Humane neutrophile Granulozyten töten B. anthracis mit einem α-Defensin abhängigen Mechanismus

Deckblatt - Impressum Zitat Table of contents Abbreviations Introduction Literature survey Materials and Methods Results Discussion Summary References Zusammenfassung Acknowledgment SelbständigkeitserklärungThe environmentally highly resistant B. anthracis spores cause natural infections in man and animals but have also been used as biological weapons. B. anthracis infection begins through intradermal inoculation, ingestion or inhalation of spores. Human pulmonary and gastric infections with Bacillus anthracis are almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Interestingly neutrophils are recruited to cutaneous but not other forms of B. anthracis infections, raising the possibility that neutrophils kill B. anthracis. Neutrophils are the first cells of the innate immune system, which are recruited to a site of infection. Other cells of the innate immune system such as macrophages and dendritic cells have been implicated in the dissemination of B. anthracis and in the pathology of the infection, but not in its resolution. The role neutrophils play in B. anthracis infection has not been extensively studied. B. anthracis is characterized by its two virulence plasmids encoding the two major virulence factors: the tripartite anthrax toxin and the poly-D-glutamate capsule. The toxin induces oedema and shock-like symptoms; the capsule was reported to have antiphagocytic properties. In this study we infected human neutrophils with B. anthracis spores or vegetative bacteria expressing either of the two major virulence factors. As studied by electron microscopy, human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then killed. The killing of vegetative bacteria either expressing the toxin or the capsule was also examined. Both B. anthracis strains were phagocytosed and efficiently killed. Through inhibition of neutrophil phagocytosis with cytochalsin D we showed that the killing mechanism was of both intracellular and extracellular nature. Further, the killing of the vegetative cells was not due to reactive oxygen species but a result of the activity of a human neutrophil granule extract. This was elucidated by the use of an inhibitor of ROS production. We fractionated the neutrophil granule extract (hNGE) by high performance liquid chromatography with a combination of C4/ C4/ C18 reverse phase columns. -defensins were identified by MALDI TOF TOF analysis as the component responsible for B. anthracis killing. -defensins are constituents of the neutrophil azurophil granules, which have also been implicated in the neutralization of the anthrax toxin. These data show that human neutrophils induce germination of B. anthracis spores and consequently kill the vegetative cells. This and the pathohistology of human anthrax cases suggest that the timely recruitment of neutrophils in the human cutaneous form of the disease can control B. anthracis infections. This is supported by the fact that pulmonary infections in pigs and dogs show massive neutrophil infiltration, resulting in survival of the host.B. anthracis infiziert seinen Wirt durch intradermale sowie orale Aufnahme oder durch Inhalation von Sporen. Humane pulmonale und gastrointestinale Verlaufsformen weisen eine sehr hohe Mortalität auf. Im Gegensatz dazu ruft die dermale Form lokale Hautveränderungen hervor und heilt in 90% aller Fälle spontan. Interessanterweise werden bei der dermalen Form neutrophile Granulozyten an den Infektionsherd rekrutiert, während diese Infiltration in den zwei anderen klinischen Formen selten beschrieben wird. Dies eröffnet die Frage ob neutrophile Granulozyten B. anthracis töten und dadurch eine ungeahnte und drastische Auswirkung auf das Krankheitsgeschehen haben können. Während dieser Studie wurden neutrophile Granulozyten mit entweder B. anthracis Sporen oder vegetativen Bakterien infiziert, die einen der beiden Hauptvirulenzfaktoren exprimieren. Die humanen neutrophilen Granulozyten phagozytieren die B. anthracis Sporen, diese germinieren intrazellulär und werden von den neutrophilen Granulozyten getötet. Die Interaktion neutrophiler Granulozyten mit toxin- oder kapselproduzierenden vegetativen Bakterien wurde auch untersucht. Beide Stämme werden phagozytiert und effizient getötet. Der Tötungsmechanismus ist unabhängig von reaktiven Sauerstoffspezies, aber abhängig von einem aus humanen neutrophilen Granulozyten gewonnenem Granulaextrakt. Das Extrakt wurde mit High Performance Liquid Chromatography (HPLC) fraktioniert und α-Defensine als eine der zuständigen anti-B. anthracis Komponenten identifiziert. Unsere Daten, im Zusammenhang mit der Pathohistologie humaner Milzbrandfälle, implizieren, dass die rechtzeitige Einwanderung neutrophiler Granulozyten während des kutanen Milzbrand zur lokalen Eindämmung dieser Infektion führt. Diese These wird durch den Umstand unterstützt, dass die pulmonale Form bei Hunden und Schweinen mit massiver neutrophiler Granulozyteninfiltration einhergeht, welches zum Überleben dieser Wirte führt

