Studies on the diagnosis, development and distribution of drug-resistant trypanosomes in cattle herds from selected sites of East and West Africa

Titelseite und Inhaltsverzeichnis 1\. Vorwort Publikationen, die Bestandteil der Habilitationsschrift sind Einleitung und Ziel der Untersuchungen 2\. Zusammenfassende Darstellung der Ergebnisse 3\. Übergreifende Diskussion 4\. Schlussfolgerungen 5\. Zusammenfassung 6\. Summary 7\. Literaturverzeichnis 8\. Abkürzungen und Definitionen 9\. Darstellung des eigenen Anteils an den Arbeiten 10\. Danksagung 11\. Anhang (Mitteilungen 1-14)Die von Tsetsefliegen zyklisch übertragenen Trypanosomosen der Rinder (Nagana) bedrohen die landwirtschaftliche Entwicklung in weiten Teilen Afrikas südlich der Sahara. Seit über 40 Jahren werden bei Rindern das Diamidin Diminazenaceturat zur Therapie und das Phenanthridinderivat Isometamidiumchlorid zur Prophylaxe eingesetzt. Die Entwicklung eines Impfstoffes scheiterte bisher an der ausgeprägten Fähigkeit der Trypanosomen zur Antigenvarianz. Hinzu kommt, dass trotz des Vorhandenseins effizienter Methoden die Nachhaltigkeit der Vektorenkontrolle (Tsetsefliegenbekämpfung) nach wie vor nicht gegeben ist. Auch zukünftig werden deshalb therapeutische und prophylaktische Behandlungen eine bedeutende Rolle in der Bekämpfung der Nagana spielen. In jüngster Zeit häufen sich jedoch Berichte über Resistenzen bei Trypanosomen. Bisher fehlen aber verlässliche Daten über das Vorkommen und die regionale Verbreitung von Resistenzen. Daher war das Ziel dieser Untersuchungen, die regionale Verbreitung und Bedeutung von Medikamentenresistenzen bei Trypanosomeninfektionen in Rinderherden an vier Standorten Afrikas exemplarisch zu bestimmen. Diese Standorte waren in Ostafrika Metekel (Nordwest-Äthiopien), Upper Didessa Valley (West-Äthiopien), Mukono County (Südost-Uganda) und in Westafrika Kénédougou Province (Südwest- Burkina Faso). In Gebieten mit erhöhtem Infektionsrisiko wurden Behandlungsstudien durchgeführt, einmal um den Verdacht auf Resistenz zu bestätigen und zum zweiten um die Häufigkeit resistenter Trypanosomeninfektionen zu bestimmen. Verschiedene in-vivo- und in-vitro- Methoden wurden zur Charakterisierung der Medikamentenempfindlichkeit von Trypanosomenfeldstämmen eingesetzt. Die Polymerase-kettenreaktion (PCR) wurde vergleichend auf ihre diagnostischen Möglichkeiten und Grenzen zur Kontrolle von prophylaktischen und therapeutischen Behandlungen bei Trypanosomen- infektionen überprüft. In Dörfern mit einem erhöhten Infektionsrisiko ergaben Blockbehandlungsstudien von Rinderherden mit Isometamidiumchlorid in Metekel, Upper Didessa Valley und Kénédougou Hinweise für ein weit verbreitetes, aber in seiner Häufigkeit sehr variables Vorkommen von Isometamidium-Resistenzen. Diese konnten bei weiteren Untersuchungen, sowohl bei der Therapie experimentell infizierter Rinder, Ziegen und Mäuse als auch unter Einsatz von in-vitro-Methoden für Isometamidium nicht nur bestätigt, sondern auf Diminazen ausgeweitet werden. Klone von Trypanosoma congolense-Populationen aus Metekel und Kénédougou zeigten in Mäusen ausgeprägte Resistenzen gegenüber beiden Wirkstoffen. Bei einem gehäuften Vorkommen mehrfachresistenter Trypanosomeninfektionen erscheint die ausschließliche Kontrolle der Nagana durch Trypanozide langfristig nicht mehr gerechtfertigt. Die Untersuchungen in Rinderherden von Mukono ergaben keine Hinweise auf Resistenzen bei Trypanosomen gegenüber Isometamidium und Diminazen. Die zunehmenden Berichte über Resistenzen aus anderen Regionen sollten allerdings auch in Mukono Anlass für einen restriktiven Einsatz von Trypanoziden geben. Die Polymerasekettenraktion (PCR) erwies sich als eine hochempfindliche und spezifische Methode zur Überprüfung des prophylaktischen und therapeutischen Erfolgs bei Trypanosomeninfektionen. Unter der Voraussetzung ausreichender finanzieller Mittel und entsprechender Laboreinrichtungen stellt sie ein geeignetes diagnostisches Hilfsmittel in epidemiologischen Untersuchungen dar, sowohl zur verbesserten Risikoabschätzung in Endemiegebieten als auch bei Erfolgsuntersuchungen in regionalen Bekämpfungsprogrammen. Da aufgrund der erheblichen Kosten mittelfristig nicht mit der Entwicklung und Zulassung neuer trypanozider Wirkstoffe für den afrikanischen Nutztiermarkt zu rechnen ist, sollte zur Verringerung der Gefahr von Resistenzentwicklung ein Medikamenteneinsatz auf klinisch erkrankte Tiere beschränkt