Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung’s Disease

Hirschsprung’s disease (HSCR) is characterized by aganglionosis from failure of neural crest cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung’s-associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs. We hypothesize that gastrointestinal (GI) mucosal immune defects occur in HSCR that may contribute to enterocolitis. EdnrB was deleted from the neural crest (EdnrB ^NCC-/-) resulting in mutants with defective NCC migration, distal colonic aganglionosis and the development of enterocolitis. The mucosal immune apparatus of these mice was interrogated at post-natal day (P) 21–24, prior to histological signs of enterocolitis. We found that EdnrB ^NCC-/- display lymphopenia of their Peyer’s Patches, the major inductive site of GI mucosal immunity. EdnrB ^NCC-/- Peyer’s Patches demonstrate decreased B-lymphocytes, specifically IgM⁺IgD^hi (Mature) B-lymphocytes, which are normally activated and produce IgA following antigen presentation. EdnrB ^NCC-/- animals demonstrate decreased small intestinal secretory IgA, but unchanged nasal and bronchial airway secretory IgA, indicating a gut-specific defect in IgA production or secretion. In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrB ^NCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes. EdnrB ^NCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone. Taken together, these findings suggest impaired GI mucosal immunity in EdnrB ^NCC-/- animals, with the spleen as a potential site of the defect. These findings build upon the growing body of literature that suggests that intestinal defects in HSCR are not restricted to the aganglionic colon but extend proximally, even into the ganglionated small intestine and immune cells.     

A Dynamical Study of a Cellular Automata Model of the Spread of HIV in a Lymph Node

We conduct a mathematical study of a cellular automata model of the spread of the HIV virus in a lymph node. The model was proposed by Zorzenon dos Santos and Coutinho and captures the unique time scale of the viral spread. We give some rigorous mathematical results about the time scales and other dynamical aspects of the model as well as discuss parameter and model changes and their consequences.     

The Role of Lipoprotein-Associated Phospholipase A? in a Murine Model of Experimental Autoimmune Uveoretinitis

Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A₂ (Lp-PLA₂), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA₂ may limit macrophage activation and protect the tissue. Utilising Lp-PLA₂ gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA₂ (SB-435495) we aimed to determine the effect of Lp-PLA₂ suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1–20 or 161–180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA₂ enzyme activity in Lp-PLA₂ KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45⁺ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA₂ depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA₂ KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA₂ suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA₂ activity.     

Evidence for a “Wattle and Daub” Model of the Cyst Wall of Entamoeba

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).     

A Mechanistic Investigation of the Algae Growth “Droop” Model

In this work a mechanistic explanation of the classical algae growth model built by M. R. Droop in the late sixties is proposed. We first recall the history of the construction of the "predictive" variable yield Droop model as well as the meaning of the introduced cell quota. We then introduce some theoretical hypotheses on the biological phenomena involved in nutrient storage by the algae that lead us to a "conceptual" model. Though more complex than Droop's one, our model remains accessible to a complete mathematical study: its confrontation to the Droop model shows both have the same asymptotic behavior. However, while Droop's cell quota comes from experimental bio-chemical measurements not related to intra-cellular biological phenomena, its analogous in our model directly follows our theoretical hypotheses. This new model should then be looked at as a re-interpretation of Droop's work from a theoretical biologist's point of view.     

Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ��A1122

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.     

A Food and Drug Administration-approved Asthma Therapeutic Agent Impacts Amyloid β in the Brain in a Transgenic Model of Alzheimer Disease

Interfering with the assembly of Amyloid β (Aβ) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aβ-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aβ monomers into higher-order oligomers and fibrils in vitro without affecting Aβ production. In vivo, the levels of soluble Aβ are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aβ in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aβ economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.     

Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis

Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia.     

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)₄ complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)₂ lobes joined with D₂ symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)₄ complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)₄ kinase core, because a β₄ subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.     

Probing the Conformation of the Fibronectin III1�C2 Domain by Fluorescence Resonance Energy Transfer