Distribution of Leptospira Serogroups in Dogs from Berlin, Germany

Leptospirosis is a bacterial zoonosis in which dogs can act as a reservoir for human infection. The annual vaccination of dogs can prevent leptospirosis caused by serovars included in the vaccine. To date, all available vaccines in Germany include only the serovars Icterohaemorrhagiae and Canicola, the most commonly found serovars prior to the introduction of the leptospirosis vaccines. Yet, the involvement of additional serovars in the clinical presentation of leptospirosis in dogs has been described. The objective of this sero-epidemiological study was to examine the different Leptospira serovars currently circulating in a population of dogs suspicious for leptospirosis from Berlin. In 329 dogs presenting at the Small Animal Clinic in Berlin, the predominant serogroup was Australis (24%), followed by Grippotyphosa (20%) and Pomona (9%). A total of 18% of the dogs were diagnosed with clinical leptospirosis; here the most prevalent serogroups were also Australis (28%), Grippotyphosa (18%), and Pomona (14%). The serovar prevalence data presented here confirm that a change of pattern of infecting Leptospira serovars in dogs has taken place in Berlin. This data corresponds to further sero-epidemiological studies from other regions in Germany. To ensure human and canine health, available vaccines should be adapted to include the most important circulating serovars

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix- Assisted Laser Desorption/Ionization Mass Spectrometry

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology

Longitudinal Study of Selected Bacterial Zoonoses in Small Ruminants in Tana River County, Kenya

Brucellosis, Q fever, and leptospirosis are priority zoonoses worldwide, yet their epidemiology is understudied, and studies investigating multiple pathogens are scarce. Therefore, we selected 316 small ruminants in irrigated, pastoral, and riverine settings in Tana River County and conducted repeated sampling for animals that were initially seronegative between September 2014 and June 2015. We carried out serological and polymerase chain reaction tests and determined risk factors for exposure. The survey-weighted serological incidence rates were 1.8 (95% confidence intervals [CI]: 1.3–2.5) and 1.3 (95% CI: 0.7–2.3) cases per 100 animal-months at risk for Leptospira spp. and C. burnetii, respectively. We observed no seroconversions for Brucella spp. Animals from the irrigated setting had 6.83 (95% CI: 2.58–18.06, p-value = 0.01) higher odds of seropositivity to C. burnetii than those from riverine settings. Considerable co-exposure of animals to more than one zoonosis was also observed, with animals exposed to one zoonosis generally having 2.5 times higher odds of exposure to a second zoonosis. The higher incidence of C. burnetii and Leptospira spp. infections, which are understudied zoonoses in Kenya compared to Brucella spp., demonstrate the need for systematic prioritization of animal diseases to enable the appropriate allocation of resources

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa

BackgroundThe awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent.MethodologyWe searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients.FindingsA total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections.ConclusionThe small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies

Longitudinal Study of Selected Bacterial Zoonoses in Small Ruminants in Tana River County, Kenya

Brucellosis, Q fever, and leptospirosis are priority zoonoses worldwide, yet their epidemiology is understudied, and studies investigating multiple pathogens are scarce. Therefore, we selected 316 small ruminants in irrigated, pastoral, and riverine settings in Tana River County and conducted repeated sampling for animals that were initially seronegative between September 2014 and June 2015. We carried out serological and polymerase chain reaction tests and determined risk factors for exposure. The survey-weighted serological incidence rates were 1.8 (95% confidence intervals [CI]: 1.3-2.5) and 1.3 (95% CI: 0.7-2.3) cases per 100 animal-months at risk for Leptospira spp. and C. burnetii, respectively. We observed no seroconversions for Brucella spp. Animals from the irrigated setting had 6.83 (95% CI: 2.58-18.06, p-value = 0.01) higher odds of seropositivity to C. burnetii than those from riverine settings. Considerable co-exposure of animals to more than one zoonosis was also observed, with animals exposed to one zoonosis generally having 2.5 times higher odds of exposure to a second zoonosis. The higher incidence of C. burnetii and Leptospira spp. infections, which are understudied zoonoses in Kenya compared to Brucella spp., demonstrate the need for systematic prioritization of animal diseases to enable the appropriate allocation of resources