werden. Bei Nachweis mehrfachresistenter Trypanosomen-populationen ist von einem Einsatz von Trypanoziden abzuraten. In solchen Regionen sind Maßnahmen zur Vektorenkontrolle zu intensivieren. Außerdem sollten Einkreuzungen mit genotypisch trypanotoleranten Rinderrassen angeregt werden.Tsetse transmitted bovine trypanosomosis or nagana is a major constraint to rural development in much of sub-Saharan Africa. In all these regions the main control method is to cure the disease in cattle through the administration of anti-trypanosomal molecules such as the diamidine diminazene aceturate and the phenanthridine derivative isometamidium chloride. These drugs are in use for more than 40 years. Due to the phenomenon of antigenic variation no vaccine has been developed until now. Furthermore, vector control even though technically feasible, is inherently confronted with problems of sustainability. Therefore, control of the disease will continue to depend largely on the use of trypanocidal drugs for the foreseeable future. However, there have been several reports in recent times indicating an increase of the drug resistance incidence in trypanosomes. The number of case reports on drug resistance is growing, yet there is lack of reliable data at regional level on its true prevalence. The present study aimed to assess the development and distribution of drug-resistant trypanosomes in cattle herds from selected sites of East Africa (Metekel, north-west Ethiopia; Upper Didessa Valley, west Ethiopia; Mukono County, south-east Uganda) and West Africa (province of Kénédougou, south-west Burkina Faso). Longitudinal field studies were conducted to estimate the incidence of trypanocidal drug resistance in high risk areas. Several in vivo and in vitro tests were used to characterize the drug sensitivity of trypanosome field stocks. The polymerase chain reaction (PCR) was evaluated and its diagnostic potential to monitor the efficacy of prophylactic and curative treatments tested. Block treatment of cattle with isometamidium in high trypanosomosis risk villages in Metekel, Upper Didessa Valley and Kénédougou indicated that isometamidum resistance in Trypanosoma congolense was widespread but varied in its incidence between villages, indicating a relation with trypanosomosis risk and, possibly, other factors. Field-derived indicators of isometamidium resistance were supported by in vivo and in vitro demonstration of resistance to both isometamidium and diminazene. Clones originating from cattle primary isolates of T. congolense from Metekel and Kénédougou and tested in mice expressed a high level of resistance to both isometamidium and diminazene. It was concluded that chemotherapeutic agents per se would not provide a viable option for the control of trypanosomosis at Metekel and Kénédougou on a long-term basis if such resistance at clonal level was highly prevalent. In contrast, there appeared to be no resistance of trypanosomes to the common used drugs in cattle of Mukono. All the same, given the apparent development of resistance to trypanocidal drugs that has occurred in other tsetse-infested areas, anti-trypanosomal compounds should be judiciously applied in Mukono County as well. PCR proved to be a highly sensitive and specific tool to monitor the therapeutic and prophylactic efficacy and disease progression in bovine trypanosomosis. Depending on the availability of financial support, PCR could provide an ideal tool to assess trypansomosis risk in endemic areas and to monitor the success of tsetse and trypanosomosis control programmes. Since there is no indication that new trypanocidal drugs will become available in the near future, it is of greatest importance that measures be taken to avoid or delay the development of resistance and to maintain the efficacy of the currently available drugs. The most efficient way is the reduction in the number of treatments in order to reduce the selection pressure caused by these drugs. Mass treatments at short intervals should be avoided, particularly in areas of high challenge. Drug use should be reduced through vector control and by decreasing host-vector contact. If resistance to both isometamidium and dimiazene is present at the level of individual trypanosomes, trypanocidal drug use should be terminated, except for the treatment of clinical cases. Vector control activities should be maximised and cross-breeding with trypanotolerant cattle should be encouraged

