ESBL-Type and AmpC-Type Beta-Lactamases in Third Generation Cephalosporin-Resistant Enterobacterales Isolated from Animal Feces in Madagascar.

Third generation cephalosporin-resistant (3GCR) Enterobacterales are known to be prevalent in Madagascar, with high colonization or infection rates in particular in Madagascan patients. Extended spectrum beta-lactamases (ESBLs) have been reported to be the predominant underlying resistance mechanism in human isolates. So far, little is known on antimicrobial resistance and its molecular determinants in Enterobacterales and other bacteria causing enteric colonization of Madagascan wild animals. To address this topic, swabs from 49 animal stool droppings were collected in the Madagascan Tsimanapesotsa National Park and assessed by cultural growth of bacterial microorganisms on elective media. In addition to 7 Acinetobacter spp., a total of 31 Enterobacterales growing on elective agar for Enterobacterales could be isolated and subjected to whole genome sequencing. Enterobacter spp. was the most frequently isolated genus, and AmpC-type beta-lactamases were the quantitatively dominating molecular resistance mechanism. In contrast, the blaCTX-M-15 gene, which has repeatedly been associated with 3GC-resistance in Madagascan Enterobacterales from humans, was detected in a single Escherichia coli isolate only. The identification of the fosfomycin-resistance gene fosA in a high proportion of isolates is concerning, as fosfomycin is increasingly used to treat infections caused by multidrug-resistant bacteria. In conclusion, the proof-of-principle assessment indicated a high colonization rate of resistant bacteria in stool droppings of Madagascan wild animals with a particular focus on 3GCR Enterobacterales. Future studies should confirm these preliminary results in a more systematic way and assess the molecular relationship of animal and human isolates to identify potential routes of transmission.

Molecular phylogenetics of the sucking louse genus Lemurpediculus (Insecta: Phthiraptera), ectoparasites of lemurs, with descriptions of three new species.

Sucking lice live in intimate association with their hosts and often display a high degree of host specificity. The present study investigated sucking lice of the genus Lemurpediculus from six mouse lemur (Microcebus) and two dwarf lemur (Cheirogaleus) species endemic to the island of Madagascar, considered a biodiversity hotspot. Louse phylogenetic trees were created based on cytochrome C oxidase subunit I (COI), elongation factor 1α (EF1α) and internal transcribed spacer 1 (ITS1) sequences. While clustering according to host species was generally observed for COI and ITS1, suggesting high host specificity of the examined lice, EF1α sequences alone did not distinguish between lice of different Microcebus species, possibly due to rather recent divergence. As bootstrap support for basal tree structure was rather low, further data are necessary to resolve the evolutionary history of louse-mouse lemur associations. Three new species of sucking lice are described: Lemurpediculus zimmermanni sp. Nov. From Microcebus ravelobensis, Lemurpediculus gerpi sp.nov. from Microcebus gerpi, and Lemurpediculus tsimanampesotsae sp. nov. from Microcebus griseorufus. These new species are compared with all known congeneric species and identifying features are illustrated for all known species of Lemurpediculus.

Ticks and tick-borne pathogens in animals and humans in the island nations of Southeast Asia: A review.

Ticks are blood-feeding ectoparasites and major vectors of pathogens that cause infectious diseases in humans and animals worldwide including mammals, birds and reptiles. Despite the growing scientific effort in the 20th century, there is still limited information on ticks and tick-borne pathogens in Southeast Asia, especially concerning medical, veterinary, socioeconomic and agricultural aspects in the island nations. This review provides an overview of the current state of knowledge of ticks and their pathogens in the island nations of Southeast Asia and peninsular Malaysia. We aim to stimulate further research studies on ticks and tick-borne pathogens of human and veterinary importance in this geographical region.

Molecular detection of Rickettsia spp., Borrelia spp., Bartonella spp. and Yersinia pestis in ectoparasites of endemic and domestic animals in southwest Madagascar.

Little is known about the presence of vector-borne bacteria in southwest Madagascar. Anthropogenic alteration of natural habitats represents an important driver for the emergence of new diseases. Especially the involvement of livestock and the involuntary maintaining of invasive synanthropic animals (particularly rats) facilitate disease transmission from wildlife to humans and associated animals and vice versa. The dissemination or acquisition of ectoparasites is most likely in regions where human/wildlife contact is increasing. Little is known about the presence of vector-borne bacteria in southwest Madagascar. In 2016 and 2017, ectoparasites were collected from various introduced (cattle and goats, cats, dogs and chicken, rats and mice) and native animal species (mouse lemurs [Microcebus griseorufus], Grandidier's mongooses [Galidictis grandidieri], bastard big-footed mice [Macrotarsomys bastardi], greater hedgehog tenrecs [Setifer setosus] and lesser hedgehog tenrecs [Echinops telfairi]) in the northern portion of Tsimanampetsotsa National Park and the adjacent littoral region. Thirteen species of blood-feeding ectoparasites (235 individuals of ticks [5 species], 414 lice [4 spp.] and 389 fleas [4 spp.]) were investigated for the presence and identity of rickettsiae, borreliae, bartonellae and Yersinia pestis using PCR techniques. Rickettsia spp. were detected in every single ectoparasite species (Amblyomma variegatum, A. chabaudi, Rhipicephalus microplus, Haemaphysalis simplex, Argas echinops, Ctenocephalides felis, Echidnophaga gallinacea, Pulex irritans, Xenopsylla cheopis, Haematopinus quadripertusus, Linognathus africanus, L. vituli, Lemurpediculus verruculosus). Lice and ticks were found harboring rickettsiae identified as Rickettsia africae, while Rickettsia felis-like bacteria were associated with fleas. Borrelia spp. were detected in 5% of H. simplex and 1% of R. microplus ticks. Bartonella spp. were detected in 40% of H. quadripertusus pools and in 5% of L. verruculosus pools. Y. pestis was detected in X. cheopis and E. gallinacea fleas collected from a rat. This study presents the detection of a broad spectrum of vector-borne bacteria including potential pathogens, and an unexpected finding of Y. pestis far off the known plague foci in Madagascar.

Ectoparasites of endemic and domestic animals in southwest Madagascar.

Human encroachment of natural habitats bears the threat of disease transmission between native and introduced species that had not come into contact before, thus promoting the spread of new diseases in both directions. This is a matter of concern especially in areas where human-wildlife contact has not been intense in the recent past. In southwest Madagascar, we collected ectoparasites from various mammalian hosts and chicken, and examined their host preferences and their prevalence in relation to season and habitat degradation. Field-work took place in the northern portion of Tsimanampetsotsa National Park and the adjacent coastal strip (littoral) in the dry and in the rainy season of 2016/2017. Endemic mammals were trapped with live traps placed in habitats of different degrees of degradation: 1) relatively pristine forest, 2) degraded forest, 3) cultivated and shrub land. Rats and mice were also trapped in 4) villages. We identified 17 species of ectoparasites (296 individuals of ticks [5 species], 535 lice [7 spp.], 389 fleas [4 spp.] and 13 mites [1 sp.]) collected from 15 host species. There was no indication for seasonal or habitat effects on parasite infection. A large portion of the parasites was host-specific. Some ectoparasite species were shared either by several endemic or by several introduced species, but apart from the introduced flea species Echidnophaga gallinacea (collected from six different hosts including the endemic carnivore Galidictis grandidieri) no other ectoparasite species was shared between endemic and introduced host species.

Short and hyperthermic torpor responses in the Malagasy bat Macronycteris commersoni reveal a broader hypometabolic scope in heterotherms.

The energy budgets of animal species are closely linked to their ecology, and balancing energy expenditure with energy acquisition is key for survival. Changes in animals' environments can be challenging, particularly for bats, which are small endotherms with large uninsulated flight membranes. Heterothermy is a powerful response used to cope with changing environmental conditions. Recent research has revealed that many tropical and subtropical species are heterothermic and display torpor with patterns unlike those of "classical" heterotherms from temperate and arctic regions. However, only a handful of studies investigating torpor in bats in their natural environment exist. Therefore, we investigated whether the Malagasy bat Macronycteris commersoni enters torpor in the driest and least predictable region in Madagascar. We examined the energy balance and thermal biology of M. commersoni in the field by relating metabolic rate (MR) and skin temperature (Tskin) measurements to local environmental characteristics in the dry and rainy seasons. Macronycteris commersoni entered torpor and showed extreme variability in torpor patterns, including surprisingly short torpor bouts, lasting on average 20 min, interrupted by MR peaks. Torpid MR was remarkably low (0.13 ml O2 h-1 g-1), even when Tskin exceeded that of normothermia (41 °C). Macronycteris commersoni is thus physiologically capable of (1) entering torpor at high ambient temperature and Tskin and (2) rapidly alternating between torpid and normothermic MR resulting in very short bouts. This suggests that the scope of hypometabolism amongst heterothermic animals is broader than previously assumed and underlines the importance of further investigation into the torpor continuum.

Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar.

The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flaB sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar.

Tick (Amblyomma chabaudi) infestation of endemic tortoises in southwest Madagascar and investigation of tick-borne pathogens.

Little is known about the role of endemic ticks as vectors for bacterial and protozoan pathogens for animals and humans in Madagascar and their interaction in anthropogenic habitats where humans, their livestock and native Malagasy species (vectors and hosts) come into more frequent contact than in natural forest ecosystems. The aims of the study were (1) to test whether habitat degradation is associated with increased infestation of tortoises by ticks and (2) to investigate whether ticks carried Babesia, Borrelia or Rickettsia species that might be pathogenic for humans and livestock. We studied hard ticks of two endemic Malagasy tortoises, Astrochelys radiata and Pyxis arachnoides in March and April 2013 in southwest Madagascar. Two tortoise habitats were compared, the National Park of Tsimanampetsotsa and the adjacent degraded pasture and agricultural land at the end of the wet season. Ticks were screened for protozoan and bacterial pathogens via PCR on DNA isolated from ticks using genus-specific primers. Only one out of 42 A. radiata collected from both habitats had ticks. The low prevalence did not allow further analyses of the effect of habitat degradation. Forty-two P. arachnoides were found in the anthropogenic habitat and 36 individuals in the national park. Tick infestation rates of P. arachnoides differed significantly between the two study sites. Tortoises inside the park had lower tick prevalence than outside (8 of 36 (22%) versus 32 of 42 individuals (76%)) and infected animals tended to have fewer ticks inside than outside the park. All ticks collected in both habitats were adults of the ixodid tick Amblyomma chabaudi, which is supposed to be a host-specific tick of P. arachnoides. Screening for Borrelia sp. and Babesia sp. was negative in all ticks. But all A. chabaudi ticks were infected with Rickettsia africae, known to cause spotted fever in humans. Thus, habitat degradation seems to be linked to higher infestation of tortoises with ticks with possible consequences for humans and their livestock.