Molecular and serological diagnosis of multiple bacterial zoonoses in febrile outpatients in Garissa County, north-eastern Kenya

Bacterial zoonoses are diseases caused by bacterial pathogens that can be naturally transmitted between humans and vertebrate animals. They are important causes of non-malarial fevers in Kenya, yet their epidemiology remains unclear. We investigated brucellosis, Q-fever and leptospirosis in the venous blood of 216 malaria-negative febrile patients recruited in two health centres (98 from Ijara and 118 from Sangailu health centres) in Garissa County in north-eastern Kenya. We determined exposure to the three zoonoses using serological (Rose Bengal test for Brucella spp., ELISA for C. burnetti and microscopic agglutination test for Leptospira spp.) and real-time PCR testing and identified risk factors for exposure. We also used non-targeted metagenomic sequencing on nine selected patients to assess the presence of other possible bacterial causes of non-malarial fevers. Considerable PCR positivity was found for Brucella (19.4%, 95% confidence intervals [CI] 14.2–25.5) and Leptospira spp. (1.7%, 95% CI 0.4–4.9), and high endpoint titres were observed against leptospiral serovar Grippotyphosa from the serological testing. Patients aged 5–17 years old had 4.02 (95% CI 1.18–13.70, p-value = 0.03) and 2.42 (95% CI 1.09–5.34, p-value = 0.03) times higher odds of infection with Brucella spp. and Coxiella burnetii than those of ages 35–80. Additionally, patients who sourced water from dams/springs, and other sources (protected wells, boreholes, bottled water, and water pans) had 2.39 (95% CI 1.22–4.68, p-value = 0.01) and 2.24 (1.15–4.35, p-value = 0.02) times higher odds of exposure to C. burnetii than those who used unprotected wells. Streptococcus and Moraxella spp. were determined using metagenomic sequencing. Brucellosis, leptospirosis, Streptococcus and Moraxella infections are potentially important causes of non-malarial fevers in Garissa. This knowledge can guide routine diagnosis, thus helping lower the disease burden and ensure better health outcomes, especially in younger populations

Surveillance of climate-sensitive zoonotic diseases: Leptospirosis at livestock slaughterhouses in three regions of Uganda

Leptospirosis is an important bacterial zoonosis worldwide and is disproportionately associated with low-income settings and with extreme weather events due to climate change. Transmission to humans often occurs when infected rodents and domestic animals contaminate the environment via urine as the bacteria preferentially colonise kidneys. Surveillance of leptospirosis at slaughterhouses can therefore be useful in providing information on vast areas of a country and screening for diseases that are not considered during animal inspections. We determined the prevalence of Leptospira bacteria in the kidneys of 2,030 livestock kidney samples (820 cattle, 761 pigs, 335 goats, 114 sheep), and 117 small mammals by realtime PCR in a cross-sectional survey of slaughter facilities in three regions in Uganda. We extracted DNA and performed real-time polymerase chain reaction (PCR) tests targeting the lipL32 gene for pathogenic leptospires. Positive samples with cycle threshold values below 38 were further characterised using single locus sequence typing (SLST) to determine likely genomospecies. PCR products were sequenced by Eurofins Genomics (Ebersberg, Germany) and identification of genomospecies was done using the basic local alignment search tool (BLAST). Multi-locus sequencing typing (MLST) was performed on selected SLST-positive samples to determine sequence types (ST) and likely serogroups. The allelic profiles were analysed using Bionumerics software and the sequence types were determined using the PubMLST database. An overall prevalence of pathogenic leptospires of 2.58 % (95 % confidence intervals [CI]: 1.89–3.42) was observed, with sheep having the highest prevalence (6.12 % CI: 2.69–12.89), followed by cattle (4.25 %, 95 % CI: 2.91–5.98), goats (2.08 %, 95 % CI=0.91–4.38), and pigs (0.46 %, 95 % CI = 0.12–1.31) in decreasing order. The genomospecies L. borgpetersenii (11 in cattle and one in goat), L. kirschneri (five in cattle and four in sheep) and L. interrogans (one in a pig) were determined. Preliminary MLST results on one sample reveal L. kirschneri ST62 which is related to serogroup Grippotyphosa. Surveillance of important zoonoses using slaughterhouses as sentinels has the potential to offer essential information on the epidemiology of important zoonotic diseases in Uganda