Developing person-centred analysis of harm in a paediatric hospital: a quality improvement report.

The provision of safe care is complex and difficult to achieve. Awareness of what happens in real time is one of the ways to develop a safe system within a culture of safety. At Great Ormond Street Hospital, we developed and tested a tool specifically designed for patients and families to report harm, with the aim of raising awareness and opportunities for staff to continually improve and provide safe care. Over a 10-month period, we developed processes to report harm. We used the Model for Improvement and multiple Plan, Do, Study, Act cycles for testing. We measured changes using culture surveys as well as analysis of the reports. The tool was tested in different formats and moved from a provider centric to a person-centred tool analysed in real time. An independent person working with the families was best placed to support reporting. Immediate feedback to families was managed by senior staff, and provided the opportunity for clarification, transparency and apologies. Feedback to staff provided learning opportunities. Improvements in culture climate and staff reporting were noted in the short term. The integration of patient involvement in safety monitoring systems is essential to achieve safety. The high number of newly identified 'near-misses' and 'critical incidents' by families demonstrated an underestimation of potentially harmful events. This testing and introduction of a self-reporting, real-time bedside tool has led to active engagement with families and patients and raised situation awareness. We believe that this will lead to improved and safer care in the longer term

Suspected autochthonous Thelazia callipaeda infection in a dog in northern Germany

A 12-year old Elo dog was presented with recurring symptoms of conjunctivitis in November 2019. A single whitish nematode was found upon inspection of the eye and identified as aThelazia callipaedamale. The morphological identification of the eye worm was supported by analysis of a partial cytochrome c oxidase I (cox1) gene sequence. The dog lived in Lower Saxony, northwestern Germany, and had not visited regions known to be endemic forT. callipaeda.This suggests that a local transmission cycle of this zoonotic nematode may exist in Germany

The Effect of Deltamethrin-Treated Net Fencing Around Cattle Enclosures on Outdoor-biting Mosquitoes in Kumasi, Ghana.

Classic vector control strategies target mosquitoes indoors as the main transmitters of malaria are indoor-biting and -resting mosquitoes. However, the intensive use of insecticide-treated bed-nets (ITNs) and indoor residual spraying have put selective pressure on mosquitoes to adapt in order to obtain human blood meals. Thus, early-evening and outdoor vector activity is becoming an increasing concern. This study assessed the effect of a deltamethrin-treated net (100 mg/m(2)) attached to a one-meter high fence around outdoor cattle enclosures on the number of mosquitoes landing on humans. Mosquitoes were collected from four cattle enclosures: Pen A - with cattle and no net; B - with cattle and protected by an untreated net; C - with cattle and protected by a deltamethrin-treated net; D - no cattle and no net. A total of 3217 culicines and 1017 anophelines were collected, of which 388 were Anopheles gambiae and 629 An. ziemanni. In the absence of cattle nearly 3 times more An. gambiae (p<0.0001) landed on humans. The deltamethrin-treated net significantly reduced (nearly three-fold, p<0.0001) culicine landings inside enclosures. The sporozoite rate of the zoophilic An. ziemanni, known to be a secondary malaria vector, was as high as that of the most competent vector An. gambiae; raising the potential of zoophilic species as secondary malaria vectors. After deployment of the ITNs a deltamethrin persistence of 9 months was observed despite exposure to African weather conditions. The outdoor use of ITNs resulted in a significant reduction of host-seeking culicines inside enclosures. Further studies investigating the effectiveness and spatial repellence of ITNs around other outdoor sites, such as bars and cooking areas, as well as their direct effect on vector-borne disease transmission are needed to evaluate its potential as an appropriate outdoor vector control tool for rural Africa

