and to determine where vaping products are acquired. DESIGN AND METHODS: A voluntary online survey of individuals aged 16 and over within the UK was conducted using a convenience sample. Data were collected on respondent demographics, smoking/vaping history and acquisition of e-liquids/products. RESULTS: A total of 2478 responses were included. Median age 45 years (interquartile range 35-57). Prevalence of current vaping of nicotine-based e-liquids, nicotine-free e-liquids and cannabinoid-based products was 14.4%, 11.2% and 5.49%, respectively. Current nicotine-based and nicotine-free vaping was most prevalent in 25-34 years olds (22.4% and 19.2% of respondents). Current cannabinoid-based vaping was most prevalent in 16-24 years olds. The most common 'ever' used cannabinoid-based products were cannabidiol oil/cannabigerol oil and cannabis leaves (4.8%). Specialist vaping stores were the most common source of 'ever' acquisition for all products. 36.8% and 40.5% of respondents who had ever vaped nicotine-based and nicotine-free e-liquids reported prior acquisition from informal sources. CONCLUSION: This survey reported a higher prevalence of current cannabinoid-based vaping within the UK (5.5%) than previously reported in the USA (2.0%). In addition to the informal acquisition of vaping products as demonstrated within the survey, these results highlight potential underestimation of the risk of EVALI within the UK.

The Emerging Cloud: a survey of vapers, their health and utilization of healthcare within the UK.

BACKGROUND: Recent work in the UK estimated the prevalence of current cannabinoid-based vaping to be higher than in the USA, a factor previously associated with e-cigarette or vaping-associated lung injury (EVALI). Research in the USA has demonstrated that attendances to emergency departments relating to e-cigarettes began to rise before the EVALI outbreak, suggesting that vapers also experience milder forms of vaping-related illness. AIM: Quantify symptom prevalence and healthcare utilization amongst current UK vapers. DESIGN: Voluntary online survey of individuals aged 16 and over within the UK. METHODS: Anonymized data were collected on demographics, vaping/smoking status and vaping substances used. Current vapers were asked about the presence of 10 prevalent symptoms from previous US EVALI case series, healthcare attendances and diagnoses given. Risk-ratios were calculated to compare the likelihood of symptoms and attendances between substances. RESULTS: A total of 2477 complete responses were analysed. In all, 397 respondents were current vapers. Symptom prevalence within the previous 12 months ranged from 3.8% to 30.5% (bloody sputum, cough). Healthcare attendances per symptomatic respondent ranged from 0.1 to 1.4 (bloody sputum, shortness of breath). Current vapers of cannabinoid-based products (alone/in combination) had the most attendances per symptomatic respondent for 9/10 symptoms and were more likely to report symptoms aside from 'cough' (nicotine-free e-liquids [risk ratio = 1.7]). Clinicians reportedly never diagnosed vaping-related illness. CONCLUSIONS: UK vapers experience symptoms previously reported in EVALI cases for which they also seek healthcare. Users of cannabinoid-based products were more likely to report symptoms and accounted for a higher healthcare burden. UK vapers may also experience vaping-related illness that does not meet EVALI case criteria.

Isotonitazene, a novel psychoactive substance opioid, detected in two cases following a local surge in opioid overdoses.

BACKGROUND: Isotonitazene is a novel opioid that was first reported in Europe in 2019. There have been no reports of the detection of isotonitazene in patients presenting to the emergency department with acute drug toxicity. AIM: There was an increase in presentations to our emergency department with acute opioid toxicity in August 2021. We aim to describe this outbreak and provide detail on two cases in which isotonitazene was quantified in serum samples. METHODS: Serum samples were available for comprehensive toxicological analysis for two presentations. Written consent was obtained and the samples were analysed through a Thermo XRS ultrahigh-performance liquid chromatography system, interfaced to a Thermo Q Exactive high-resolution accurate mass spectrometer, operating in heated positive ion electrospray mode. Acquired data were processed using Toxfinder software (Thermo) against a regularly updated in-house database. RESULTS: There was an increase in acute opioid presentations to our emergency department from a median of 10 per month to 36 in August 2021. Twenty were treated with naloxone, and 23 were admitted to the hospital for observation and treatment. Serum sample analysis from two patients with acute opioid toxicity responsive to naloxone detected the presence of isotonitazene (0.18 and 0.81 ng/ml). CONCLUSION: We report a cluster of acute opioid toxicity presentations to our Emergency Department with detection of isotonitazene in two cases. Analytical screening is important in determining the presence of novel psychoactive substances (NPS) and to help inform the public health of the implications of NPS use, particularly during clusters of acute recreational drug toxicity presentations.

Emergency radiology II--Head trauma.