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III₁ and III₂, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III_1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III_1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III_1–2.Fibronectin (FN)³ is a 500-kDa modular dimeric protein and a major component of the extracellular matrix. It exists in the blood and other body fluids as a soluble compact molecule and undergoes cell-mediated assembly to form an insoluble three-dimensional fibrillar matrix (reviewed in Ref. 1). The process of FN matrix assembly has been implicated in embryonic development, wound healing, and cancer (2–4). FN is composed of type I–III modules, and sets of these modules comprise binding domains for cells and for other extracellular matrix components (see Fig. 1A). Three of these binding domains are essential for matrix assembly (1). Integrin receptor interactions with the cell-binding domain tether disulfide-bonded FN dimers to the cell surface, where FN-FN interactions involving the N-terminal assembly domain form dimers into fibrils. In addition to these essential domains, other FN-binding sites have been implicated in assembly. In particular, the III_1–2 FN-binding domain plays a regulatory role in matrix assembly. Within this domain reside a cryptic FN-binding site in III₁ and a site available for FN binding in the native form of III₂ (5–8). Recombinant FN lacking III₁ is assembled into a matrix at wild-type levels, but that lacking the III_1–2 domain results in short immature FN fibrils (8). Peptides derived from the III_1–2 domain or antibodies against III_1–2 block matrix assembly by cultured cells (9–11). Furthermore, FN binding to this region is enhanced when FN is mechanically stretched (12). Taken together, these results suggest that conformational changes in the III_1–2 domain may control its interactions during FN assembly.Open in a separate windowFIGURE 1.The FN III_1–2 FRET conformational sensor. A, representation of the domain structure of FN and major interaction sites. FN is composed of repeating modules that form binding domains for other FN molecules, cell receptors, and other extracellular matrix components as indicated. The first two type III modules III₁ and III₂ (black), have FN-binding sites and regulate FN matrix assembly. The N-terminal 70-kDa region contains a matrix assembly domain with FN-binding activity. The cell-binding domain (cell), the heparin-binding domain (heparin), the dimerization site (SS), and the alternatively spliced type IIIA (A), IIIB (B), and variable regions (V) are indicated. 70kD, N-terminal 70-kDa FN fragment. B, schematic of proposed model of III_1–2 domain conformation. Panel i, in solution, the FN-binding sites in III₁ and III₂ (hatched areas) are sequestered through domain orientations that are facilitated by the linker between modules (thin line). Panel ii, binding sites are exposed through conformational changes resulting from cell-mediated extension of FN (arrows). The length of the linker and the height and width of the modules are drawn to scale for a linear peptide and published data on FN type III modules, respectively. C, ribbon diagram representation of CIIIY, a FRET sensor of the model in B (panel i), oriented with N and C termini 50 Å apart. CIIIY consists of the III_1–2 domain with CFP at the N terminus and YFP at the C terminus.To more fully understand the roles of native and cryptic FN-binding sites in matrix assembly, the conformational dynamics of III_1–2 must be characterized. One approach to this problem is to tag III_1–2 with fluorescent probes, which, in conjunction with fluorescent resonance energy transfer (FRET), create a molecular conformational sensor. FRET involves the radiationless transfer of energy from an excited donor fluorophore to an acceptor fluorophore, a process that is very sensitive to the distance between the two fluorophores (13–15). Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), are highly related to green fluorescent protein (GFP). Because the emission spectrum of CFP is well matched to the excitation spectrum of YFP, these two fluorophores have been widely used as a donor-acceptor pair in FRET studies (13–15).In this study, we describe a FRET conformational sensor designed to test the idea that intramolecular interactions between III₁ and III₂ sequester key FN-binding and assembly sites. We show that III_1–2 with CFP and YFP fused to the N and C termini, respectively, displays a clear FRET signal, indicating that the attached fluorescent proteins and thus the ends of III_1–2 are in close proximity. FRET data from III_1–2 mutants support the presence of a stabilizing intermodule salt bridge that regulates FN-binding activity.     

Beneficial Effects of the Activation of the Angiotensin-(1–7) Mas Receptor in a Murine Model of Adriamycin-Induced Nephropathy

Angiotensin-(1–7) [Ang-(1–7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT₁ receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT₁ receptor antagonist, Losartan. AT₁ and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas^+/+) and Mas knockout (Mas ^−/−) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas^+/+, but not in Mas ^−/− mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT₁ receptor blockade. Medications which target specifically the ACE2/Ang-(1–7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.     

<Emphasis Type="Italic">“Pseudoalteromonas januaria”</Emphasis> SUT 11 as the Source of Rare Lipodepsipeptides

The marine bacterium “Pseudoalteromonas januaria” SUT 11 isolated from a seawater sample produced the rare cell-bound cyclic lipodepsipeptides A/A′, B/B′, and C/C′. The matrix-assisted laser desorption/ionization mass spectra indicated that one bromine atom presented in the peptides B/B′ and C/C′, whereas the component A/A′ contained no bromine atom. The acyldepsipeptides A/A′–C/C′ have an identical amino acid sequence, Thr-Val-Asn-Asn-Leu/allo-Ile, but differed in C-terminal amino acid and acyl moieties. Peptides A–C have Leu as a C-terminal amino acid, whereas peptides A′-C′ have allo-Ile. Acyl moieties in peptides A/A′, B/B′, and C/C′ have been found to consist of 11-(4′-hydroxyphenyl)-undeca-2,4,6,8,10-pentaenic acid, 9-(3′-bromo-4′-hydroxyphenyl)-nona-2,4,6,8-tetraenic acid, and 11-(3′-bromo-4′-hydroxyphenyl)-undeca-2,4,6,8,10-pentaenic acid, respectively. The structure of a main pair of peptides B/B′ with molecular masses 843/845 Da has been determined by means of ultraviolet, infrared, and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that tandem nano-electrospray ionization mass spectrometry is a very efficient way for the fast and sensitive investigation of lipopeptides A/A′ and C/C′ with molecular masses 791 and 869/871 Da, respectively, which have been isolated in small amounts.     

A Transcultural Model of the Centrality of “Thinking a Lot” in Psychopathologies Across the Globe and the Process of Localization: A Cambodian Refugee Example

We present a general model of why “thinking a lot” is a key presentation of distress in many cultures and examine how “thinking a lot” plays out in the Cambodian cultural context. We argue that the complaint of “thinking a lot” indicates the presence of a certain causal network of psychopathology that is found across cultures, but that this causal network is localized in profound ways. We show, using a Cambodian example, that examining “thinking a lot” in a cultural context is a key way of investigating the local bio-cultural ontology of psychopathology. Among Cambodian refugees, a typical episode of “thinking a lot” begins with ruminative-type negative cognitions, in particular worry and depressive thoughts. Next these negative cognitions may induce mental symptoms (e.g., poor concentration, forgetfulness, and “zoning out”) and somatic symptoms (e.g., migraine headache, migraine-like blurry vision such as scintillating scotomas, dizziness, palpitations). Subsequently the very fact of “thinking a lot” and the induced symptoms may give rise to multiple catastrophic cognitions. Soon, as distress escalates, in a kind of looping, other negative cognitions such as trauma memories may be triggered. All these processes are highly shaped by the Cambodian socio-cultural context. The article shows that Cambodian trauma survivors have a locally specific illness reality that centers on dynamic episodes of “thinking a lot,” or on what might be called the “thinking a lot” causal network.     

A Modular BAM Complex in the Outer Membrane of the α-Proteobacterium Caulobacter crescentus

Mitochondria are organelles derived from an intracellular α-proteobacterium. The biogenesis of mitochondria relies on the assembly of β-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an α-proteobacterium, and the BAM (β-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A ∼150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.     

Recent advances in the design of RAR α and RAR β agonists as orally bioavailable drugs. A review

Retinoic acid receptors (RARs) α, β, and γ are members of the nuclear receptor superfamily. Compounds which bind to and activate the RARs are termed retinoids which regulate a wide variety of biological processes such as vertebrate embryonic morphogenesis and organogenesis, cell growth arrest, differentiation, and apoptosis, as well as their disorders. Although many synthetic selective RARα, RARβ, and RARγ agonists have been designed and prepared, these have generally been lipophilic acids without good drug-like properties and with low oral bioavailability. Recently this has been changing and drug design approaches to highly potent and selective RARα and RARβ agonists with low lipophilicity that are orally bioavailable and less toxic have been developed, that have a range of potential therapeutic uses. This review covers these new advances.     

Environmental Impact of the Production of Mealworms as a Protein Source for Humans – A Life Cycle Assessment

The demand for animal protein is expected to rise by 70–80% between 2012 and 2050, while the current animal production sector already causes major environmental degradation. Edible insects are suggested as a more sustainable source of animal protein. However, few experimental data regarding environmental impact of insect production are available. Therefore, a lifecycle assessment for mealworm production was conducted, in which greenhouse gas production, energy use and land use were quantified and compared to conventional sources of animal protein. Production of one kg of edible protein from milk, chicken, pork or beef result in higher greenhouse gas emissions, require similar amounts of energy and require much more land. This study demonstrates that mealworms should be considered a more sustainable source of edible protein.     

Land-Applied Goat Manure as a Source of Human Q-Fever in the Netherlands, 2006–2010

Studies have shown a link between Q-fever positive farms (QFPFs) and community cases of human Q-fever. Our study is the first to investigate the potential role of contaminated land-applied manure in human Q-fever, based on a large set of nationwide notification and farm management data. Time between manure application and disease onset in geographically linked notified human cases coincided with the incubation period of Q-fever. Proximity of contaminated land parcels predicted human cases better than proximity of QFPFs (80% vs. 58%, 0–5 km in 2009). Incidence around QFPFs and contaminated land parcels decreased with distance, but not around non-contaminated land parcels. Incidence was higher around contaminated land parcels than non-contaminated land parcels (RR = [10],95%CI = [7], [1]–[14,2]). Our findings deliver evidence that, apart from QFPFs, land-applied contaminated manure may be another source of human Q-fever.