Mouse models of Loa loa

Elimination of the helminth disease, river blindness, remains challenging due to ivermectin treatment-associated adverse reactions in loiasis co-infected patients. Here, we address a deficit in preclinical research tools for filarial translational research by developing Loa loa mouse infection models. We demonstrate that adult Loa loa worms in subcutaneous tissues, circulating microfilariae (mf) and presence of filarial biomarkers in sera occur following experimental infections of lymphopenic mice deficient in interleukin (IL)-2/7 gamma-chain signaling. A microfilaraemic infection model is also achievable, utilizing immune-competent or -deficient mice infused with purified Loa mf. Ivermectin but not benzimidazole treatments induce rapid decline (>90%) in parasitaemias in microfilaraemic mice. We identify up-regulation of inflammatory markers associated with allergic type-2 immune responses and eosinophilia post-ivermectin treatment. Thus, we provide validation of murine research models to identify loiasis biomarkers, to counter-screen candidate river blindness cures and to interrogate the inflammatory etiology of loiasis ivermectin-associated adverse reactions.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI) Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code Data collection There were no commercial, open source and custom code used to collect the data in this study. All data was collected from samples and clearly reported in graphs as single data points or central tendancy with variance.

Data analysis
All tests were performed in GraphPad Prism software at a significance level of 5% and significance is indicated P<0.05* P<0.01** P<0.001***. Heatmap analysis was undertaken in Excel using conditional formatting with -10 fold change being color-coded as blue and +10 fold change being reported as red. Data on differential cell counts on either thick smears or immunohistological material were collected in a blinded manner and represent mean counts for 100 leucocytes and 10 quadrants of 120μmx200μm per sample respectively.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All data generated or analysed during this study are included in this published article (and its supplementary information files).

Field-specific reporting
Please select the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/authors/policies/ReportingSummary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample size was based on previous studies of a similar design and double checked with by a power analysis test.
Data exclusions No data was excluded from the analysis; it indeed helps reflecting natural data heterogeneity.

Replication
Reproducibility of the data was tested by experimental repeats accordingly (as stated in the manuscript) and data were tested for homoscedasticity and distribution before being gathered.
Randomization For drug treatments, mice were randomly allocated to treatment groups, stratified on time of Loa mf infusion.

Blinding
Investigators involved in parasitological analysis of results were blinded to group allocation during data collection. An Excel spreadsheet has been used to report group allocations and was accessible for investigators who performed mice cull at studies end points. Unblinding of the investigators was done after data analysis.
In addition, the revised version of the manuscript details an additional FACS analysis performed on samples as follow: Mouse peritoneal cells were collected via a peritoneal cavity wash with 10mL PBS-5% FCS. Cells were subsequently centrifuged (800 rpm, 5min, 4°C) and a Fc blocking step (using α-CD16/32, eBioscience) was performed prior to the application of the following cocktail: viability dye eFluor450 (eBioscience), anti-mouse SiglecF-APC (clone E50-2440, BD Bioscience) and anti-mouse Ly6G-FITC (clone RB6-8C5, eBioscience). All samples were subsequently acquired using a BD LSR II flow cytometer (BD Bioscience) and analysed on FloJo Software. Peritoneal eosinophils were gated on live cells as SiglecFhigh Ly6G-and peritoneal neutrophils on live cells as SiglecF-Ly6Ghigh.

Validation
Validation has been performed following manufacturer's instructions, using standards and quality controls provided in the kit.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research quarantined for a period of two months during which they were pre-screened for a panel of natural infections (loiasis, other blood-borne parasites, and intestinal worms). Each animal was observed daily by the veterinary staff to ensure that they were healthy, and any animal found to be ill was immediately given appropriate treatment, both in the quarantine period and during the main study period. The animals were housed individually in large custom built cages that allowed the animals to move about freely and be allowed to display their normal repertoire of locomotor behavior (walking, climbing, running, jumping and swinging) by providing them with vertical climbing surfaces and perches. Horizontal surfaces were also provided to allow them to rest comfortably and perform their social interactions such as sprawling during grooming. The housing facility was well aerated and equipped with a system that provided water ad libitum for each animal. Each baboon's behavior was regularly monitored to identify any indications of poor welfare. Baboons received a diet of food that mimicked their natural diet (leaves, grass, roots, bark, flowers, fruit, lichens, tubers, seeds, mushrooms, corms, and rhizomes). They were also fed a supplement of a nutritionally complete commercial-available diet. The health and well-being of the baboons were regularly assessed during the study by an animal welfare officer who advised on matters such as disease prophylaxis, zoonoses, anesthe-sia, and methods of humane euthanasia and provision of health certificates. All measures were taken to minimize suffering during capture, captivity and experimentation. Health screening of workers in contact with the baboons was performed regularly to prevent animal losses from diseases transmitted from humans to baboons as well as zoonotic transmission of disease from baboons to workers." Ethical and administrative clearances for the use of baboons in this study were obtained from the Ministry of Scientific Research and Innovation of Cameroon (Research permit #028/MINRESI/B00/C00/C10/C12) and the Animal Care Committee at REFOTDE. Procedures adhered to the NIH Guide for the Care and Use of Laboratory Animals.
The study also involved wild Chrysops silacea (a fly, vector of the parasite) being captured via baited traps in a known hyperendemic area Field-collected samples Flies were dissected to allow release of any L. loa L3 infective stages. Infective doses of 100 to 200 L3 per 200μL medium (DMEM + 10% FCS) were loaded in 25G 1mL syringes and subcutaneously injected into mice.