This is the second of three self-assessment articles which aim to further readers' understanding of diagnostic imaging, and its integral role in the management of patients with emergency conditions, through a series of case studies.

Emergency radiology III--Chest and limb trauma.

This is the final of three self-assessment articles which aims to further readers' understanding of diagnostic imaging, and its integral role in the management of patients with emergency conditions, through a series of case studies.

Self-assessment exercises in Emergency Radiology I -- abdominal trauma.

This is the first of three self-assessment articles which aim to further readers' understanding of diagnostic imaging and its integral role in the management of patients with emergency conditions, through a series of case studies.

Pervasive translation in Mycobacterium tuberculosis.

Most bacterial ORFs are identified by automated prediction algorithms. However, these algorithms often fail to identify ORFs lacking canonical features such as a length of >50 codons or the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to identify actively translated ORFs in Mycobacterium tuberculosis. Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. The newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. Codon usage of the newly discovered ORFs suggests that most have not been subject to purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify 90 new ORFs (median length of 52 codons) that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs in Mycobacterium tuberculosis serves as a rich source for the evolution of new functional proteins.

How can you predict which proteins an organism can make? To answer this question, scientists often use computer programs that can scan the genetic information of a species for open reading frames ­ a type of DNA sequence that codes for a protein. However, very short genes and overlapping genes are often missed through these searches. Mycobacteria are a group of bacteria that includes the species Mycobacterium tuberculosis, which causes tuberculosis. Previous work has predicted several thousand open reading frames for M. tuberculosis, but Smith et al. decided to use a different approach to determine whether there could be more. They focused on ribosomes, the cellular structures that assemble a specific protein by reading the instructions provided by the corresponding gene. Examining the sections of genetic code that ribosomes were processing in M. tuberculosis uncovered hundreds of new open reading frames, most of which carried the instructions to make very short proteins. A closer look suggested that only 90 of these proteins were likely to have a useful role in the life of the bacteria, which could open new doors in tuberculosis research. The rest of the sequences showed no evidence of having evolved a useful job, yet they were still manufactured by the mycobacteria. This pervasive production could play a role in helping the bacteria adapt to quickly changing environments by evolving new, functional proteins.

Children's Oncology Group Trial AALL1231: A Phase III Clinical Trial Testing Bortezomib in Newly Diagnosed T-Cell Acute Lymphoblastic Leukemia and Lymphoma.

PURPOSE: To improve the outcomes of patients with T-cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LL), the proteasome inhibitor bortezomib was examined in the Children's Oncology Group phase III clinical trial AALL1231, which also attempted to reduce the use of prophylactic cranial radiation (CRT) in newly diagnosed T-ALL. PATIENTS AND METHODS: Children and young adults with T-ALL/T-LL were randomly assigned to a modified augmented Berlin-Frankfurt-Münster chemotherapy regimen with/without bortezomib during induction and delayed intensification. Multiple modifications were made to the augmented Berlin-Frankfurt-Münster backbone used in the predecessor trial, AALL0434, including using dexamethasone instead of prednisone and adding two extra doses of pegaspargase in an attempt to eliminate CRT in most patients. RESULTS: AALL1231 accrued 824 eligible and evaluable patients from 2014 to 2017. The 4-year event-free survival (EFS) and overall survival (OS) for arm A (no bortezomib) versus arm B (bortezomib) were 80.1% ± 2.3% versus 83.8% ± 2.1% (EFS, P = .131) and 85.7% ± 2.0% versus 88.3% ± 1.8% (OS, P = .085). Patients with T-LL had improved EFS and OS with bortezomib: 4-year EFS (76.5% ± 5.1% v 86.4% ± 4.0%; P = .041); and 4-year OS (78.3% ± 4.9% v 89.5% ± 3.6%; P = .009). No excess toxicity was seen with bortezomib. In AALL0434, 90.8% of patients with T-ALL received CRT. In AALL1231, 9.5% of patients were scheduled to receive CRT. Evaluation of comparable AALL0434 patients who received CRT and AALL1231 patients who did not receive CRT demonstrated no statistical differences in EFS (P = .412) and OS (P = .600). CONCLUSION: Patients with T-LL had significantly improved EFS and OS with bortezomib on the AALL1231 backbone. Systemic therapy intensification allowed elimination of CRT in more than 90% of patients with T-ALL without excess relapse.

Republished paper: assessment of doctors' consultation skills in the paediatric setting: the Paediatric Consultation Assessment Tool.

OBJECTIVE: To determine the utility of a novel Paediatric Consultation Assessment Tool (PCAT). DESIGN: Developed to measure clinicians' communication behaviour with children and their parents/guardian, PCAT was designed according to consensus guidelines and refined at a number of stages. Volunteer clinicians provided videotaped real consultations. Assessors were trained to score communication skills using PCAT, a novel rating scale. SETTING: Eight UK paediatric units. PARTICIPANTS: 19 paediatricians collected video-recorded material; a second cohort of 17 clinicians rated the videos. MAIN OUTCOME MEASURES: Itemised and aggregated scores were analysed (means and 95% confidence intervals) to determine measurement characteristics and relationship to patient, consultation, clinician and assessor attributes; generalisability coefficient of aggregate score; factor analysis of items; comparison of scores between groups of patients, consultations, clinicians and assessors. RESULTS: 188 complete consultations were analysed (median per doctor = 10). 3 videos marked by any trained assessor are needed to reliably (r > 0.8) assess a doctor's triadic consultation skills using PCAT, 4 to assess communication with just children or parents. Performance maps to two factors - "clinical skills" and "communication behaviour"; clinicians score more highly on the former (mean (SD) 95% CI 0.52 (0.075)). There were significant differences in scores for the same skills applied to parent and child, especially between the ages of 2 and 10 years, and for information-sharing rather than relationship building skills (2-tailed significance < 0.001). Conclusions The PCAT appears to be reliable, valid and feasible for the assessment of triadic consultation skills by direct observation.

BrachyView: development of an algorithm for real-time automatic LDR brachytherapy seed detection.

BrachyView is a novel in-body imaging system developed to provide real-time intraoperative dosimetry for low dose rate prostate brachytherapy treatments. Seed positions can be reconstructed after in-vivo implantation using a high-resolution pinhole gamma camera inserted into the patient rectum. The obtained data is a set of 2D projections of the seeds on the image plane. The 3D reconstruction algorithm requires the identification of the seed's centre of mass. This work presents the development and techniques adopted to build an algorithm that provides the means for fully automatic seed centre of mass identification and 3D position reconstruction for real-time applications. The algorithm presented uses a local feature detector, speeded up robust features, to perform detection of brachytherapy seed 2D projections from images, allowing for robust seed identification. Initial results have been obtained with datasets of 30, 96 and 98 I-125 brachytherapy seeds implanted into a prostate gel phantom. It can detect 97% of seeds and correctly match 97% of seeds. The average overall computation time of 2.75 s per image and improved reconstruction accuracy of 22.87% for the 98 seed dataset was noted. Elimination processes for initial false positive detection removal have shown to be extremely effective, resulting in a 99.9% reduction of false positives, and when paired with automatic frame alignment and subtraction procedures allows for the effective removal of excess counts generated by previously implanted needles. The proposed algorithm will allow the BrachyView system to be used as a real-time intraoperative dosimetry tool for low dose rate prostate brachytherapy treatments.

Long-term follow-up of owned, free-roaming dogs in South Africa naturally exposed to Babesia rossi.

Babesia rossi is an important, tick-borne intraerythrocytic protozoan parasite; however, its natural history and epidemiology is poorly understood. Babesia rossi is the most virulent Babesia sp. in domestic dogs and is generally considered to cause severe babesiosis, which is fatal if left untreated. However, subclinical infections and mild disease from B. rossi have been reported, although the clinical progression of these cases was not reported. Therefore, to better understand B. rossi under field conditions, we evaluated its clinical progression and seroprevalence in an owned, free-roaming dog population in Zenzele, South Africa, where the parasite is endemic and prevention is not routine. The entire dog population in Zenzele was monitored intensively at the individual level from March 2008 until April 2014, primarily for a longitudinal study on rabies control. Subsequent evaluation of B. rossi comprised analyses of clinical and laboratory data collected from the Zenzele dog population during the 6 year study period. A substantial proportion (31% (n = 34)) of 109 dogs (randomly selected from every available dog in February/March 2010 older than ~6-8 weeks (n = 246)) tested by Indirect Fluorescent Antibody Test had seroconverted strongly to B. rossi. All 34 dogs were generally consistently healthy adults, determined from regular clinical examinations between March 2008 and April 2014. Blood smear examinations at multiple time points between July 2009 and February 2011 were also undertaken for almost all of these (34) seropositive dogs and all those tested were consistently negative for Babesia spp. Subclinical infections and mild disease were also the main findings for a separate group of 18 dogs positive for Babesia spp. on blood smear examination and confirmed to be infected with B. rossi by Polymerase Chain Reaction - Reverse Line Blot. Almost all of these dogs were positive at only one time point from repeat blood smear examinations between July 2009 and February 2011. We suggest that these observations are consistent with immunity acquired from repeated, low-level exposure to the parasite, generating transient subclinical infections or mild disease. Should this be the case, the use of tick control, particularly in adult dogs in free-roaming populations in B. rossi endemic regions, should be carefully considered.