Artificial Feeding of All Consecutive Life Stages of Ixodes ricinus

The hard tick Ixodes ricinus is an obligate hematophagous arthropod and the main vector for several zoonotic diseases. The life cycle of this three-host tick species was completed for the first time in vitro by feeding all consecutive life stages using an artificial tick feeding system (ATFS) on heparinized bovine blood supplemented with glucose, adenosine triphosphate, and gentamicin. Relevant physiological parameters were compared to ticks fed on cattle (in vivo). All in vitro feedings lasted significantly longer and the mean engorgement weight of F0 adults and F1 larvae and nymphs was significantly lower compared to ticks fed in vivo. The proportions of engorged ticks were significantly lower for in vitro fed adults and nymphs as well, but higher for in vitro fed larvae. F1-females fed on blood supplemented with vitamin B had a higher detachment proportion and engorgement weight compared to F1-females fed on blood without vitamin B, suggesting that vitamin B supplementation is essential in the artificial feeding of I. ricinus ticks previously exposed to gentamicin

Physical limits of semiconductor laser operation: A time-resolved analysis of catastrophic optical damage

The early stages of catastrophic optical damage (COD) in 808 nm emitting diode lasers are mapped by simultaneously monitoring the optical emission with a 1 ns time resolution and deriving the device temperature from thermal images. COD occurs in highly localized damage regions on a 30 to 400 ns time scale which is determined by the accumulation of excess energy absorbed from the optical output. We identify regimes in which COD is avoided by the proper choice of operation parameters

Molecular detection of tick‐borne pathogens in bovine blood and ticks from Khentii, Mongolia

Recent studies reported the detection of DNA from tick‐borne pathogens (TBPs) of veterinary relevance such as Anaplasma marginale, Babesia bigemina, Babesia bovis and Theileria orientalis in bovine blood samples from Mongolia. These findings were unexpected, as the known tick vectors of these pathogens are not known to occur in Mongolia. We therefore conducted a study in May and June 2013 in six districts of Khentii province where DNA of the said TBPs was previously found. Ticks collected from the vegetation and rodents, as well as blood samples from cattle, were screened for the presence of TBPs by reverse line blot (RLB) hybridization. Tick larvae collected from rodents were pooled. A total of 310 adult ticks were collected from the vegetation, and 249 tick larvae were collected from 24 rodents. Adult ticks (n = 2,318) and blood samples were collected from 481 heads of cattle. All adult ticks were identified as Dermacentor nuttalli. DNA from Rickettsia raoultii (252/310; 81.3%), an uncharacterized Anaplasma species preliminary named Anaplasma sp. Mongolia (26/310; 8.4%), Candidatus Midichloria sp. (18/310; 5.8%), Theileria equi (16/310; 5.2%), Babesia caballi (5/310; 1.6%), T. orientalis (1/310; 0.3%), Borrelia afzelii (1/310; 0.3%) and Candidatus Neoehrlichia mikurensis (1/310; 0.3%) was detected in ticks collected from the vegetation. DNA of R. raoultii (27/28; 96.4%) and Midichloria sp. (2/28; 7.1%) was detected in the pooled tick larvae. Anaplasma sp. Mongolia, a species related to Anaplasma ovis based on a multi‐locus analysis, was also detected in 153/481 (31.8%) of the bovine blood samples. DNA of B. bovis, B. bigemina and A. marginale was not detected in the ticks or bovine blood samples from Khentii district

Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci

Theileria parva is a tick‐transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti‐transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south‐western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen‐encoding genes that are targets of CD8+T‐cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south‐western Uganda. Cattle‐derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved